production of alkaline protease by streptomyces indicus.

ISBN: 978-81-931973-8-7
PRODUCTION OF ALKALINE PROTEASE
BY STREPTOMYCES INDICUS.
Dr. M.GURAVAIAH
DARSHAN PUBLISHERS
First published in India in 2016
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ISBN: 978-81-931973-8-7
Printed by: Darshan Publishers, Rasipuram, Namakkal, Tamil Nadu, India
PRODUCTION OF ALKALINE PROTEASE BY
STREPTOMYCES INDICUS.
Authored by
Dr. M. Guravaiah
Assistant Professor,
Department of Microbiology and Biotechnology (P.G),
J.K.C College , Guntur-6,
Andhra Pradesh, India.
EDITION
Published By
Darshan Publishers,
No: 8/173, Vengayapalayam, Kakkaveri, Rasipuram,
Namakkal, Tamil Nadu,
India – 637406.
www.darshanpublishers.com
e-mail:darshanpublishers12@gmail.com
ABOUT AUTHOR:-
Dr. M.GURAVAIAH did his M.Sc. (2005, Biotechnology),M Phil.(2008,
Biotechnology) and Ph.D. (2013, Biotechology) from Acharya
Nagarjuna
University (ANU). Guntur, India. Currently he is working as a Assistant Professor
in Department of Microbiology and Biotechnology (P.G),J.K.C College
,GUNTUR-6,ANDHRA PRADESH, INDIA.
ABOUT BOOK:Alkaline protease is one of the most important industrial enzymes. Which account
for about 60% of total enzymes used in world wide. Alkaline proteases and have
wide range of industrial applications. Alkaline proteases produced by micro
organisms are of main interest from a biotechnological prospective and are
investigated mostly in scientific field of protein chemistry and protein engineering
also in applied field such as detergents, food, leather and pharmaceuticals
industries. This book is very simple and easily understandable with all
experimental illustrations.
DEDICATED TO MY BELOVED
PARENTS,
WIFE and DAUGHTERS
PREFACE
We are pleased to present a new book on “Production of alkaline protease by
Streptomycin indicus” designed to meet the requirement of students .a full course in
Microbiology and Biotechnology has been introduced at Bachelor degree and Master degree
level to familiarize the modern science to the students .this book contain 8 chapters covering
all the topics prescribed in the syllabus of under graduate and master graduate students .this
book will cover all Practical’s of Microbiology and Biotechnology students .Our effort was
to make it very simple and well illustrated to develop interest about science in life sciences
particular .
Working past 11 years in field of Microbiology and Biotechnology ,we have
been developing new courses syllabus like M.Sc Animal Biotechnology and forensic sciences
.Now we are presenting the students a comprehensive book to meet the requirement of course
.As researcher and teacher of Biotechnology and Microbiology for the last 10 years ,it is our
humble endeavor to provide student latest information on this frontier area of biology and
students will find latest information on this such as DNA Sequencing ,Native-PAGE,
Zymograph and SDS-PAGE method .all the chapters are arranged as a continuous and
progressive theme for clear understanding of the modern students.
We hope students and teachers find this new book on “Production of alkaline
Protease by Streptomycin indicus” useful .suggestions to improve the book is always
welcomed and entertained wholeheartedly.
ACKNOWLEDGEMENT
I have great pleasure in expressing my profound sense of gratitude and
sincere thanks to my Guide and Teacher Dr. T.Prabhakar, Professor, University College of
Pharmaceutical Sciences, Andhra University, Visakhapatnam for his invaluable suggestions,
incredible patience, utmost care, keen interest and constant encouragement in shaping this
research work and to bring it to completion.
I express my sincere thanks to Dr.P.Sudhakar, Coordinator,
Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar for having
provided the necessary support for the completion of the research work.
I express my sincere thanks to Prof. K.R.S. Sambasiva Rao,
Professor, Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar
for having provided the necessary support for the completion of the research work.
I express my sincere thanks to Dr. A. Krishna Satya, Assistant
Professor, Dr. K. Kasturi. Assistant Professor, Dr. P. Udaya Sri . Assistant Professor,
Miss. N. Vijaya Sree. Assistant Professor, Dr. K. Haritha, Assistant Professor and Prof.Y.
Vikram Kumar, Honorary Visiting Professor.Acharya Nagarjuna University, Nagarjuna
Nagar for having provided the necessary support for the completion of the research work.
I express my sincere thanks to Dr.G.Girija Shankar, Associate Professor,
University College of Pharmaceutical Sciences, Andhra University, and Visakhapatnam for
having provided the necessary support for the completion of the research work.
I am very grateful to Prof. D. Gouri Sankar, Professor, Department of
Pharmaceutical Analysis, Andhra University, and Visakhapatnam for his timely help in my
doctoral program.
I am immensely grateful to the Management of J.K.C College, Guntur
for providing me the required infrastructure for the completion of the proposed work.
I am very much grateful to Sri. S.R.K. Prasad, Director, J.K.C.
College for providing the necessary leave of absence and permission for carrying out this
research work.
I am very grateful to my beloved parents, my sisters, my brothers, my
brother in-laws and friends who have given me the moral support during my course of study.
I would fail in my duties if I forget to thank my wife Smt. CH.
Bramaramba and my daughters M. Guru Prasanna , M. Guru Monish and M.Guru
Venkat Mahith Sai
who have been a constant and continuous source of delight and
encouragement during my research work. I would like to thank each and every one who are
directly or indirectly associated with this research work and without whose contributions this
dream could not have materialized.
I am equally thankful to The God Almighty for blessing me and giving
me the courage, strength, health and patience for completing this research work.
CONTENTS
Page No.
Preface
1
CHAPTER-I
Introduction
3
Objectives of the present investigation
4
Enzyme Technology
5
Production of Enzymes
12
References
13
CHAPTER-II
Review of literature
Proteases
14
Alkalophilic microorganisms
17
Application of alkaline proteases
23
Production of alkaline proteases
27
Isolation and purification of alkaline protease
34
Actinomycetes
41
References
46
CHAPTER-III
Page No
Analytical methods
60
Screening and isolation of protease producing actinomycetes
66
Taxonomic studies
78
Identification and characterization of selected isolates
86
References
98
CHAPTER-IV
Strain improvement studies
100
References
110
CHAPTER-V
Purification and partial characterization studies
111
References
142
CHAPTER-VI
Optimization of bioparameters for alkaline protease production by GAS-4
144
Experimental design and Optimization of protease production by
Plackett-Burmann design
155
References
165
CHAPTER-VII
Summary and Conclusions
168
Appendix
170
CHAPTER-I
INTRODUCTION
PREFACE
Proteases or proteolytic enzymes are degradative enzymes which catalyze the
hydrolysis of proteins and specifically act on the internal peptide bonds of proteins and
peptides (Bayoudh et al. 2000). Proteases are of the most important industrial enzymes
accounting for nearly 60% of total worldwide sale (Ward OP et al. 1985; Kalisz HM,
1988). Among the proteases alkaline proteases find wide industrial applications in the
detergent, food, pharmaceutical and leather industries as they have a high level of
activity over a broad range of pH. For this reason considerable attention has been paid to
the isolation of alkaliphilic microorganisms and study of their proteases.
Actinomycetes are biotechnologically important as they are prolific producers of
secondary metabolites like antibiotics. Recently they are being investigated for their
potential to produce bioactive compounds have anti inflammatory, hypertensive,
immunosuppressive and other bioactivities.
Actinomycetes are abundant in terrestrial soils and other natural habitats.
Actinomycetes producing alkaline proteases have been isolated from alkaline soils and
extreme environments such as soda lakes in Ethiopia ( Amare Gessesse et al. 2003).
In the present investigation an attempt has
been made to isolate protease
producing actinomycetes from soil samples collected from milk processing units and
other soils, optimize the bioprocess variables for submerged fermentation and finally to
characterize and evaluate the potential of the isolated enzyme for industrial use.
1
INTRODUCTION
Proteases are hydrolytic enzymes found in every organism to undertake important
physiological functions. These include: Cell division, regulating protein turnover,
activation of zymogenic performs, blood clotting, lysis of blood clot, processing and
transport of secretory proteins across membrane, nutrition, regulation of gene expression
and in pathogenic factors. Proteases differ in their specific activity, substrate specificity,
pH and temperature optima and stability, active site and catalytic mechanisms. All these
features contributed in diversifying their classification and practical applications in
industries involving protein hydrolysis.
Proteases are classified based on chemical nature of the active site, the reaction
they catalyze, their structure and composition (Rao et al. 1998). The major classes are
again classified into subclasses based on pH, catalytic site on polypeptide, occurrence
and so on.
Based on the catalytic site on the substrate, proteases are mainly classified into
endoproteases and exoproteases. Endoproteases preferably act at the inner region of the
polypeptide chain. By contrast, exoproteases preferentially act at the end of the
polypeptide. Exoproteases are further classified into aminopeptidases (those proteases
which act at the free N-terminus of the polypeptide substrate) and the carboxypeptidases
are those proteases which act at the free C-terminal of the polypeptide chain.
2
OBJECTIVE OF THE PRESENT INVESTIGATION
To isolate Protease producing actinomycetes from different terrestrial and water
samples, strain improvement and optimization of medium constituents and process
variables for maximizing protease production.
The objectives of the present study include:
1. Isolation of actinomycetes from different terrestrial and natural substrates.
2. Primary screening of the isolates to evaluate their protease producing capability.
3. Secondary screening of the isolates showing promising activity for selection of
the production media and determining the concentration of the constituents.
(a). Taxonomical studies for determination of morphological and biochemical
properties, cell wall composition and 16S rRNA.
4. Strain improvement studies using physical and chemical mutagens and selection
of the highest protease producing mutants.
5. Utilization of statistical methods for optimization of medium constituents and
process variables in submerged fermentation.
6. Purification and Characterization of the enzyme and determination of its
structure.
3
ENZYME TECHNOLOGY
With the development of the science of biochemistry, has come, a fuller
understanding of the wide range of enzymes present in living cells and their mode of
action. Although enzymes are formed only in living cells, many can be isolated without
loss of catalytic function in vitro. This unique ability of enzymes to perform their
specific chemical transformations in isolation has led to an ever-increasing use of
enzymes in industrial processes, collectively termed ‘enzyme technology’.
Microbial enzymes and coenzymes are widely used in several industries, notably
in detergent, food processing, brewing and pharmaceuticals. They are also used for
diagnostic, scientific and analytical purposes. Since ancient times they have been used in
the preparation of fermented foods, especially in oriental countries (Reed, 1975). At
present the economically most important enzymes are proteases, glucoamylases, glucose
isomerase, and pectinases. Over 300 tons of each enzyme is being produced annually.
Some of the microbial enzymes used industrially are shown in Table1.1 (Kumar, 1991).
It may be noted that most of these are hydrolases.
Most industrially important enzymes are extra cellular i.e secreted by the cells
into the ambient medium, from where they have to be recovered by removal and
separation from the cellular and other solid material.
Determination of enzyme activity
The enzyme activity is determined by the concentrations of enzyme, substrate, cofactors ,
allosteric effectors, the concentration and type of inhibitors, ionic strength, pH,
temperature and initial reaction time etc.
4
Table 1.1 Microbial Sources of some representative enzymes used industrially
Enzyme
Source
Amylase
Aspergillus oryzae, B. licheniformis, B.cereus. |B. megaterium, B.polymyxa
Cellulase
Aspergilus niger, trichoderma reeesi
Dextranase
Penicillium sp., Trichoderma sp.
Glucoamylase A.niger, Rhizopus sp.
Glucose
Bacillus coagulans, Actinoplanes sp., Arthrobacter sp, Streptomyces sp,
Isomerase
Some other Bacillus sp.
Invertase
Saccharomyces Cerecisiae
Lactase
Kluyveromyces fragilis, K.lactis, a. Niger
Lipase
Rhizopus sp, Candida lipolytica, Geotrichum candidum.
Pectinase
Aspergillus sp.
Protease
Aspergillus ap., Bacillus sp., Streptomyces griseus
Rennet
Mucor pusillus, Endothia Parasitica.
Many assay procedures for measurement of enzyme activity are available. The
rate of substrate conversion serves as a measure of the activity. The knowledge of
enzyme activity is necessary: to follow the production and isolation of enzymes, to
understand and determine the properties of commercial preparations and to ascertain the
correct amount of enzyme to be added to a particular commercial process.
5
The first step in deciding on a suitable assay is to choose the
appropriate substrate. Some of the substrates that have been used for the assay of
hydrolases are as follows (Collier, 1970).
Amylases and amyloglucosidases: Raw or soluble and modified starch of known
dextrose equivalent.
Cellulases: Cellulose power, cellular phosphate, filter paper and ground bran.
Pectinases: Pectic acid, pectin, pectinic acid and freeze-dried fruit purees.
Proteases: Casein, egg albumin, gelatin, hemoglobin, milk powder and raw meat.
Once the substrate is selected, the assay is carried out under
predetermined temperature, pH and incubation period. At the end of incubation period,
the reaction is readily stopped by the use of pH change or heat or by adding sufficient
enzyme inhibitor. The extent of reaction is then determined by a suitable chemical or
physical method.
Chemical methods
In assay of proteases (where casein or azocasein or any other
suitable substrate is used), the addition of trichloro acetic acid (TCA) causes the
precipitation of large peptide molecules, which can be removed by filtration or
centrifugation. Then the supernatant can be read at 280 nm.
 The unit of enzyme activity is defined in different ways by different authors. For
example
 One unit of enzyme activity represents the amount of enzyme required to liberate one
g of tyrosine per min per ml under standard assay conditions, where enzyme activity
is calculated by measuring g of tyrosine released, by comparing with the standard
(Tsuchida et al. 1986; Nehra et al. 1998).
 An increase of 0.1 absorbance unit against blank without enzyme under standard assay
conditions is considered as one unit of proteolytic activity. (Romero et al. 2001).
 One unit of enzyme activity corresponds to the increase in optical density by 0.1
against a blank, where enzyme is first treated with trichloro acetic acid and then
substrate is added under the standard assay conditions (Chrzanowdka, 1993).
Sometimes, it is desirable to prepare a control blank, where
enzyme solution is first mixed with TCA and casein is added next. This is done to correct
6
any color formation due to undigested proteins or incomplete termination of the reaction
by TCA.
Physical Methods
Most proteases cause milk to clot, the clotting time being
inversely proportional to the enzyme activity. The substrate is usually prepared from
dried milk, which is easier to standardize than fresh milk. Enzyme is added to the
solution of fat free milk, which is already at the correct temperature, pH and calcium
concentration.
The time taken to form visible curd flake is noted (It should be
between 1 to 5 minutes). The initial production of these flakes is accurately seen if the
reaction solution is well mixed and if only a thin film of this continually blowing liquid
is observed.
Sources of Enzymes
Enzymes can be obtained from plant, animal and microbial sources.
Plant source: β-amylase, papain, bromelain, urease, ficin, polyphenol oxidase
(tyrosinase), lipoxygenase etc.
Animal Source: Pepsin, lipase, lysozyme, rennin, trypsin, phosphor-mannase,
chymotrypsin etc.
Microbial Source: α-amylase, pencillin acylase, protease, invertase, lactase, dextranase,
pectinase, pullulanase etc.
In general, the enzymes from plant and animals are considered to
be more important than those from microbial sources, but for both technical and
economical reasons, microbial enzymes are considered to be more important. Therefore
increasing efforts are being persuaded to produce enzymes by microbial fermentation.
Advantages of microbial enzymes
 Animal sources for enzymes are very limited. Microorganisms are attractive because
of their biochemical diversity.
 They have short generation time and require smaller area, 20kg of rennin is produced
in 12 hrs by B. subtilis with liter fermentor where as one calf stomach gives 10 kg after
several months.
 Feasibility of bulk production and ease of extraction.
 Use of inexpensive media.
 Ease of developing simple screening procedures.
7
 Strain development by genetic engineering to produce abnormal amounts.
 Synthesis of foreign enzymes by genetically engineered microorganisms.
 Absence of seasonal variations.
Until 1985, about 2500 enzymes were known, out of which only
250 enzymes find commercial applications and another 200 were available for use in
genetic engineering. These include restriction endonucleases, ligases and editing
enzymes (editases).
Only a handful enzymes have attained the status of being
industrially significant by virtue of their role in well established and well defined
commercial applications (e.g bacterial α-amylase, amyloglucosidase, alkaline protease,
urease, papain, pencillin acylase, glucose isomerase etc.). While some other enzymes are
awaiting the status of significant enzymes (e.g. lipase fungal α-amylase, acid protease
etc.).
With the advent of biotechnological methods in the manipulation
of proteins, the classical biocatalysts, enzymes have metamorphosed into an important
tool, finding wide range of industrial applications. The advantage of adopting enzymes
as industrial reagents is because of their efficiency, precision, specificity, convenience
and economics. They are replacing chemical catalysts in many reactions where value
added products are produced.
The prospects for enzymes application have improved due to developments
in the following areas:
 High yields can be obtained by genetic manipulation. Hansenula polymorpha, yeast
has been genetically modified, so that 35% of its total protein consists of the enzyme
alcohol oxidase.
 Optimization of fermentation conditions via induction of enzymes production, use of
low cost nutrients and introduction of fed batch fermentation.
 Release of enzymes from cells by means of new cell breaking methods.
 Modern purification methods such as affinity chromatography, ion-exange
chromatography and precipitation.
 Development of processes for the immobilization of enzymes and for their recycling. The proportion of enzyme cost in some processes becomes only a few percent.
 Continuous enzyme production in special reactors, which minimizes the cost for a
new system in continuous operations.
8
 The following enzymes are currently produced commercially: amylase, protease,
penicillin acylase, isomerases, catalases etc.
 Analytical enzymes used for analytical purpose: glucose oxidase, cholesterol
oxidase etc.
 Enzymes used in medicine: proteases, streptokinase, asparaginase etc.
Table 1.2.The current applications of enzymes and their sources.
Enzyme
Source
Region of application
Dextranase
Penicillium sp. Trichoderma sp.
Dental hygiene
(cosmetic/healthcare)
Proteases
Papain
Papaya Latex
Meat tenderization (food industry)
Latex of ficus
Latex of Ficus carica
Dissolves scrap film to recover the
silver.
Trypsin
Beef pancreas
Mucolytic action, wound cleaning
(therapeutics)
Chymotrypsin
Beef pancreas
Along with trypsin treatment
Pepsin
Beef stomach
Digestive agent (therapeutics)
Renin
Beef
stomach,
Bacterial- Curdling
B.subtilis, Fungal- A . Oryzae
of
milk
manufacture
for
(food
cheese
&
food
(food
&
drink
(food
&
drink
processing)
Pectinases
Aspergillus niger, A. wentii
Fruit
juices,
industry)
Lipases
Rhizopus sp., Candida lipolytica.
Fat
synthesis
industry).
Penicillin
E.coli. Penicillium sp.
penicillins.
acylase
Invertase
In the preparation of semisynthetic
Saccharomyces cerevisiae
Confectionary
(food
&
drink
industry)
Cellulase
A.niger, Trichoderma reesei.
Cellulase
production
(food
industry).
Glucose
Oxidase
A.niger., P.amagasakiense
Blood
Glucose
estimation
(diagnostics), antioxidant (food &
9
drink industry)
Amylases
B.amyloliquifaciens
Maltose production, baking (food
processing).
B.subtilis, B. polymyxa
Paper making, alcohol production
(chemical industry)
A.oryzae
Degumming
of
silk
(textile
industry)
Glucose
Bacillus
sp.,
isomerase
Actinoplanes sp.,
B.
coagulans, Fructose production (food & drink
industry)
10
PRODUCTION OF ENZYMES
There are three basic techniques by which enzymes can be produced:
1. Semi-solid culture
2. Submerged culture
3. Multi- stage continuous submerged culture.
Submerged batch culture is more important of these two, since
most commercially import enzymes are growth associated .Multistage culture is only
applicable to those cases where product formation is non-growth- associated.
The following are the factors of importance in enzyme production:

Microbial strain and its metabolic behaviour.

Growth rate.

Medium components.

Culture conditions: temperature, pH aeration and addition of surfactants.

Regulatory mechanisms: Induction, feedback repression and catabolic repression.
11
REFERENCES:
Amare Gessesse, Hatti-Kaul, R., Berhanu A. Gashe and Mattiasson, B. (2003). Novel
Alkaline protease fom alkaliphilic bacteria grown on chicken feather. Enz. Microb.
Technol.32:519-527.
Bayoudh, a., Gharsallah, N., Chamkha, M.Dhouib, A.; Ammar, S. and Nasri, M. (2000):
Purification and characterization of an alkaline protease from Pseudomonas aeruginosa
MNI. J. of Industrial Microbiology & Biotechnology. 24: 291-295.
Chrzanowdka, J., Kolaezkowak, M. And Polanowski, A.( 1993). Production of
exocellular
proteolytic enzymes by
various
species
of Penicillium
Enzyme
Microb.Technol. 15:140-143.
Collier, b.(1970), Thiol-dependent dissociation of a fraction of toxin into enzymically
active and inactive fragments.Process Biochem.5:39-40.
Kalisz, HM.(1983). Microbial Proeinasses. Adv Biochem. Eng. Biotechnol. 36:1-65.
Kumar, H.D. (1991). A text Book of Biotechnology Affilaited East-West. Press(P)Ltd,
ed ,New Delhi.
Nehra, K.S., Santhosh, D., Kamala, C. and Randhir, S.(1998). Production of alkaline
protease by Aspergillus sp. under submerged and solid substrate fermentation .Indian J.
Microbio. 38:153.
Rao, M.B., Tanksale, A.M., Ghatge, M.S and Deshpande, V.V , (1998). Molecular and
Biotechnological aspects of microbial proteases. Microbiol, Mol.Bol. Rev.62:597-635.
Reed, G.(1975). Enzymes in Food Processing., (2nd edition), Academic Press, Orlando.
Romero, F.J., Garcia, L.A., Salas, J. A., Diaz, M. And Quiors, L. M.(2001). Production,
purification and partial characterization of two extracellular proteases from Serratia
marcescens grown in whey .Process Biochem.36:507.
Tsuchida, O., Yamagota, Y., Ishizukz, J., Arai, J., Yamada, J., Takeuchi, M.and
Ichishima, E.(1986). An alkaline proteinase of an alkalophilicBacillus sp. Curr.
Microbiol. 14:7.
Ward, O. P. (1985) Proteolytic Enzymes., In: Blanch HW, Drew S, Wang DI, eds.
Comprehensive Biotechnology, Oxford, UK: Pergamon Press. 3: 789-818.
12
CHAPTER-II
REVIEW OF LITERATURE
PROTEASES
Among the large number of microbial enzymes, proteases occupy
a pivotal position owing to their wide applications. The current estimated value of the
worldwide sales of microbial enzymes is$ I billion and proteases alone account for about
60% of the total sales and they were the first enzymes to be produced in bulk (Meenu et
al. 2000; Neurath, 1989; Manject Kaur et al. 1998).Milk clotting enzymes have been
used to transform milk into products such as cheese since about 5000 BC. Pancreatic
proteases were used for dehairing of hides and as pre-soak detergents since about
1910.Now; pancreatic proteases are largely replaced by microbial proteases.
Alkaline proteases are a physiologically and commercially
important group of enzymes which are primarily used as detergent additives. They play a
specific catalytic role in the hydrolysis of proteins. In 1994, the total market for
industrial enzymes account for approximately $400 million, of which enzymes worth
$112 million were used for detergent purposes (Hodgson, 1994).In Japan, 1994, alkaline
proteases sales were estimated at15,000million yen (equivalent to $116million
)(Horikoshi,1996).This enzyme accounts for 40%of the total worldwide enzyme sales. It
is expected that there will be an upward trend in the use of alkaline proteases in the
future.
Proteases are broadly classified into two groups – peptidases and
proteinases. Peptidases hydrolyze peptide bonds from either N or C terminal end of the
protein chain, or in other words, hydrolyze bonds of amino acids, which are outside.
Peptidases were formerly called as exopeptidases. The proteinases hydrolyze peptide
bonds within the protein chain or in other chain or in other words, hydrolyze amino acids
in the middle of the chain. They were formerly referred to as endopeptidases.
A more rational system of proteases classification is based on a
comparison of active sites, mechanism of action and 3-D structure (Rawlings and Barret,
1993).
Proteases can also be classified on the basis of
a) pH
b) Substrate specificity
c) Similarity in action to well characterized enzymes like trypsin, chymotrypsin and
elastase
13
d) Active site amino acid residue and catalytic mechanism.
More conventionally, proteases are classified into 4 important
groups like serine, cysteine, aspartic and metallo proteases.
Serine proteases
Serine proteases are the most widely distributed group of
proteolytic enzymes of both microbial and animal origin (salvesen and Nagase, 1983).
The enzymes have a reactive serine residue in the active site and are generally inhibited
by diisopropyl fluorophosphate (DFP) and phenyl methyl sulphonyl fluoride (PMSF).
Most of the proteases are also inhibited by some thiol reagents, such as Pchloromercuric benzoate(pCMB).These are generally active at neutral and alkaline pH,
with an optimum pH, between 7-11. They have broad substrate specificities, including
considerable esterolytic activity towards many ester substrates, and are generally of low
molecular weight (18.5-35 kDa).
Cysteine proteases
Cysteine proteases are sensitive to sulphydryl reagents, such as
pCMB, Tosyllysine Chloromethyl Ketone (TLCK), iodoacetic acid, iodoacetamide,
heavy metals, and are activated by reducing agents such as potassium cyanide or
cysteine,dithiotheitol, and ethylene diamine traacetic acid (EDTA). The occurrence of
cysteine proteases has been reported in only a few fungi (Kalisz, 1988).Intracellular
enzymes with properties similar to cysteine proteinase have been reported in Trichosporn
species, Oidiodendron kalrai and Nannizzia fulva. Extracellular cysteine proteases have
been observed in Microsporium species, Aspergillus oryzazae, and Sporotrichum
pulverulentum .
Aspartic proteases
Aspartic proteases are characterized by maximum activity at low
pH (3-4) and insensitivity to inhibitors of the other three groups of enzymes.They are
widely distributed in fungi, but are rarely found in bacteria or protozoa. Most aspartic
proteases have molecular weights in the range 30-45 kDa and their isoelectric points are
usually in the pH range of 3.4-4.6.
Metalloproteases
All these enzymes have pH optima between pH 5-9 and are
sensitive to metal chelating reagents, such as EDTA, but are unaffected by serine
protease inhibitors or sulphydryl agents (Salvesen and Nagase, 1983). Many of the
EDTA-inhibited enzymes can be reactivated by ions such as zinc, calcium, and cobalt.
14
These are widespread, but only a few have been reported in fungi. Most of the bacterial
and fungal metalloproteases are zinc-containing enzymes, with one atom of zinc per
molecule of enzyme. The zinc atom is essential for enzyme activity. Calcium is required
to stabilize the protein structure.
Based on their optimal pH proteases are also classified as:
1) Acid proteases
Acid proteases are proteases which are active in the pH ranges of 2-6 (Rao et al.
1998) and are mainly of fungal in origin (Aguilar et al. 2008). Common examples
in this subclass include aspartic proteases of the pepsin family. Some of the
metalloprotease and cystein proteases are also categorized in as acidic proteases.
2) Neutral proteases
Neutral proteases are proteases which are active at neutral, weakly alkaline or
weakly acidic pH .Majority of the cystein proteases, metalloproteases, and some
of the serine proteases are classified under neutral proteases. They are mainly of
plant in origin, except few fungal and bacterial neutral proteases (Aguilar et al.
2008).
3) Alkaline proteases
Alkaline proteases are optimally active in the alkaline range (pH 8-13), though
they maintain some activity in the neutral pH range as well (Horikoshi, 1996).
They are obtained mainly from neutralophilic and alkaliphilic microorganisms
such as Bacillus and Streptomyces species. In most cases the active site consists
of a serine residue, though some alkaline proteases may have other amino acid
residue in their active site (Rao et al. 1998).
15
ALKALOPHILIC MICROORGANISMS
All microorganisms follow a normal distribution pattern based on
the pH dependence for their maximum growth, and the majority of these microorganisms
are known to proliferate well at near neutral pH values. As the pH moves away from this
neutral range the number of microorganisms decreases. However, some neutrophilic
organisms are capable of growth even at extreme pH conditions. This is primarily due to
the special physiological and metabolic systems, enabling their survival and
multiplication under such adverse conditions (Krulwich et al. 1990; Krulwith and
Guffanti, 1989). Such microorganisms may also be referred to as pH dependent
extremophiles.
Alkalophilic microorganisms constitute a diverse of group that
thrives in highly alkaline environments. They have been further categorized into two
broad groups, namely alkalophiles and alkalotolerants. The term alkalophiles is used for
those organisms that were capable of growth above pH 10, with an optimal growth
around pH 9, and are unable to grow at pH 7 or less (Krulwich, 1986). On the other
hand, alkalotolerant organisms are capable of growing at pH values 10, but have an
optimal growth rate nearer to neutrality (Hodgson, 1994). The extreme alkalophiles have
been further subdivided into two groups, namely facultative and obligate alklophiles.
Facultative alkalophiles have optimal growth at pH 10 or above but can grow well at
neutrality, while obligate alkalophiles fail to grow at neutrality .
Isolation and Screening
Vonder (1993) has reported the isolation of obligate alkalophilic
organisms from human and animal feces in 1993. He briefly described these organisms
and proposed the name Bacillus alcalophilus for his strains and also stated that he had
been able to prove that life exists that not only tolerates, but also depends on, a highly
alkaline pH. Today, many of these alkalophilic Bacillus strains and other alkalophiles are
of considerable industrial importance, particularly for use of their proteases in laundry
detergents (Aunstrup et al. 1972). Normal garden soil was reported to be a preferred
source for isolation, presumably because of the various biological activities that generate
transient alkaline conditions in such environment (Grant et al. 1990). These organisms
were also isolated from nonalkaline habitats, such as neutral and acidic soils, and thus
appear to be fairly widespread.
One of the most important and noteworthy features of many
alkalophiles is their ability to modulate their environment. They can convert neutral
16
medium or high alkaline medium to optimize external pH for growth (Krulwich and
Guffanti, 1983).
In natural environments, sodium carbonate is generally the major
source of alkalinity. Its addition to the isolation media enhances the growth of
alkalophilic microorganisms (Grant et al. 1979). The addition of sodium carbonate to the
medium for the isolation of alkalophilic. Actinomycetes results in brown color and
cracking of the medium (Kitada et al.1987). At temperatures >70 C, agar based media
usually lose their gel strength, making them useless for isolation of thermophiles . As a
result, the need for gelling agents with good thermal stability led to the discovery of
agents, such as Gelrite TM (Deming and Baross,1986) and an optimized concentration (3
&w/v) of bacteriological grade agar.
Isolation media
The primary stage in the development of an industrial fermentation
process is to isolate strain (s) capable of producing the target product in commercial
yields. This approach results in intensive screening programs to test a large number of
strains to identify high producers having novel properties. In the course of designing a
medium for screening proteases, it is essential that the medium should contain likely
inducers of the product and be devoid of constituents that may repress enzyme syntheses.
Normally, alkalophilic organisms are isolated by surface plating on a highly alkaline
medium and subsequent screening for the desired characteristics. The organisms are
further grown on specific media for estimating proteolytic activities using appropriate
substrates such as skimmed milk or casein. The isolates, exhibiting desired level of
activity are chosen and maintained on slants for further use. The most commonly used
general medium for the isolation of alkalophiles has been described by Horikoshi (1971).
Several types of defined media have also been used for their isolation, which include
nutrient agar (Jashi and Ball, 1993),glucose-yeast extract-asparagine agar (GYA) (Sen
and Satyanarayana, 1993),MYGP agar (Srinivasa et al. 1983),peptone- yeast extractglucose (PPYG)media (Gee et al. 1980), wheat meal agar (Fujiwara and Yamamoto,
1987).the medium composition was varied by several workers to isolate microorganisms
of choice, such as those with high proteolytic activity or those that were thermostable.
For any type of medium, a high pH value is essential to isolate the obligate alkalophiles
(Grant and Tindall, 1980).
Extra cellular alkaline protease producing Streptomyces species is
an isolated from soil which was characterized and tentatively identified as Sterptomyces
17
aurantiogriseus EGS-5 was cultivated in production medium investigated by Rao and
Narasu (2007).
Alkalophilic microorganisms which have been screened for use in
various industrial applications, predominantly, members of the genus Bacillus and other
species were found to be prolific source of alkaline proteases. The different alkaline
protease-producing Bacillus species and strains are summarized in Table 2.1 (Steele et
al. 1992).
Several fungi have also been reported to produce extracellular
alkaline proteases (Matsubara and Feder, 1971). The different alkaline proteases
producing fungal species are summarized in Table 2.2 similarly, some yeasts were also
reported to produce alkaline protease, which include Candida lipolytica (Tobe et al.
1976); Yarrowia lipolytica (Ogrydziak, 1993), and Aureobasidium Pullulans (Donaghy
and McKay, 1993).
Halophiles that were described to produce alkaline proteases
included Holobacterium sp. Alkaline proteases are also produced by some rare
actinomycetes. Some of the commercially exploited microorganisms for alkaline
proteases are shown in Table 2.3.
18
Table 2.1 Some alkaline protease producing Bacillus species
Bacillus sp.and their strains
References
B.firmus
Moon and parulekar, 1991;
B.alcalophilus
Sharma et al. 1994
B.alcalophilus subsp.halodurans KP1239
Takii et al. 1990
B.amyloliquefaciens
Malathi and Chakoshi,1991
B.licheniformis
Horikoshi,1987
B.proreolyticus
Boyer and Byng,1996
Bacillus alcalophilus ATCC
21522
Horiloshi,1996
(Bacillus sp.No. 221)
B.subtilis
Chu et al. 1992
B.thuringiensis
Hotha and Banik,1997
Bacillus sp.Ya-B
Tsai et al. 1983
Bacillus sp.B21-2
Fujiwara and Yamamoto, 1987
Bacillus sp. Y
Shimogaki et al. 1991
Bacillus sp. KSM-K16
Kobayashi et al. 1996
19
Table 2.2 Alkaline protease producing fungal species
Fungal species
References
A.flavus
Chakraborty and Srinivasan, 1993
A.fumigatus
Monod et al. 1991; Larcher et al. 1996
A. melleus
Luisetti et al. 1991
A.sulphureus
Danno, 1970
A.niger
Barthomeuf et al. 1992
A.oryzae
Nakadai et al. 1973
Cephalosporium sp. KSM 388
Tsuchiya et al. 1987
Chrysosporium Keratinophilum
Dozie et al. 1994
Entomophthora coronata
Jonsson, 1968
Fusarium graminearum
Phadatare et al. 1993
Penicillium griseofulvum
Dixit and Verma, 1993
Fusarium sp.
Kitano et al. 1992
P.lilacinus
Den Belder et al. 1994
20
Table 2.3 Commercial producers of alkaline proteases
Organism
Trade name
Manufacturer
Bacillius licheniformis
Alcalase
Novo Nordisk, Denmark
Protein engineered variant
Durazym
Novo Nordisk, Denmark
Of Savinase ®
Protein engineered variant
Maxapem
Solvay Enzymes GmbH, Germany
Of alklophilic Bacillus sp.
Alkalophilic Bacillus sp.
Savinase, Esperase
Novo Nordisk,Denmark
Alkalophilic Bacillus sp.
Maxacal, Maxatase
Alkalophilic Bacilus sp.
Opticlean, ptimase
Solvsy Enzymes GmbH,,Germany
Alkalophilic Bacillus sp.
Proleather
Amano Pharmaceuticals Ltd. Japan
Aspergillus sp.
Protease P
Amano pharmaceuticals Ltd. Japan
Gist-Brocades, The Netherlands
21
APPLICATIONS OF ALKALINE PROTEASES
Alkaline proteases are robust enzymes with considerable industrial
potential in detergents, leather processing, silver recovery, and medical purposes, food
processing, feeds, and chemical industries, as well as waste treatment. The different areas
of applications currently using alkaline proteases are:
Detergent Industry
The detergent industry has now emerged as the single major
consumer of several hydrolytic enzymes acting in the alkaline pH range. Detergents
containing different enzymes: proteases, amylases and lipases are available in the
international markets under several brand names. The use of different enzymes as
detergent additives arises from the fact that proteases can hydrolyze proteinaceous stains
and amylases are effective against starch and other carbohydrate stains while lipases are
effective
against oily or fat at alkaline pH and it should also be compatible with
detergents. (Aunstrup and Andersen, 1974)
The interest in using alkaline enzymes in automatic dishwashing
detergents has also increased recently (Charyan, 1986; Glover, 1985). The enzyme
detergent preparations presently marked for cleaning of membrane systems are Alkazym
(Novodan A/S, Copenhagen, Denmark), Terg-A-Zyme (Alconox, Inc, New York, USA)
and Ultrasil 53 (Hankel kGaA, Dusseldorf, Germany). In addition, contact lens cleaning
solution containing alkaline protease derived from a marine shipworm bacterium was
used for the cleaning of contact lens at low temperatures.In India, one such enzyme
based optical cleaner (available in the form of tablets containing Subtilopeptidase is
presently marketed by M/S Bausch and Lomb (India) Ltd.
Leather Industry
Another industrial process, which has received attention, is the
enzyme-assisted dehairing of animal hides and skin in the leather industry. Traditionally,
this process is carried out by treating animal hides with a saturated solution of lime and
sodium sulphide, besides being expensive and particularly unpleasant to carry out, a
strongly polluting effluent is produced. The alternative to this process is
enzyme-
assisted dehairing. Enzyme- assisted dehairing is preferentially possible if proteolytic
enzymes can be found that are stable and active under the alkaline conditions (pH 12) of
tanning.
22
Early attempts using a wide variety of enzyme were largely
unsuccessful, but proteases from certain bacteria which are alkalophilic in nature have
been shown to be effective in assisting the hair removal process (Taylor et al.
1987).several alkaline proteases from alkalophilic actinomycetes have also been
investigated for this purpose. Some as hair, feather, wool, etc. at alkaline pH and may
have commercial applications (Horikoshi and Akiba, 1982).
Silver recovery
Alkaline proteases find potential application in the bioprocessing
of used X-ray films for silver in its gelatin layers. The conventional practice of silver
recovery by burning film causes a major environment pollution problem. Thus, the
enzymatic hydrolysis of the gelatin layers on the X-ray film enables not only silver, but
also the polyester film base, to be recycled.
Medicinal uses
Collagenases with alkaline protease activity are increasingly used
for therapeutic application in the preparation of slow- release dosage forms. A new semialkaline protease with high collagenolytic activity was produced by Aspergillus niger
LCF9.the enzyme hydrolyzed various collagen types without amino acid release and
liberated low molecular weight peptides of potential therapeutic use (Barthomeuf et al.
1992).
Food Industry
Alkaline proteases can hydrolyse proteins from plants fish or
animals to produce hydrolysates of well- defined profile. The commercial alkaline
protease, Alcalase has a broad specificity with some preference for terminal hydrophobic
amino acids. Using this enzyme, a less bitter hydrolysate (Adler Nissen, 1986) and a
debittered enzymatic whey protein hydrolysate (Nakamura et al. 1993) were produced.
Very recently, another alkaline protease from B.amyloliquefaciens
resulted in
the production of a methionine-rich protein hydrolysate from chickpea
protein (George et al. 1997).The protein hydrolysates commonly generated from casein,
whey protein and soya protein find major application in hypoallergenic infant food
formulations
( American Academy of pediatrics Committee on Nutrition, 1989). They
can also be used for the fortification of fruit juices or soft drinks and in manufacturing of
protein rich therapeutic diets (Adamson and Reynolds, 1996; Parrado et al. 1991).
In addition, protein hydrolysates having angiotensin-1 converting
enzyme inhibitory activity were produced from sardine muscle by treatment with a
23
B.lichemformis alkaline protease. These protein hydrolysates could be used effectively
as a physiologically functional food that plays an important role in blood pressure
regulation (Matsui et al. 1993)
Further, proteases play a prominent role in meat tenderization;
especially of beef. An alkaline elastase (Takagi et al. 1992) and alkaline protease
(Wilson et al. 1992) have proved to be successful and promising meat tenderizing
enzymes, as they possess the ability to hydrolyze connective tissue proteins as well as
muscle fiber proteins. A method has been developed in which the enzyme is introduced
directly in the circulatory system of the animal, shortly before slaughter (Bernholdi,
1975) or after stunning the animal to cause brain death (Warren, 1992).
A potential method used a specific combination of neutral and
alkaline proteases for hydrolyzing raw meat. The resulting meat hydrolysate exhibited
excellent organoleptic properties and can be used as a meat flavoured additive to soup
concentrates. Hydrolysis of over 20% did not show any bitterness when such
combinations of enzymes were used. The reason for this may be that the preferential
specificity was favorable when metalloproteinase and serine proteinase were used
simultaneously (Pedersen et al. 1994).
Waste treatment
Alkaline proteases provide important application for the
management of wastes from various food processing industries and household activities.
These proteases can solubilize wastes through a multistep process to recover liquid
concentrates or dry solids of nutritional value for fish or livestock (Shoemaker,
1986;Shih and Lee,1993).
Dalev (1994) reported an enzymatic process using a B.subtilis
alkaline protease in the processing of waste feathers from poultry slaughter houses. The
end product was a heavy, grayish powder with a very high protein content, which could
be used as a feed additive.
Similarly, many such other keratinolytic alkaline proteases were
used in food technology (Dhar and Sreenivasulu,1984; Chandrasekharan and Dhar,1986;
Bockle and Miller,1997) for the production of amino acids or peptides(Kida et
al.1995),for degrading waste keratinous material in household refuse (Mukhopadhay and
Chandra,1992), and as a depilatory agent to remove hair in bath tub drains, which caused
bad odors in houses and in public places(Takami et al.1992).
24
Chemical Industry
It is now firmly established that enzymes in organic solvents can
expand the application of biocatalysts in synthetic chemistry. However, a major
drawback of this approach is the strongly reduced activity of enzymes under anhydrous
conditions. Thus, it is of practical importance to discover ways to activate enzymes in
organic solvents. Some studies have demonstrated the possibility of using alkaline
proteases to catalyze peptide synthesis in organic solvents (Chen et al. 1991; Nagashima
et al.1992; Gololobov et al. 1994). In addition, many efforts to synthesize peptides
enzymatically have employed proteases immobilized on insoluble supports (Wilson et al.
1992).
A sucrose-polyester synthesis was done in anhydrous pyridine
using Proleather, a commercial alkaline protease preparation from Bacillus sp. (Patil et
al.1991). The Proleather also catalyzes the transesterification of D-glucose with various
acyl donors in pyridine (Watanabe et al. 1995).
Further, the enzyme Alcalase acted as catalyst for resolution of Nprotected amino acid esters (Chen et al. 1991) and alkaline proteases from Conidiobolus
coronatus was found to replace subtilisin Carlsberg in resolving the racemic mixtures of
DL-phenylalanine and DL-phenylglycine (Sutar et al;1992).
25
PRODUCTION OF ALKALINE PROTEASES
In industrial strain development, strain potential is certainly the
most important factor, but not the only one to consider. The best potential of a strain is
realized only under the best – regulated process regimen. In the absence of the latter, it is
possible to get the best strain, but end up with mediocre fermentation performance. Thus
production of a metabolite in excess of normal is also determined by the nutritional and
environmental conditions during the growth.
Media development
The appropriate selection of medium components based on both
aspects of regulatory effects and economy is the goal in designing the chemical
composition of the fermentation media, where the nutritional requirement for growth and
production must be met. Fast formation and high concentration of the desired product are
the criteria for the qualitative and quantitative supplement of nutrients and other
ingredients.
Further a continuing study of fermentation conditions should be
done as an important part of a strain development program as new mutant strains will be
obtained that may perform better, under conditions other than those originally developed
from the parent culture. Thus in any enzyme fermentation, the principle aim would be to
minimize the cost of manufacture by optimizing both the fermentation and recovery
processes using high producer.
This it is important to recognize that the development of strain for
fermentation process requires a triangular interaction among culture improvement,
development of media and optimization of process conditions. Any improvement made
in one of these areas will suddenly lead to numerous opportunities in the other two areas
.This triangular interaction on is s an endless cycle. The reward of running this cycle is
increased productivities, decreased costs and a more readily available supply of health
and life-saving pharmaceuticals.
Most alkalophilic microorganisms produce alkaline proteases,
though interest is limited only to those that yield substantial amounts. It is essential that
these organisms be provided with optimal growth conditions to increase enzyme
production. The Culture conditions that promote protease production were found to be
significantly different from the culture conditions promoting cell growth (Moon and
Parulekar, 1991). In the industrial production of alkaline proteases, technical media were
usually employed that contained very high concentrations (100-150 g dry wt
of
26
complex carbohydrates, pro-teins, and other media components). With a view to improve
an economically feasible technology, research efforts are mainly focused on: (1)
Improvement in the yields of alkaline proteases and (ii) Optimization of the fermentation
medium and production conditions.
Improvement of Yield
Strain improvement plays a potential role in the
commercial development of microbial fermentation processes. As a rule the wild
strains usually produce limited quantities of the desired enzyme to be useful for
commercial application (Glazer and Nikaido, 1995). However,
in
most cases, by adopting
simple selection methods, such as spreading of the culture on specific media, it is
possible to pick colonies that show substantial increase in yield (Aunstrup, 1974).
Conventional physical and chemical mutagens are used for screening of high yielding
strains (Sidney and Nathan, 1975).
Strain improvement to overproduce a given product relies
heavily on random mutagenesis and the subsequent selection of, or screening for
overproducing mutants. The development of mutants by actinomycetes has long been
recognized. These were considered as special type of variants. The formation of new
strains through the mutation of a culture, however, is more fundamental and hereditary.
White strains were obtained from blue-pigmented forms; strains free from aerial
mycelium, from those producing such mycelium; red strains from orange-yellow forms.
These mutations were accompanied by changes in morphological, cultural and
physiological characters which differentiated the new strains from the parent cultures.
The difference thus obtained may be so distinct as to give the new strain a characteristic
of a species. In recent years, extensive use has been made of the mutagenic effects of
irradiation and of certain chemical agents. These have found extensive application in
obtaining special strains of organisms.
Jensen reported that, under the influence of
ultraviolet rays, strains of Nocardia, isolated from Australian soils, gave rise to new
forms, some of these resembled typical species of Streptomyces and others were closely
related to the mycobacteria.A strain of Streptomyces griseus kept for a long time (more
than 30 years) in the culture collection and which was inactive antibiotically was
induced to form a mutant that produced streptomycin .The exposure of spores of
Streptomycin sp. to ultraviolet and gamma rays results in hereditary changes affecting
27
colony morphology and pigmentation. These changes are largely associated with
instabilities that result in further variation during colony growth and spore formation,
These instabilities persist indefinitely, giving rise to new variants having their own
patterns of instability .These changes differ from gene mutations in that they can be
induced with much greater frequency UV-sensitive mutants were isolated from
Streptomyces coelicolor and S. clavuligerus .Which showed a hyper mutable phenotype.
Optimization Of Fermentation Medium
Nutritional and environmental conditions optimization by the
classical method of changing one independent variable (nutrient, antifoam, pH,
temperature, etc.) while fixing all the others at a certain level can be extremely time
consuming and expensive for a large number of variables. To make a full factorial
search, which would examine each possible combination of independent variable at
appropriate levels, could require a large number of experiments xn, where x is the number
of levels and n is the number of variables. Other alternative strategies of conventional
medium optimization must, therefore, be considered which allow more than one variable
to be changed at a time. These methods have been discussed by several investigators
(Greasham and Inamine, 1986; Hicks, 1993; Bull et al. 1990 ; Veronique et al. 1983;
Nelson, 1982; Hendrix, 1980; Stowe and Mayer 1966 ).
When more than five independent variables are to be investigated,
the Plackett and Burman (1946) design may be used to find out the most important
variables in a system, which are then optimized in further studies. Das and Giri (1996)
studied the effects and interactions of the factors in factorial experiments using response
surface design. Dunn et al. (1994) used modeling expressed in sets of mathematical
equations.
Alkaline proteases are mostly produced by submerged
fermentation. In addition, solid state fermentation processes have also been exploited to
a lesser extent for production of these enzymes (George et al.1995;Chakraborty and
Srinivasan, 1993) .Efforts have been directed mainly towards:(i)Evaluation of the effects
of various carbon and nitrogenous nutrients as cost effective substrates on the yield of
enzymes, (ii) Requirement of divalent metal ions in the fermentation medium; and (iii)
Optimization of environmental and fermentation parameters such as pH, temperature,
aeration and agitation.
28
In addition, no defined medium has been established for the best
production of alkaline proteases from different microbial sources. Each organism or
strain has its own special conditions for maximum enzyme production.
Carbon source
Studies have indicated a reduction in protease production due to catabolite
repression by glucose (Kole et al. 1988; Frankena et al. 1986; Frankena et al. 1985;
Hanlon et al. 1982). On the other hand, (Zamost et al. 1990) have correlated the low
yields of protease production with the lowering of pH brought about by the rapid growth
of the organism. In commercial practice, high carbohydrate concentrations repressed
enzyme production. Therefore, carbohydrate was added, either continuously or in small
amounts through out the fermentation to supplement the exhausted component and
keep the volume minimum and thereby reduce the power requirements (Aunstrup, 1980).
Increased yields of alkaline proteases were reported by several
workers who used different sugars such as lactose (Malachi and Chakraborty, 1991),
maltose (Tsuchiya et al. 1991), sucrose (Phadatare et al. 1993), and fructose (Seri and
Satyanarayana, 1993). However, a repression in enzyme synthesis was observed with
these ingredients at high concentrations. Whey, a waste byproduct of the dairy industry
containing mainly lactose and salts, has been demonstrated as a potential substrate for
alkaline protease production (Donaghy and McKay, 1993). Various organix acids, such as
acetic acid ( lKeda et al. 1974), methyl acetate ( Kitada and Horikoshi, 1976), and citric
acid or sodium citrate (Kumar et al. 1997; Takii et al. 1990) have been demonstrated to
increase the production of proteases at alkaline pH. The use of these organic acids was
interesting in view of their economy as well as their ability to control pH variations.
Nitrogen source
In most microorganisms, both inorganic and organic forms of
nitrogen are metabolized to produce amino acids, proteins, and cell wall components.
The alkaline protease comprises 15.6% sources in the medium (Kole et al. 1988).
Althogh complex nitrogen sources are usually used for alkaline protease production, the
requirement for a specific nitrogen supplement differs from organism to organism. Low
levels of alkaline protease production were reported with the use of inorganic nitrogen
29
sources in the production medium (Sen and Satyanarayana, 1993). Enzyme synthesis was
found to be repressed by rapidly metabolizble nitrogen sources, such as amino acids or
ammonium ions in the medium (Frankena et al. 1986), indicated repression in the
protease activity with the use of ammonium salts (Nehete et al. 1986). Sinha and
Satyanarayana (1991) have observed an increase in protease production by the addition
of ammonium sulphate and potassium nitrate. Similarly, sodium nitrate (0.25%) was
found to be stimulatory for alkaline protease production (Banerjee and Bhattacharyya,
1992b). On the contrary, several reports have demonstrated the use of organic nitrogen
sources leading to higher enzyme production than the inorganic nitrogen sources.
Fujiwara and Yamamoto (1987) have recorded maximum enzyme yields using a
combination of 3 % soyabean meal and 1.5 %bonito extract. Soyabean meal was also
reported to be a suitable nitrogen source for protease production (Cheng et al. 1995; Sen
and Satyanarayana, 1993; Tsai et al. 1988; Chandrasekharan and Dhar, 1983).
Corn steep liquor (CSL) was found to be a cheap and suitable
source of nitrogen by some workers (Sen and Satyanarayana, 1993; Fujiwara and
Yamamoto, 1987). Tryptone (2 %) and casein (1-2 %) also serve as excellent nitrogen
sources (Phadatare et al. 1993; Ong and Gaucher, 1976). Addition of certain amino
compounds was shown to be effective in the production of extracellular enzymes by
alkalophilic Bacillus sp. However, glycine appeared to have inhibitory effects on both
amylase and protease production. Casamino acids were also found to inhibit protease
production (Ong and Gaucher, 1976). Oil cakes (as nitrogen source) were found to
stimulate the production of enzymes. In some studies, use of oil cakes did not favor
enzyme production (Sen and Satyanarayana, 1993; Sinha and Satyanarayana, 1991).
Metal ion requirement
Divalent metal ions, such as calcium, cobalt, copper, boron, iron,
magnesium, manganese, and molybdenum are required in the fermentation medium for
optimum production of alkaline proteases. However the requirement for specific metal
ions depends on the source of enzyme. The use of AgNO3 at a concentration of 0.05
mg/100ml or ZnSO4 at a concentration of 0.1.25 mg/100 ml resulted in an increase in
protease activity by RhiZopus oryzae (Banerjee and Bhattacharyya, 1992b). Potassium
phosphate has been used as a source of phosphate in most studies (Mao et al. 1992;
Moon and Parulekar, 1991). This was shown to be responsible for buffering the medium.
Phosphate at a concentration of 2 g/1 was found to be optimal for protease production.
30
However, amounts in excess of this concentration showed an inhibition in cell growth
and repression in protease production (Moon and Parulekar, 1991). When the phosphate
concentration was 4 g/1, precipitation of the medium on autoclaving was observed
(Moon-and Parulekar, 1993). This problem, however, could be overcome by the
supplementation of the disodium salt of EDTA in the medium (Chaloupka, 1985). In at
least one case the salts did not have any effect on the protease yields (Phadatare, 1993).
pH and temperature
The important characteristic of most alkalophlic microorganisms
is their strong dependence on the extracellular pH for cell growth and enzyme
production. For increased protease yields from these alkalophiles, the pH of the medium
must be maintained above 7.5 throughout the fermentation process (Aunstrup, 1980).
The culture pH also strongly affects many enzymatic processes and transport of various
components across the cell membrane (Moon and Parulekar, 1991). When
ammonium ions were used the medium turned acidic, while it turned alkaline when
organic nitrogen, such as aminoacids or peptides were consumed (Moon and Parulekar,
1993). The decline. in the pH may also be due to the production of acidic products
(Moon and Parulekar, 1991). In view of a close relationship between protease synthesis
and the utilization of nitrogenous compounds, pH variations during fermentation may
indicate kinetic information about the protease production, such as the start and end
of the protease production period.
Temperature is yet another critical parameter that has to be
controlled and varied from organism to organism. The mechanism of temperature
control of enzyme production is not well understood (Chaloupka, 1985). However,
studies by Frankena et al. (1986) have shown that a link existed between enzyme
synthesis and energy metabolism in Bacilli, which was controlled by temperature and
oxygen uptake.
Aeration and agitation
During fermentation the aeration rate indirectly indicates the
dissolved oxygen level in the fermentation broth. Different dissolved oxygen profiles can
be obtained by: (i) Variations in the aeration rate, (ii) Variations in the agitation speed of
the bioreactor; or (iii) Use of oxygen rich or oxygen deficient gas phase (appropriate air
oxygen or air-nitrogen mixtures) as the oxygen source (Moon and Parulekar, 1991;
Michalik et al. 1995). The variation in the agitation speed influences the extent of
31
mixing in the shake flasks or the bioreactor and also affects the nutrient availability.
Optimum yields of alkaline protease are produced at 200 rpm
for B. subtilis ATCC 14416 (Chu et al. 1992) and B. licheniformis (Sen and
Satyanarayana, 1993). In one study, Bacillus sp.B21-2 produced increased enzyme
titres when agitated at 600 rpm and aerated at 0.5 vvm (Fujiwara and Yamamoto,
1987). Similarly, Bacillus firmus exhibited maximum enzyme yields at an aeration rate
of 7.0 l/min (Mao et al. 1992) and an agitation rate of 360 rpm. However, lowering
the aeration rate to 0.1 1/min caused a drastic reduction in the protease yields (Moon
and Parulekar, 1991). This indicates that a reduction in oxygen supply is an important
limiting factor for growth as well as protease synthesis.
32
ISOLATION AND PURIFICATION OF ALKALINE PROTEASES
When isolating enzymes on industrial scale for commercial
purposes the prime consideration is the cost of production in relation to the
value of the end product. Crude preparations of alkaline' proteases are generally
employed for commercial use. Nevertheless the purification of alkaline proteases is
important from the perspective of developing a better understanding of the functioning of
the enzyme .
Recovery
After successful fermentation, when the fermented medium leaves
the controlled environment of the fermenter, it is exposed to a drastic change in
environmental conditions. The removal of the cells, solids, and colloids from the
fermentation broth is the primary step in enzyme downstream processing, for which
vacuum rotary drum filters and continuous disc centrifuges are commonly used. To
prevent the losses in enzyme activity caused by imperfect clarification or to prevent the
clogging of filters, it is necessary to perform some chemical pretreatment of the
fermentation broth before commencing separation ( Mukhopadhyay et al. 1990;
Aunstrup, 1980). Changes in pH may also be suitable for better separation of solids (Tsai
et al. 1983). Furthermore the fermentation broth solids are often colloidal in nature
and are difficult to remove directly. In this case, addition of coagulating or
flocculating agents becomes vital (Boyer and Byng, 1996). Flocculating agents
are generally employed to effect the formation of larger flocs or agglomerates, which,
in turn, accelerate the solid-liquid separation. Cell flocculation can be improved by
neutralization of the charges on the microbial cell surfaces, which includes changes
in pH and the addition of a range of compounds that alter the ionic environment.
The flocculating agents, commonly used are inorganic salts,
mineral hydrocolloids, and organic polyelectrolytes. For example the use of a
polyelectrolyte Sedipur TF 5 proved to be an effective flocculating agent at 150 ppm and
pH 7.0-9.0, and gave 74 % yield of alkaline protease activity (Sitkey et al. 1992). In
some cases, it becomes necessary to add a bioprocessing filter aid, such as diatomaceous
earth, before filtration (Boyer and Byng, 1996).
Concentration
Because the amount of enzyme present in the cell free filtrate is
usually low, the removal of water is a primary objective. Recently, membrane separation
processes have been widely used for downstream processing (Strathmann, 1990).
33
Ultrafiltration (UF) is one such membrane process that has been largely used for the
recovery of enzymes (Bohdziewicz, 1994; Bohdziewicz, 1996) and formed a preferred
alternative to evaporation. This pressure driven separation process is expensive,
results in tittle loss of enzyme activity, and offers purification and concentration
(Sullivan et al. 1984), as well as diafiltration, for salt removal or for changing the
salt composition (Boyer and Byng, 1996). However, a disadvantage underlying this
process is the fouling or membrane clogging due to the precipitates formed by the final
product. This clogging can usually be alleviated or overcome by treatment with
detergents, proteases, or acids and alkalies. Han et al (1995) used a temperature-sensitive
hydrogel ultrafiltration for concentrating an alkaline protease. This hydrogel comprised
poly (N- isopropylacrylamide), which changed its volume reversibly by the changes in
temperature. The separation efficiency of the enzyme was dependent on the temperature
and was 84 % at temperatures of 15°C and 20T. However, at temperatures above 25T, a
decrease in the separation efficiency was observed.
Precipitation
Precipitation is the most commonly used method for the isolation
and recovery of proteins from crude biological mixtures (Bell et al. 1983). It also
performs both purification and concentration steps. It is generally affected by the
addition of reagents such as salt or an organic solvent, which lowers the solubility of the
desired proteins in an aqueous solution. Although precipitation by ammonium sulphate
has been used for many years, it is not the precipitating agent of choice for detergent
enzymes. Ammonium sulphate was found wide utility only in acidic and neutral pH
values and it developed ammonia under alkaline conditions (Aunstrup, 1980). Hence, the
use of sodium sulphate or an organic solvent was the preferred choice.
Despite better precipitating qualities of sodium sulphate over ammonium sulphate, the
poor solubility of the salt at low temperatures restricted its use for this purpose (Shih et
al. 1992).
Many reports revealed the use of acetone at different volume
concentrations: 5 volumes (Horikoshi, 1971), 3 volumes (Kim et al. 1996; Tsujibo et al.
1990), and 2.5 volumes (Kumar et al. 1997), as a primary precipitation agent for the
recovery of alkaline proteases. Precipitation was also reported by various workers
with acetone at different concentrations: 80 % (v/v) (Kwon et al. 1994), 66 % (v/v)
(Yamagata et al. 1995) or 44, 66, and 83 % (v/v) (El-Shanshoury et al. 1995), followed
by centrifugation and/or drying. Precipitation of enzymes can also be achieved by the use
34
of water soluble, neutral polymers such as polyethylene glycol (Larcher et al. 1996).
Ion-exchange chromatography (IEC)
Alkaline proteases are generally positively charged and are not
bound to anion exchangers (Tsai et al. 1983; Kumar et al. 1997; Fujiwara et al.
1993). However, cation exchangers can be a rational choice and the bound molecules
are eluted from the column, by an increasing salt or pH gradient.
Affinity chromatography
Reports on the purification of alkaline proteases by
different affinity chromatographic methods showed that an affinity adsorbent
hydroxyapatite was used to separate the neutral protease (Keay and Wildi, 1970) as well
as to purify the alkaline protease from a Bacillus sp. (Kobayashi et al. 1996). Other
affinity matrices used were Sephadex-4-phenylbutylamine (Ong and Gaucher, 1976),
casein agarose (Bockle et al. 1995; Manachini et al. 1998), or N-benzoyloxycarbonyl
phenylalanine immobilized on agarose adsorbents (Larcher et al. 1996). However, the
major limitations of affinity chromatography are the high cost of enzyme supports and
the labile nature of some affinity ligands, which make them unrecommendable for use as
a process scale.
Aqueous two-phase systems
This technique has been applied for purification of alkaline
proteases using mixtures of polyethylene glycol (PEG) and dextran or PEG and salts
such as H3PO4, MgSO4 (Lee and Chang, 1990; Sharma et al. 1994; Sinha et al. 1996,
Hotha and Banik, 1997). In addition, other methods, such as the use of reversed micelles
for liquid-liquid extraction (Rahman et al. 1988), affinity precipitation (Pecs et al. 1991),
and foam fractionation (Banerjee et al. 1993) have also been employed for the recovery
of alkaline proteases.
Stabilization
The enzyme preparations used commercially are impure and are
standardized to specified levels of activity by the addition of diluents and carriers.
Further, the conditions for maximum stability of crude preparations may be quite
different than for purified enzymes. Because loss of activity is encountered during
storage m the factory, shipment to chent(s) and /or storage in client's facilities, storage
stability is of prime concern to enzyme manufacturers. Protease solutions are subjected
to proteolytic and autolytic degradation that results in rapid inactivation of enzymatic
35
activity. To maintain the enzyme activity and provide stability, addition of stabilizers
like calcium salts, sodium formate, borate, propylene glycol, glycerine or
betaine polyhydric alcohols, protein preparations, and related compounds has proved
successful;; (Weijers and Van't, 1992; Eilertson et al. 1985; Schmid, 1979). Also, to
prevent contamination of the final commercial crude preparation during
storage, addition of sodium chloride at 18-20 % concentration has been advised (Shetty
et al.1993; Aunstrup, 1980). The handling of dry enzymes possesses potential health
hazards and therefore, it is customary to maintain the enzyme preparations in
stabilized liquid form.
The stabilization of alkaline proteases and/or subtilisins has also
been made possible through use of protein engineering and numerous examples have
been illustrated in literature. The alkaline and thermal stabilities of subtilisin BPN9 were
improved by random mutagenesis followed by application of proper screening assays
(Cunningham and Wells, 1987; Bryan et al. 1986). Site-directed mutagenesis is often
based on specific protein design strategies, including change of electrostatic potential
(Erwin et al. 1990;Pantaliano et al. 1987), introduction of disulfide brid ges (Mitchinson
and Wells, 1989; Takagi et al. 1990), replacement of oxidation labile residues (Estell et
al.1985), modification of side chain interactions (Braxton and Wells, 1991),
improvement of internal packaging (Imanaka et al. 1986), strengthening of metal ion
binding (Pantaliano et al. 1988), reduction in unfolding entropy (Pantaliano et al. 1989;
Mattews et al. 1987), residue substitution or deletion based on homology (Yonder et al.
1993 ;Takagi et al. 1992) and modification of substrate specificity (Takagi et al. 1.997;
Takagi et al. 1996).
Properties of Alkaline Proteases
The enzymatic and physicochemical properties of alkaline
proteases from several microorganisms have been studied extensively.
Optimum pH and temperature
The optimum pH range of alkaline proteases is generally
between pH 9 and 11, with a few exceptions of higher pH optima of 11.5 (Yum et al.
1994; Tobe et al. 1975, Takami et al. 1990), pH 11-12 (Horikoshi, 1996; Kumar, 1997),
and pH 12-13 (Fujiwara et al. 1993). They also have high isoelectric points and are
generally stable between pH 6 and 12. The optimum temperatures of alkaline proteases
range from 50 to 70°C. In addition, the enzyme from an alkalophilic Bacillus sp. B18
showed an exceptionally high optimum temperature of 85°C.
36
Molecular masses
The molecular masse of alkaline proteases range from 15 to 30
kDa (Fogarty et al. 1974) with few reports of higher molecular masses of 31.6 kDa
(Freeman et al. 1993), 33 kDa (Larcher et al. 1996), 36 kDa (Tsujibo et al. 1990) and 45
kDa (Kwon et al. 1994). However, an enzyme from Kurthia spiroforme had an
extremely low molecular weight of .8 kDa. (Steele et al. 1992). In some
Bacillus sp. multiple electrophoretic forms of alkaline proteases were observed
(Kumar, 1997; Kobayashi et al. 1996; Zuidweg et al. 1972). The multiple forms of these
enzymes were the result of nonenzymatic, irreversible deamination of glutamine or
asparagine residues in the protein molecules, or of autoproteolysis (Kobayashi et al.
1996).
Metal ion requirement and inhibitors
Alkaline proteases require a divalent cation like Ca
Mn
+2
+2
Mg+2, and
or a combination of these cations, for maximum activity. These cations were also
found to enhance the thermal stability of a Bacillus alkaline protease (Palowal et al.
1994). It is believed that these cations protect the enzyme against thermal denaturation
and play a vital role in maintaining the active conformation of the enzyme at high
temperatures .In addition, specific ca 2+ binding sites that influence the protein activity
and stability apart from the catalytic site were described for protease K (Bajorath et
al. 1988).
Inhibition studies give insight into the nature of the enzyme, its
cofactor requirements, and the nature of the active site (Sigma and Moser, 1975). In
some of the studies, catalytic activity was inhibited by Hg+2 ions (Shimogaki et al. 1991).
In this regard, the poisoning of enzymes by heavy metal ions has been well documented
in the literature (Vallee and Ulmer, 1972).
Alkaline proteases are completely inhibited by phenylmethyl
sulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP). In this regard, PMSF
sulfonates the essential serine residue in the active site and results in the complete loss of
activity (Gold and Fahmey, 1964). This inhibition profile classifies these proteases as
serine hydrolases (Morihara, 1974). In addition, some of the alkaline proteases were
37
found to be metal ion dependent in view of their sensitivity to metal chelating agents,
such as EDTA (Steele et al. 1992; Dhandapani and Vijayaragavan, 1994; Shevchenko et
al. 1995). Thiol inhibitors have little effect on alkaline proteases of Bacillus sp. although
they do affect the alkaline enzymes produced by Streptomyces sp. (Yum et al. 1994; ElShanshoury et al. 1995).
Substrate specificity
Although alkaline proteases are active against many synthetic
substrates and native proteins, reaction rates vary widely. The alkaline proteases and
subtilisins are found to be more active against casein than against haemoglobin
or bovine serum albumin.
Alkaline proteases are specific against aromatic or
hydrophobic amino acid residues such as tyrosine, phenylalanine, or leucine at the
carboxyl side of the splitting point, having a specificity similar to, but less stringent than
a chymotrypsin (Morihara, 1974). With the B-chain of insulin as substrate, the bonds
most frequently cleaved by a number of alkaline proteases were Glu 4 - His 5, Ser 9 -His 10, Leu. 15 -Tyr 16, Tyr 16 -- number of alkaline proteases were Glu 4 - His 5,
Ser 9 -- His 10, Leu. 15 -Tyr 16, Tyr 16 --Leu 17, Phe 25 -Tyr 26, Tyr 26 -Thr 27 and
Lys 29 -Ala 30(Yamagata et al. 1995; Larcher et al. 1996; Peek et al. 1992;
Matsuzawa et al. 1988;Tsai et al. 1988; Tsuchiya et al. 1993). In addition to elucidated
that an alkaline elastase from Bacillus sp. Ya-B cleaved both the oxidized insulin
A- and B-chains in a block cutting manner.
Tsai et al (1984) observed that the alkaline elastase from Bacillus
sp. Ya-B also hydrolysed elastin and elastase specific substrates like succinyl-Ala3-pnitroanilide and succinyl-Ala-Pro- Ala-p-mitroariflide at a faster rate. This enzyme
showed a preference for aliphatic amino acid residues, such as alam*ne,, that are present
in elastin. It is considered that the elastolysis was initiated by the formation of an enzyme
substrate complex through electrostatic interaction between positively charged residues
of the elastase and negatively charged residues of the elastin in a pH range below 10.6
(Tsai et al. 1984). In keratin, the disulfide bonds form an important structural
feature and prevent the proteolytic degradation of the most compact areas of the
keratinous substrates. A thermostable alkaline protease from an alkalophific Bacillus
sp. no. AH101 exhibiting keratinolytic activity showed degradation of human hair
keratin with 1 % thioglycolic acid at pH 12 and 70°C, and the hair was solubilized within
1 hr (Takami et al. 1992). Similarly, enhanced keratin degradation after addition of DTT
38
has also been presented for alkaline proteases of Streptomyces sp. (Bockle et al.
1995).
39
ACTINOMYCETES
Actinomycetes are widely distributed in nature. Soils and
composts are particularly favourable for their development, where they are found in
great abundance, both in numbers and in kinds. Globig,(1988) was among the first to
draw attention to the occurrence of actinomycetes in the soil. Beijerinck (1900)
established that actinomycetes occur in great abundance in the soil. They found that
the season of year and soil treatment had a great influence upon the numbers of these
organisms. This was followed by the works of numerous other investigators, notably
that of Waksman (1920). The role of actinomycetes in the breakdown of organic
residues in the soil, methods for determining their presence and abundance in the soil, and
the recognition of the presence of numerous types of actinomycetes received considerable
attention in these works. A large number of studies reported the abundance of
Streptomyces and Micromonospora, the two actinomycete genera
in
soil. They have
proved to be prolific sources of antibiotics, enzymes and enzyme inhibitors. They are
relatively easy to isolate and can be included with little difficulty in high throughput
screening programs which evolved accordingly (Cross, 1982).
Some general properties of actinomycetes ascribing their
fungal as well as bacterial properties were reviewed earlier (Becker et al .1965).
Like fungi, actinomycetes form hyphae with true branching. True bacteria have no
vegetative thallus and actinomycetes are morphologically similar to filamentous fungi,
which have a vegetative thallus during at least part of their life cycle. Bacterial
endospores are not known to be formed by actinomycetes but the formation of
endospore-like structures may occur in mycobacteria.The actinomycetes have many
bacterial properties as well. The diameter of their hyphae falls within the bacterial order
of magnitude of 1um Cytological similarities between true bacteria and actinomycetes
include the types of flagella formed. Eucaryotic organisms have flagella formed of
11 fibrils, each of which has about the diameter of a bacterial or actinomycete flagella.
Other bacterial properties shared by actinomycetes include lack of sterols, sensitivity
to
antibacterial
antibiotics
and
phages,
lysine
synthesis
through
the
diaminopimelic acid pathway and cell walls containing mucopeptides (Becker et
al .1965).
Proteolytic actinomycetes
Waksman first established that various actmomycetes, mostly
members of the genus Streptomyces possess strong, proteolytic activities. Some cultures
40
are able to decompose very efficiently proteins in gelatin, egg white and blood serum.
This is equally applicable for both saprophytic and pathogenic types. They vary greatly,
in this respect, both qualitatively and quantitatively, as tested by the process of gelatin
liquefaction or casein decomposition in ordinary plates. The degree and rapidity of
proteolysis varied with individual species. Stapp found that out of 477 freshly
isolated cultures of streptomycetes, only one failed to liquefy gelatin.The liquefying
actions of the others were characterized by varying degrees of rapidity. The quantitative
ability to secrete proteolytic enzymes could be measured by the degrees of gelatin
of gelatin liquefaction and of casein hydrolysis.
The proteolytic activities of the various species of actinomycetes
are so marked that waksman.A (1920) suggested the use of this property for diagnostic
purposes. However, Lieske stated that proteolysis is not a constant property and cannot
be used for characterization of the organisms. Various forms of gelatin were used for
the test. The results were always identical. A strain that dissolved gelatin rapidly when
first isolated continued to do so after 1, 2, 3, 4 and 5 years of cultivation. The proteolysis
enzymes of actinomycetes are more resistant to the effect of higher temperatures than
are corresponding animal enzymes. Sterile culture filtrates of certain species of
actinomycetes were found to exert a marked effect not only upon animal proteins but
also upon proteins derived ftom, soybeans, peanut meal, and corn meal. According to
Simon (1955), Streptomyces griseus produced a protease in a medium containing 2
percent soybean meal. An active enzyme preparation with potency equal to that of
pancreatin was obtained in the culture. Casein, soybean, fibrin and peptone could be
used as substrates for the enzymes. The optimum pH reaction for the enzyme activity
was found to be pH 8.2. An aqueous solution of the enzyme was inactivated at 60°C in
30 min.
Enzymes produced by actinomycetes seem to be very promising as
immobilized preparations for use in routine clinical diagnostic tests. Using
enzymes from actinomycetes a number of methods have been developed for
rapid enzymic determinations of clinically important compounds in biological fluids,
such as the determination of,the total level of cholesterol using cholesterol oxidase and
cholesterol esterase, and uric-acid and L-glutainate by application of urate oxidase and
L-glutamate respectively and phospholipids by the concerted action of phospholipase D
and choline oxidase. There are also possibilities for the use of such enzymes as Lasparginase, pronase and urate oxidase as therapeutic agents There is an ever increasing
41
interest in the use of actinomycete enzymes in bio-organic chemistry. For example,
synthesis of biologically active oligopeptides have been performed by means of some
proteases, while resolution of racemic mixtures have been achieved by acylases
and chiral components obtained by stereospecific reduction of appropriate substrates
performed by oxidoreductases. Actmomycete enzymes with high substrate specificit y
find application in molecular biology for the structural analysis of complex
glycopeptides, polysaccharides and proteins.
Actinomycetes produce a large number of proteolytic enzymes.
Proteolytic enzymes are produced by different species of Streptomycetes. Among the
Streptomyces strains studied which showed varying degree of proteolytic activities were
S. griseus (5strains), S. griseoflavus (2strains), S.cellulosae (5strains), S. fulvissimus
(3strains), S.olivaceous (5strains), S.violaceous niber (3strains), S.diastatochromogenus
(3strains), S.bobiliae (2strains),
S.aureus (2strains), S.pheochromogenus and S.
erythrochromogenus.
Bechtereva et al (1958) studied the course of accumulation of
active proteolytic enzymes by Streptomyces violaceus and S. lavendulae. The
period of intensive accumulation of proteolytic enzymes in a simple synthetic medium
and in a corn-extract medium, was found to be related to the decomposition of the cells .
Actinomycete proteases are applied in laboratory practice in the
structural determination of protein-composed macromolecules in removing proteinaceous
material during purification of certain biopreparations. For commercial purposes
they are routinely obtained as by products formed during biosynthesis of antibiotics
mostly from the fermentation broths of Streptomyces fradiae,Streptomyces griseus and
Streptomyces rimosus(Morihara et al, 1967) .Of all the actinomycete protease
complexes available, the enzyme pronase has gained considerable interest in the recent
past. Pronase is a mixture of several peptidases, ten of which have been purified to
homogeneity and characterized. Most of the components of Pronase are serffie- and
metalloproteases. The molecular weights of pronase enzymes were estimated to vary
from 15,000 to 30,000 daltons and they were found to display proteolytic activity under
alkaline conditions. It may also serve as a highly specific reagent for the preparation
of optically active amino acids . In the pharmaceutical industry, immobilized pronase is
used to remove impurities ftom, preparations of 6-amino peniciflanoic acid . Highly
purified preparations of Pronase, lipase and phospholipase injected into the lens capsule
42
liquefies hardened material present in age-related cataracts prior to their surgical removal
.Some mesophilic and thermophilic actinomycete proteases are proved to be
homologous with well-known microbial and mammalian endopeptidases. These
proteases classified according to their substrate specificity exhibit a collagenase-like,
elastase-like, fibrinolytic, keratiase-like, rennin-like or trypsin-like activity. A
trypsinlike activity was found not only in the enzymatic complex produced by
Streptomyces erythraeus and also by Streptomyces fradiae (Morihara, 1974). These
enzymes were most active at about pH 8 and their molecular weights were estimated to
be about 20,000 daltons. Enzymes having collagenase-like activity have been isolated
from culture filtrates of pathogenic actinomycetes such as Actinonmadura .
The production of collagenase was induced by insoluble
collagen and its macromolecular fragments, as well as by gelatin and peptone. A soil
streptomycete was reported to degrade collagen isolated from bovine achiles tendon, calf
skin, human placenta, carp swim bladder and rat-tail tendon and release appreciable
quantities of hydroxyproline (Mukhopadhyay and Chandra, 1996). A keratinase-like
activity was detected in the culture filtrates of Streptomyces fradiae. The enzyme was
strongly alkalophilic having an optimum pH of 13.0 .
Actinomycete proteases are similar in action to mammalian
proteases and find major application in the food industry for protein liquefation, milk
clotting or as meat tenderizers. Attempts have also been made to introduce actinomycete
proteases as fibrinolytic and thrombolytic agents in medial treatment. Since actinomycete
proteases are easy to obtain in a highly purified form, these can be used in model
reaction studies and for enzymatic synthesis of biologically active peptides. Protease
isolated from Streptomyces cellulosae) have been used to obtain biologically active
peptides. Some proteases of actinomycete origin are highly resistant to heat and
denaturing agents. The high thermostability of proteases isolated &om thermophilic
actinomycetes is well known. Protease isolated from Streptomyces rectos var.
proteolyticus, Thermoactinomycesalbus, (Mordarski et al, 1976), Thermomonospora
vulgris and Thermomonospora fusca . Several other actinomycetes are used on an
industrial scale. Recently, a newly isolated Streptomyces diastaticus strain SS I was
reported to produce a dier-mostable alkaline metalloprotease .
Proteolytic enzymes find widespread application in many
industries. A recent application of proteolytic enzymes in the leather industry is their use
43
as dehairing or depilation agents. Dehairing or depilation of hides and skids is an
important and unavoidable step for the manufacture of leather in the tannery. '171-te
conventional chemical method of dehairing hides and skins is the lime-sulphide process,
which is environmentally objectionable. The treatment is liable to damage the hair
or wool, which is valuable by-products of the leather In addition; dissolved
sulphide and pulped hair contribute to high C.O.D and B.O.D. of the effluent. Hence,
there is a need for an alternative method of dehairing. Enzymatic dehairing has
been widely accepted as a suitable alternative.
Proteolytic enzymes cause depilation of skins and hides by degrading the
component globular and non-fibrous proteins of the basement membrane at the epidermal
junction.
The first successful enzymic unhairing process, teimed as Arazym
process, was achieved by Rohm in Germany . Among the mold proteases, protease from
Aspergillus flavus, A.oryzae, A.parasiticus, Afiiniigatus, A.effusus, A.ochraceous,
Awentil, Penicillium griseofulvum and rhizopus oryzae exhibited marked
depilatory activities on hides and skins.Proteolytic enzymes derived from a large
number of Bacillus species were reported to be used in dehairing and bating of hides
and skins in earlier times.
Among
the
actinomycetes,
proteolytic
enzymes
from
Streptomyces sp. were reported to effectively dehair hides and skins. Recently,
keratinolytic activity of Streptomyces sp. SKI-02, was reported (Leuchtenberger et
al.1983).
Enzymes from Streptomyces sp. Such as S. moderatus NRRL
3150, S. hygroscoplcus. S. froadiae and S. griseus have potential application in dehairing
of hides and skins.
44
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properties of alkaline protease of Candida lipolytica. Agric. Biol. Chem. 40: 1087.
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58
CHAPTER-III
SCREENING AND TAXONOMIC STUDIES
ANALYTICAL METHODS
Chemicals
All the chemicals used in this study were of analytical grade.
Determination of proteolytic activity
The proteolytic activity was determined with casein as substrate.
As a matter of convenience, the hydrolytic power of the enzymes on casein was called
caseinase activity. Caseinase determination (Bergkvist, 1963)
Caseinase activity was assessed by the modified procedure of
Tsuchida et al. (1986) using 2% casein (Hammerstan casein, Merck, Germany) in 0.2 M
carbonate buffer (Varley et al, 1995) (pH 10) as substrate. Casein solution (0.5 ml) with
an equal volume of suitably diluted enzyme solution was incubated at 55°C. After 10
min, the reaction was terminated by the addition of 1 ml of 10% trichloroacetic acid. The
mixture was centrifuged and to the supernatant was added 5 ml of 0.44 M Na 2CO3 and
1ml of two-fold diluted Folin Ciocalteau reagent. After 30 min, the colour developed was
read at 660 nm against a reagent blank prepared in the same manner. Tyrosine served as
the reference standard. The optical density of these solutions was measured in a
Shimadzu (Japan) spectrophotometer. One unit of enzyme activity was defined as the
amount of enzyme that released one ug of tyrosine per ml per min.
Preparation of 0.44M Na2CO3
0.2 M solutions each of sodium carbonate anhydrous (21.2 g/L)
and sodium hydrogen carbonate (16.8 g/L) were prepared. 50 ml of 0.2 M sodium
carbonate solution was pipette into a 100 ml volumetric flask and made upto the mark
with 0.2 M sodium hydrogen carbonates.
Construction of standard graph for Tyrosine
Stock solution: 50 mg of Tyrosine was dissolved in distilled water
to make 100 ml in a volumetric flask, which resulted in 500 g/ml.
Procedure
Into a series of 10 ml volumetric flasks 1,2, 3,4, and 5 ml of
standard stock solution of tyrosine was taken and distilled water was added to make upto
10 ml mark in each volumetric flask. Mixed well and the optical density was measured at
59
660 nm after developing the colour as described above, against a reagent blank prepared
in same manner. The results are shown in the Table 3.1.
A standard curve was constructed taking concentration of
Tyrosine g/ml on X-axis and corresponding optical density on Y-axis (Fig. 3.1).
Table 3.1 Construction of standard graph for Tyrosine
Tyrosine concentration (g/ml)
Optical density
0(Blank)
0.00
20
0.18
40
0.36
60
0.55
80
0.72
100
0.90
Unit: One protease unit (PU) is defined as the amount of enzyme that released 1ug of
tyrosine per ml per minute under the above assay conditions.
60
61
Estimation of total protein:
Estimation of extracellular protein:
Extracellular protein was estimated by Lowry method (Lowry et
al. 1951). The protein reacts with the Folin Ciocalteau reagent to give a coloured
complex. The color formed is due to the reaction of alkaline copper with protein as in the
Biuret reaction and the reduction of the phosphomolybdate by tyrosine and tryptophan
present in the protein. The intensity of "the colour depends on the amount of these
aromatic amino acids and thus vary with different proteins.
Preparation of reagents
Solution A (alkaline sodium carbonate): 2% Na2CO3 in 0.1 N NaOH.
Solution B (copper sulphate solution): 1% CuSC4 5H2O in distilled water.
Solution C (Rochelle 's salt): 2% sodium potassium tartarate.
Alkaline solution (working solution): 1 ml CuSC4 5H2O solution + 1 ml Rochelle's salt +
98 ml alkaline Na2CO3 solution
Folin Ciocalteau reagent: The commercial reagent (LOBA) was used after diluting with
equal volume of water on the day of use.
Standard protein solution: Five mg of Bovine serum albumin (BSA) was dissolved in 50
ml of distilled water to get a final concentration of 0.1 mg/ml (100 g/ml).
Procedure:
To 1 ml properly diluted protein sample (10-60 fig/ml), 5 ml
alkaline solution.(working solution) was added, mixed well and incubated at 55°C for 10
min. To the above mixture 0.5 ml Folin Ciocalteau reagent was added, mixed well and
incubated at 55°C for 30 min.
The absorbence of the colour was measured at 680 nm in a
spectrophotometer. The results are presented in Table 3.2 and Fig 3.2. The amount of
protein present in the sample was calculated from the standard curve.
Table 3.2 Construction of standard graph for protein estimation
Con. of BSA(g/ml)
OD (at 680 nm)
Blank
0.000
10
0.145
20
0.306
30
0.448
62
40
0.595
50
0.757
60
0.900
63
64
SCREENING AND ISOLATION OF ALKALINE PROTEASE PRODUCING
ACTINOMYCETES
Experimental
Chemicals
All chemicals and medium constituents used in this study were of analytical
grade.
Screening program
The following samples were collected at various places from
Prakasam, Khammam and Guntur districts in Andhra Pradesh with a view to isolate
potent protease producing actinomycetes.
Sample Collection:
Sample I: Sample was collected from the underneath the compost from Ratnagiri Nagar,
Guntur.
Sample II: Sample was collected from the dumping yard of Municipal High School at
Kurnool Road, Ongole which was rich in Organic matter.
Sample III: It was a liquid sample and was collected from the vermicompost, waste water
besides Vignan College at Palakaluru, Guntur. It was turbid and blackish in color.
Sample IV: Sample was collected from rice fields, N.R.I Junior College at
Gujjanagundla, Guntur. Soil having organic matter and black in color.
Sample V: Soil sample was collected from the drainage of a slaughter house at
Ramnagar, Khammam district which was black in color.
Sample VI: The liquid sample was collected from Lam, Guntur. It is rich in organic
matter.
Sample VII: The sample was collected from the J.K.C. College waste dumping yard at
Guntur, which was black in color.
Sample VIII: The sample was liquid collected from A.P.S.R.T.C. Garage at Vijayawada.
Sample IX: Sample was collected from the dumping yard of Sugarcane industry near
Nallapadu, Guntur which was grey in color.
Sample X: Sample was liquid and collected from the dumping yard of Mother Diary
Industry at Pernamitta, Ongole.
Sample XI: Sample was collected from Sangam Diary Industry at Sangam Jaagarlamudi,
Guntur.
Sample XII: Sample was collected from sandy soil at Mangaladas nagar, Guntur.
65
All the samples were collected in sterile screw capped tubes and
care was taken to see that the points of collection had as widely varying characteristics as
possible with regard to the organic matter, particle size, colour of soil and geographical
distribution.
Isolation of Proteolytic actinomycetes from soil samples
About 1 gm of each of the above samples was taken into separate
conical flasks each containing 100 ml of sterile water. The suspension was kept on rotary
shaker for 30 min and kept aside to settle the suspending matter. One ml of the
supernatant was serially diluted with sterile water. One ml each, of these dilutions was
added to 20 ml of sterile molten starch casein-agar medium maintained at 45°C. Mixed
thoroughly and plated in 10 cm dia. sterile Petri dishes and incubated at 28°C. Antifungal
(Flucanazole-75 g/ml) and antibacterial (Refampicin-25 g/ml) agents were
incorporated to control the fungal and bacterial contamination. After 96 h of incubation,
the actinomycete colonies with clear hydrolyzed zones around them were picked up and
transferred onto starch casein agar slants. The composition of starch casein agar is (g/L):
Soluble starch, 10; casein, 3; KNO3, 2; NaCl, 2; K2HPO4, 2; MgSO4.7H2O, 0.05;
CaCO3, 0.01; FeSO4.7H2O, 0.01 and agar, 50ml in distilled water.
A total of 18 colonies were isolated from all the samples. The
number of isolates from each sample is given in Table 3.3. The slants were incubated for
48 to 96 h. Out of 18 isolates, 3 were selected based on their macroscopic characters,
eliminating those that appeared close to each other. These 3 isolates were sub-cultured
onto two different types of media: Skimmed milk agar, Starch Casein Agar and tested for
their proteolytic activity. The results are presented in Table 3.4.
Screening of isolates for proteolytic activity
Primary screening:
The selected isolates were initially screened for their proteolytic
activities i.e. caseinolytic and gelatinolytic activities.
Casein hydrolysis:
The caseinolytic activity of the isolates was evaluated using
casein-agar plate technique (Williams and Cross, 1971) as described below:
Composition of milk-agar base
Peptone
:
0.1%
Agar
:
2.0%
66
To the sterilized milk agar base, 10% of pasteurized skimmed milk
was added aseptically and this medium was transferred into 10 cm dia. sterile petridish
and kept aside for solidification. Then a loopful of each culture was streaked onto the
medium, incubated at 28°C for 96 h. The diameters of hydrolyzed zones around the
colonies and the growth zones were measured. The ratio of hydrolysis zone/growth zone
was calculated (Table 3.4) which gives a measure of the caseinolytic activities of the
isolates.
Gelatinolytic activity:
For this purpose, 20 ml of sterile nutrient gelatin agar medium
(Williams and Cross, 1971) was poured in sterile petridishes and spot inoculated with a
loop full of spores from 48 h old cultures and incubated at 28°C for 96 h. The plates were
flooded with mercuric chloride reagent with the following composition: mercuric
chloride 15% and concentrated HCl 20% in distilled water (Williams and Cross, 1971).
After treating with mercuric chloride-HCl solution, the hydrolysis zone and growth
zones were noted and the results are presented in Table 3.4.
The actinomycetes with promising proteolytic activity were selected for further
studies.
67
Table 3.3 Number of Actinomycetes from various samples
Sample No.
No. of cells present per gm. of the sample
No. of selected
isolates
I
3x105
3
II
2x105
2
III
2x105
2
IV
1x105
1
V
1x105
1
1x10
5
1
2x10
5
2
VIII
1x10
5
1
IX
1x105
1
X
1x105
1
XI
2x105
2
XII
1x105
1
VI
VII
68
Table 3.4 Growth pattern and Proteolytic activities of selected isolates
Extent of growth
Sample
Isolate No.
No.
Proteolytic activity *
(96h)
YEME
Starch
Gelatinolytic
Caseinolytic
Casein Agar
Activity
Activity
Medium
1
25
2
11
3
+
++
5.3
4.1
+
++
4.9
3.0
29
+
++
4.5
2.2
4
05
++
+++
7.1
5.6
5
07
+
++
5.4
3.8
6
02
+
+
4.8
2.9
7
10
+
+
4.9
3.0
10
23
++
+++
6.9
5.1
11
17
++
+++
6.6
4.4
69
12
02
+
+
3.2
2.0
13
15
+
++
2.1
1.2
14
33
++
++
5.1
4.5
15
06
+
++
4.4
3.4
16
24
+
++
4.6
3.8
17
19
+
++
4.8
4.0
18
28
+
++
4.7
3.1
*Ratio= Hydrolyzed zone (mm)/growth zone(mm)
70
Secondary Screening
Among the 18 isolates, 3 isolates (Nos. 04, 10 and 11) were
selected for secondary screening and designated as GAS-04, GAS-10 and GAS-11. They
were tested for extracellular protease production, in shake flasks on rotary shaker at 180
rpm. The following six different types of media were used for the study. These media
were selected based on the literature survey.
Composition of different media for protease production:
Medium
Composition (g/100ml)
Reference
No.
Glucose, 6.0; soyabean meal, 2.0; CaCl2, 0.04; MgCl2,
I
0.2.
Lee et al (1990)
Manachini et al
II
Glucose, 1.5; yeast extract, 0.5; CaCl2, 0.2.
(1988)
III
Glucose, 0.1; yeast extract, 0.5; tryptone, 0.5
Frankena et al (1986)
IV
Glucose 0.2; Caseine 0.15; salt solution, 5 ml*.
Ellaiah et al(2003)
V
Glucose 0.2; peptone 0.15; salt solution 5 ml*.
Ellaiah et al (2003)
Soluble starch, 1; Casein, 0.3;
KNO3, 0.2;
K2HPO4, 0.2; MgSO4.7H2O, 0.005;
VI
CaCC-3, 0.002; FeSO4.7H2O, 0.001.
*Salt solution: MgSO4,0.5%; KH2PO4, 0.5%; FeSO4,0.5%.
71
Shake flask fermentation:
5 ml of sterile water was added to 96 h old slant of above
isolates. The cells were scrapped from the slant into sterile water and from the resultant 5
ml cell suspension, 1 ml suspension was transferred aseptically into 250 ml Erlenmeyer
flasks containing 50 ml each of sterile medium as mentioned above. The flasks were
incubated on a rotary shaker (180 rpm) at 28°C for 96 h. The contents of the flasks were
centrifuged at 3000 rpm for 10 min and the supernatant solution were tested for
proteolytic activity by modified method of Tsuchida et al. (1986) as described earlier.
The results are presented in Table 3.5.
It is clear from the results that isolate GAS-4 is the best protease
producer and in all the six media selected .Hence this isolate GAS-4 was selected for
further studies.
Table 3.5. Production of protease (U/ml) by selected isolates in shake flask*
Medium No.
Isolate GAS-04
Isolate GAS-10
Isolate GAS-11
I
64.7
65.3
II
58.2
59.9
63.9
III
80.1
78.2
75.5
IV
53.5
63.2
52.3
V
62.9
62.4
62.1
VI
69.5
68.3
66.7
50.6
* Protease activity expressed in U/ml.
Design of suitable basal medium:
The composition of the medium I and III were slightly
changed in their glucose concentration and used for fermentative production of protease
by isolate GAS-4 to compare and design a suitable basal medium for efficient
production. The results are presented in Table 3.6. Maximum yield of 80.1 U/ml was
obtained in medium No III The composition of which was Glucose, 0.1; yeast extract,
0.5; tryptone, 0.5. This was designated as the basal medium and used for further
72
studies.Medium I and
III were slightly modified and the composition of the modified
media were given in Table 3.6
Table 3.6 Production of protease in modified media
Medium
Composition of media
Enzyme yield (U/ml)
No.
I
GAS-04
Glucose 6%, Soybean meal 2%,
70.9 ± 1.5
CaCl2 0.04% and MgCl2 0.2%
II
Glucose
1%, Soybean meal 2%,
79.7 ± 1.0
CaCl2 0.04% and MgCl2 0.2%
III
Glucose 0.1%, Yeast extract 0.5%,
78.5 ± 2.0
Tiyptone 0.5%
IV
Glucose 1%, Yeast extract 0.5%,
92.0 ±1.0
Tiyptone 0.5%
The yields of protease produced in the modified media by isolate
GAS-4 are presented in Table 3.7.It was observed that maximum protease of 92.0 U/m L
was produced in the modified media No. IV .Hence this media was selected for further
optimization studies.
Table 3.7 Production of protease in modified media
Modified media
Isolate-GAS-4
Protease U/ml
I
70.9
II
79.7
III
78.5
73
IV
92.0
Determination of type of Protease produced by the isolates
To find out whether the enzyme secreted by the isolates belong to
alkaline or acidic or neutral protease the following experiments were conducted. Protease
activity in the harvested broth was assayed by adding 0.5 ml culture broth to 0.5 ml of
2% casein solution. In order to distinguish between acid, neutral and alkaline protease,
the reactions were carried out at pH 4 (Citrate buffer), pH 7 (Phosphate buffer) and pH
10 (Carbonate buffer), by dissolving the casein in respective buffers. Buffer solutions:
pH 4.0: Commercially available buffer – Titrisol®
was used. It is a citrate /
hydrochloric acid buffer manufactured by Merck.
pH 7.0: Commercially available buffer - Convol® (pH- 7.0 ± 0.05 at 27°C) was used. It
contains potassium dihydrogen orthophosphate.
These commercially available buffers were diluted to 500 ml and then used for
preparing casein solution.
Preparation of carbonate buffer pH 10.0:
0.2 M solutions of each, anhydrous sodium carbonate (21.2 g/L)
and sodium hydrogen carbonate (16.8 g/L) were prepared. 50 ml of 0.2 M sodium
carbonate solution was pipetted into a 100 ml volumetric flask and made up to the mark
with 0.2 M sodium hydrogen carbonate.
Each enzymatic reaction was carried out in duplicate for 10 min.
at 55°C. The amount of Tyrosine released was measured by the modified method of
Tsuchida et al (1986) as described earlier.
The results are shown in Table 3.7. The results indicated that the
enzyme activities by isolate (GAS-04) was high at pH 10 indicating that the enzyme is an
alkaline protease. Further the isolate GAS-04 showed comparatively good enzyme
productivity.
74
Table: 3.8 Protease activity at different pH values:
Protease activity (U/ml)
Isolate
-
pH 9.0
pH 7.0
pH 5.0
GAS-4
96.0
70.2
26.1
The result obtained indicated that the protease produced by isolate
GAS-4 is more active at an alkaline pH of 9.0 where maximum protease of 96 U/m l was
produced, at pH 7.0 and 8.0 the yield of protease decreased to 70 U/m l and 26 U/m l
respectively .These observations suggested that isolate GAS -4 Produced an alkaline
protease.
Capacity of proteases to dehair goat skin:
The isolate GAS-4 was grown in basal medium, the culture broth
was centrifuged at 3000 rpm for 10 min, and the supernatant was used as source of
enzyme. Skin pieces (area about 4  4cm) from freshly slaughtered goat were procured
from the local meat shop. The enzyme (20 ml) in the form of a paste, after mixing with
kaolin (10 gm) and streptomycin sulphate (100 mg) was painted on the flesh side of
paired goatskin pieces and incubated for 1 h. In control experiment, water was used in
place of enzyme solution. After incubation, ease of unhairing was noted by removing-the
hairs with a blunt scalpel. The results are presented in Table 3.9 and Fig 3.3.
The promising two isolate (GAS-04) was selected for further studies and
subjected to taxonomic studies for identification.
Table 3.9 Dehairing activities of Protease produced by Isolate GAS-4
Strain
GAS-04
+
Loosening of hairs in 6 hr.
++
loosening of hair by applying maximum force.
++ loosening of hair by applying moderate force.
75
Fig. 3.3 Dehairing capacities of isolate GAS-04
Control
GAS-4
76
TAXONOMIC STUDIES
Selection of media for taxonomic studies
Culture media used for characterization and identification of
species consists of both synthetic and organic forms. Synthetic media have found
extensive application in the study of morphology, physiology and cultural properties of
the organism while organic media are used for obtaining supplementary cultural
evidence.
Media used for characterization:
Waksman (1958) and others recommended the inclusion of
following media for characterization of actinomycetes:
1. At least three synthetic media, preferably sucrose nitrate salt agar or sucrose
ammonium salt agar, glucose or glycerol asparagine agar and calcium malate or
citrate agar.
2. Two to three organic media such as nutrient agar, yeast extract malt extract agar,
potato glycerol glutamate agar or oatmeal agar.
1. Three or four complex natural media such as potato plug, gelatin or milk.
2. Peptone iron yeast extract agar for H2S production.
3. Tyrosine medium for tyrosinase reaction.
4. A synthetic medium for carbohydrate utilization.
Experimental
In the present work the morphological studies and colour determinations of the
selected isolate GAS -4 was studied by following International Streptomycetes Project
(ISP) procedures (Shirling and Gottlieb, 1966). The following media as recommended
by ISP were used for the morphological studies and colour determinations.
1. Yeast extract malt extract agar (ISP-2).
2. Oatmeal agar (ISP-3).
3. Inorganic salts starch agar medium (ISP-4) and
4. Glycerol asparagine agar medium (ISP-5)
Further, the following biochemical reactions were determined
employing the prescribed media: melanin formation, H2S production, tyrosine reaction,
gelatin hydrolysis, coagulation and peptization of milk, casein hydrolysis, starch
hydrolysis, nitrate reduction, carbon source utilization, sodium chloride tolerance, effect
77
of various nitrogen sources on growth, growth temperature range, chemical tolerance
and cell wall composition.
Preparation of inoculum:
In general, the agar media favouring abundant sporulation are those with a high
C/N ratio such as jowar starch agar, oatmeal agar (ISP) and starch-casein agar.
In the present study starch-casein agar was used for the isolates. These slants
were inoculated from the stock cultures and incubated at 28°C for 1-5 days to get
maximum sporulation. Spore suspension was prepared by transferring a loopful of spores
from these slants into sterile distilled water and shaking thoroughly. For gelatin
liquefaction, starch hydrolysis and casein hydrolysis, a loopful of spores taken from the
stock culture was used for inoculation. For all other tests, spore suspensions prepared as
above were used employing equal volumes of the suspension in each case.
Preparation of media:
Detailed compositions of all the media employed in this work are given in the
Appendix I.
Morphological and Cultural Studies
The color of aerial mycelium, substrate mycelium and soluble
pigment when grown on different media were observed and recorded. The macro and
micro-morphological features of the colonies and the color determinations of the aerial
mycelium, substrate mycelium and soluble pigment were examined after 96 h of
incubation. Macro-morphology was noted by the naked eye and by observation with
magnifying lens.
Micro-morphology:
To study the aerial mycelium and its sporulation characteristics, the following
two methods were used.
1. Direct method
Very thin layer of the respective solidified media in petridishes,
were inoculated with 0.05 ml of the spore suspension. This was placed near the edge of
the plate to serve as a pool of inoculum. Using a sterile loop, four to five equally spaced
streaks were made. A number of plates were inoculated in this manner to facilitate
observations on different days. Observations were recorded from next day onwards up to
4 days.
78
2. Inclined cover slip method (Kawato and Shinobu, 1959;Williams and Davis
1967):
Sterile cover slips were placed at an angle of 45° into solidified
agar medium in petridish such that half of the cover slip was in medium. Inoculum was
spread along the line where the upper surface of the cover slip meets the medium. After
full sporulation, the cover slips were removed and examined directly under the
microscope.
3. Electron microscopy (Williams and Davis, 1967):
For scanning electron microscopy, slides were cut into 1cm pieces
and sterilized. The pieces were dipped at an angle of 45° into solidified starch casein
agar medium. The inoculum was spread along the glass-agar medium interface. During
incubation, the organisms grew over the surface of the glass pieces. The growth from the
glass pieces were removed carefully using a brush and affixed onto the copper stud,
washed with serial grades of 30,50,70 and 90% alcohol. The studs were then kept in a
dessicator for final drying. The surface containing organisms was coated under vacuum,
with a film of gold about 150-200A thickness and observed under Scanning Electron
Microscope (PSEM 500) for spore surface ornamentation. The scanning electron
microscope was provided by Advanced Analytical Laboratory, Andhra University,
Visakhapatnam, India.
Physiological Characteristics:
1. Gram - staining
.
To study the Gram's reaction of the culture, a 48 h culture was
used. The experimental protocol as described by Salle (1948) was followed
2. H2S production (Shirling and Gottlieb, 1966):
Many organisms,
in their metabolism
of sulphur containing
organic compounds, liberate hydrogen sulphide in considerable amounts. This can be
demonstrated if sulphide producing cultures are grown on media containing salts of iron
resulting in the formation of a black or bluish black precipitate. The use of H2S
production as a taxonomic implement was suggested by many authors.
The inoculated peptone-yeast extract-iron agar (ISP-6) slants were
incubated at 28°C for 5 days. Observations for the presence of characteristic greenish
brown, brown, blackish brown, bluish black or black colour of the substrate, indicative of
H2S production, were made every 24 h upto 5 days.
79
3.Gelatin hydrolysis (Gordon and Mihm, 1957):
This represents the ability of microorganisms to hydrolyze or
liquefy gelatin. Most of the species of Streptomyces bring about liquefaction of gelatin
but the rapidity of liquefaction varies with the species. The non-pigmented forms are
most active while the pigmented forms are least active.
For this test, the isolates were grown on gelatin agar plates for two
days at 55°C. At the end of incubation period, the plates were flooded with 1 ml of the
following solution.
Mercuric chloride
:
15 g
Conc. HCl
:
20 ml
Distilled water
:
100 ml
The extent of hydrolysis was noted by comparing the width of the
clear zone around the growth. The widths of the hydrolyzed zone and growth zone were
measured and the ratio of hydrolyzed zone and growth zone was calculated.
4.. Casein hydrolysis (Salle, 1948):
The proteolytic activity was studied with milk-casein agar medium
by measuring the hydrolyzed zones after incubating the inoculated plates at 28°C for 48
h. The extra-cellular protease (caseinase) activity of the isolates was determined
qualitatively as the ratio of the diameter of the hydrolyzed zone and that of the growth on
the milk-casein agar medium.
5. Starch hydrolysis (Salle, 1948):
Numerous actinomycetes are able to hydrolyze starch rapidly by
the action of amylolytic enzymes. For this test, the selected isolates were grown for 5
days on starch agar plates. At the end of incubation period, the plates were flooded with
weak iodine solution. The width of hydrolyzed zone around the growth versus the width
of the growth was measured.
Composition of iodine solution:
Potassium iodide
: 3g
Iodine
: 1g
Distilled water
: 100mL.
6. Nitrate reduction test (Salle, 1948):
The reduction of nitrate to nitrite has been universally used among
the criteria for species differentiation. The reduction is the result of the use of NaNO3 or
KNO3 as an electron acceptor with some organisms. Nitrate (NO3) and NO2 serve as
80
sources of nitrogen for the synthesis of organic nitrogen compounds or they may
function as H+ acceptors in reactions concerned with the organisms energy metabolism.
The first step in the reduction of NO3 involves its conversion to NO2 by an enzyme
system, which is adaptive in nature and is known as nitratase.
5ml of nitrate broth medium was inoculated with a loopful of
spores and incubated at 28°C for 5 days. Controls were also run without inoculation.
After 5 days, the clear broth was tested for the presence of nitrite in the following way.
Reagents:
a)  -Naphthylamine test solution:
-Naphthylamine
:
5.0g
Conc. H2SO4
:
8.0 ml
Distilled water
:
1L
To the diluted sulphuric acid, -naphthylamine was added and stirred until solution was
effected.
b) Sulphanilic acid test solution:
Sulphanilic acid
:
8.0 g
Conc. H2SO4
:
48 ml
Distilled water
:
1L
Sulfuric acid was added to 500 ml of water. Then sulphanilic acid was added
followed by water to make up the volume.
Procedure:
To 1 ml of the broth under examination and 1 ml of control, two
drops of sulphanilic acid solution followed by two drops of a-naphthylamine solution
were added. The presence of nitrite was indicated by a pink, red or orange colour and
absence of colour change was considered as nitrite negative. In the later case presence or
absence of nitrate in the broth under examination was confirmed by adding a pinch of
zinc dust, after the addition of the reagents, when the unreduced nitrate, if present, gave a
pink, red or orange colour.
7. Growth temperature (Williams et al. 1989):
Ability of the isolates to grow at different temperatures was
studied at 15° C, 25°C, 37°C and 40°C. The selected isolates were inoculated on starch
casein agar medium and jowar starch medium slants and incubated at the different
temperatures as mentioned above. Results were recorded on 3rd day and 5th day.
81
8. Chemical tolerance pH(Williams et al, 1989):
10 ml quantities of glycerol-nutrient broth with pH levels of 5.2,
8.0, 9.0, and 10.0 were inoculated and incubated for3 to 5 days. Then the tubes were
examined for the extent of growth of the organism.
9. Sodium chloride tolerance (Pridham et al. 1956):
The ability of certain types of actinomycetes to tolerate and to
adapt to high concentrations of sodium chloride is well known. This adaptability is
particularly marked in organisms found in marine water and salt lake mud. Klevenskaya
(1960) found that Streptomyces isolated from dry and saline soils tolerated upto 7%
NaCl. Waksman's literature also indicated that different Streptomyces species vary
widely in their sodium chloride tolerance. Tressener et al. (1968) surveyed
approximately 1300 strains of Streptomyces for tolerance to sodium chloride in the
growth medium. Their results indicated that higher tolerance was found with the yellow
and white-spored Streptomycetes, whereas red series have less tolerance.
For the determination of sodium chloride tolerance, Bennet's agar
medium was supplemented with graded amounts of sodium chloride (1,4,7,10 and 13%).
The above medium was inoculated with spore suspensions of the organisms. After
incubation for 3-5 days, the maximum salt concentration supporting growth was
recorded.
10. Utilization of carbon sources (Shirling and Gottlieb, 1966):
The
ability
of
Streptomyces
species
to
utilize
various
carbohydrates, alcohols, salts of organic acids, fats and amino compounds can be of
considerable diagnostic value (Hata et al. 1953). Waksman (1961) in his early work,
employed synthetic solution with various carbon sources, while others (Shirling and
Gottlieb, 1966; Hata et al. 1953; Waksman, 1961; Benedict et al. 1955) indicated that
solid media were more suitable. Pridham and Gottleib's (1948) basal medium is widely
used for this purpose.
The ability of thermoactinomycete isolates to utilize various
carbon compounds as source of energy was studied using Pridham and Gottleib's basal
salts medium (ISP-9). The following chemically pure carbon sources were employed in
the present study: D-glucose, L-arabinose, galactose, sucrose, ribose, meso-inositol, Dmannitol, D-fructose, rhamnose, raffinose, cellulose, lactose, maltose, salicin, trehalose
and D-xylose.
82
A 10% solution of the above were prepared and sterilized except
cellulose and inositol by filtration using bacteriological filters. Cellulose and inositol
were sterilized by ether sterilization technique. Sterilized carbon sources were added to
the Pridham and Gottleib's basal mineral salts agar to give a final concentration of l%.
The inoculated tubes were incubated at 55°C and observed for growth on 3 rd and 5th day.
The results were recorded as per the extent of growth in the respective slants.
Good growth
:
+++
Moderate growth
:
++
Poor growth
:
+
Doubtful growth
:
±
No growth
:
–
11. Growth in the presence of different nitrogen sources (Williams et al. 1989):
The ability of isolates to use different nitrogen sources was studied
by the following method. Each nitrogen source was incorporated into the basal medium
at 0.1% level. The prepared slants were inoculated and incubated at 28 oC. Results were
recorded after 2-4 days. The growth on each source was compared with that on the unsupplemented basal medium and on a positive control containing L-asparagine. The
following nitrogen sources were used in the present study: L-asparagine (positive
control), L-arginine, L-cysteine HCl, L-histidine, L-valine, phenyl alanine, threonine,
hydroxyl praline, methionine, serine,arginine and potassium nitrate.
Identification and characterization of the selected isolate:
Proper identification and characterization of microorganisms is very important
because it expands the scope for exploitation of industrially important products. To
establish the novelty or otherwise of the present isolate with those of reported in the
literature, the various morphological, physiological and biochemical characteristics of
the isolate GAS-04 was done with the description cited in the literature. The literature
survey includes Bergey’s Manual of Systematic Bacteriology (1992), Bergey’s Manual
of Determinative Bacteriology (1974), Biological Abstracts, Microbiological Abstracts
and all other relevant journals. The isolate GAS-04 was further identified by MTCC,
Chandigarh and results were shown in Table 3.3
Bio-chemical characteristics:
The biochemical characteristics of the isolates were studied by
analyzing the cell wall composition and physiological characteristics.
83
1. Analysis of cell Walls:
Cell Wall compositions were analyzed according to the method of
Boone and Pine (1968). Cultures were grown for 3 days in 50 ml yeast extract malt
extract (YEME) broth in 250 ml conical flasks, the mycelia were collected by
centrifugation at 10,000 rpm for 15 min and washed thrice with sterile distilled water.
Five hundred milligram of the mycelia was extracted with 5 ml of 0. IN NaOH, in tightly
sealed screw capped tubes for 1 h, in a boiling water bath. The mixture was cooled and
centrifuged. The alkali extract was discarded. The cell walls were then re-suspended in 1
ml of water and was used for detection of sugars and amino acids. Identification of
sugars:
The cell wall sample (0.7 ml) was taken and HCl was added to it
(to give a final concentration of 2N HCl) in screw capped tubes, sealed tightly and
placed in a boiling water bath for 2 h. The hydrolyzed materials were transferred to small
beakers and dried over a boiling water bath. To this water was again added, the process
was repeated four times and the materials were finally suspended in 0.5 ml of water and
used for chromatography. One-tenth ml sample was spotted on Whatman No. 1 paper
and ascending chromatography was run using the solvent system n-butanol, acetic acid
and water (3:1:1). The chromatogram was sprayed with a solution containing 0.5 g silver
nitrate, 1 ml water and 25 ml of 95% ethanol. The papers were then dried for 2 to 3 min
until the appearance of dark spots.
2. Identification of amino acids:
The cell wall samples (0.3 ml) were taken in a sealed tube and
hydrolyzed with HC1 (to give a final concentration 6N HC1) at 110°C for 18 h. The
hydrolyzed material was dried in the same way as mentioned for sugar identification
procedure. One-tenth ml of the same sample was spotted on Whatman No. 1 paper and
ascending chromatography was run using the solvent n-butanol, acetic acid and water
(4:1:1). Amino acids were detected by spraying the chromatograms with 0.25%
ninhydrin and drying at 100°C for 5 min.
84
RESULTS AND DISCUSSIONS:
IDENTIFICATION AND CHARACTERISATION OF THE SELECTED
ISOLATE OF GAS-4
Proper identification and characterization of microorganisms is
very important because it expands the scope for exploitation of industrially important
products. In the present investigation, criteria laid down by the International
Streptomyces Project (ISP) were followed for the identification and characterization of
the selected isolates.
To establish the novelty or otherwise of the present isolates with
those reported in the literature, the various morphological, cultural and biochemical
characteristics of the isolated organisms were compared with the descriptions of the
numerous Thermoactinomyces species cited in the literature. The literature survey
includes: Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons,
1974), Bergey's Manual of Systematic Bacteriology (Williams et al. 1989), The
Actinomycetes (Vol. H) by Waksman (1961), The International Streptomyces Project
Reports (ISP) (Shirling and Gottlieb, 1966, 1968, 1969 and 1972), Biological and
Microbiological abstracts and all other relevant journals.
Fig. 3.4 growth of isolate GAS -4 on starch casein agar
85
Fig. 3.5 Screening of isolates with caseinolytic activity by skimmed milk agar
plate
Fig .3.5.1 Screening of isolates with gelatenolytic activity by gelatin agar
medium
The 18 isolates selected after primary screening for caseinolytic
activity in skimmed milk upon were employed to assess their gelatinolytic activity in
gelatin agar medium.It was observed that in general all these isolates had higher
gelatinolytic activity compared to caseinolytic activity which was evident from the extent
of the zones of the hydrolysis
Among 3 isolates isolates which exhibited significant caseinolytic
and gelatenolytic activities,isolate GAS-4.Showed
activity.Hence
most promising caseinolytic
isolate (GAS-4) was selected for detailed
taxonomic studies. The
following taxonomic properties were investigated for the characterization of the isolate
GAS-4.
86
Section A: Macroscopic appearance:
 Growth rate was moderately rapid with a colony diameter ranging from 0.5 to 1
cm.
 The colony texture was cottony to powdery to mealy and
 The color was white becoming yellowish white or pale pinkish while pale on the
reverse
Microscopic appearance:
 The hyphae were hyaline, narrow and septate
 Conidiogenous cells on the hyphae were inflated at the base and were typically
flask-shaped and terminated in a thin zigzagging filaments;
 Conidia were produced from each bending point of the filament, this type of
conidium production is called sympodial geniculate growth;
 Conidia were hyaline, one-celled and globose to ellipsoid in shape and diameter
ranges from 2 to 3 μm;
 The condiogenous cells formed dense clusters which appeared as small powdery
balls in the aerial hyphae when viewed under a dissecting microscope.
Microscopic morphology:
Mount showed hyaline and septate hyphae. Conidiophores
were globose to elliptical. The non motile, elliptical spores with smooth surface are
straight to flexuous chains with compact coils at the ends .On the test media ,aerial
mycelium color was white and no diffusible pigment produced as shown in Fig. 3.6
87
Fig. 3.6 Microscopic morphology of the isolate GAS-4 under 40x magnification
Fig.3.7 Scanning electron Microscope photograph of isolate GAS-4
A.X 6500 Magnification
B.X 9500 Magnification
Macroscopic observation:
For the macroscopic observation, the isolate GAS-4 was
inoculated on the plates containing the following differential media. Yeast extract Malt
extract, Glucose aspargine Agar, Nutrient Agar, Half strength Nutrient Agar, Gelatin
Agar, Starch Agar, L.C.Agar, Potato Dextrose Agar, Oat meal Agar, Inorganic saltsStarch Agar. The culture characteristics were shown in Table 3.10.
Table 3.10 Cultural Characteristics of isolate GAS-04
Medium
Cultural
Yeast extract-Malt extract agar (ISP-2)
Characteristics
G
: Abundant
AM
: White
R
: Nil
88
Glucose Aspargine Agar
Nutrient Agar
Half strength Nutrient Agar
Gelatin Agar
SP
: None
G
: Moderate
AM
: White
R
: Nil
SP
: None
G
: Moderate
AM
: Dull white
R
: Nil
SP
: None
G
: Moderate
AM
: White
R
: Nil
SP
: None
G
: Poor
AM
: Dull white, Little
aerial mycelia
Starch Agar
Lactobacillus casei agar (L.C.Agar)
Potato Dextrose Agar
Oat meal Agar (ISP-3)
R
: Nil
SP
: None
G
: Good
AM
: White
R
: Nil
SP
: None
G
: Good
AM
: White
R
: Nil
SP
: None
G
: Moderate
AM
: Dull white
R
: Nil
SP
: None
G
: Good
AM
: White
R
: Nil
SP
: None
89
Inorganic Salts-Starch Agar (ISP-4)
G
: Good
AM
: Dull white,
moderate aerial
mycelia
Glycerol-aspargine Agar (ISP-5)
R
: Nil
SP
: None
G
: Moderate, wrinkled
colonies
AM
: White
R
: Nil
SP
: None
G: growth, AM: Aerial mycelium, R: Reverse Color and SP: Soluble Pigment.
Table 3.11 Physiological and biochemical properties of isolate GAS-4
Reaction
Result
Gram staining
+
Growth at 150C
+
Growth at 250C
+
Growth at 370C
+
Growth at 400C
+
Growth at pH 5.2
+
Growth at pH 8.0
+
Growth at pH 9.0
+
Growth at pH 10.0
+
Growth on NaCl 2%
+
Growth on NaCl 5%
+
Growth on NaCl 7%
+
Growth on NaCl 10%
-
Starch hydrolysis
+
90
Casein hydrolysis
+
Citrate utilization test
+
Gelatin Liquefaction
+
H2S production
-
MR
+
VP
-
Nitrate Reduction
-
Indole
-
Catalase
-
Urease
-
Acid Production from
Arabinose
-
Galactose
-
Glucose
+
Mannitol
-
Raffinose
+
Salicin
-
Xylose
-
Sucrose
-
Rhamnose
-
Meso-inositol
-
Fructose
+
Cell wall composition
Cell Wall Type-I
91
Table 3.12 Carbon source utilization pattern of isolate GAS-4
Carbon sources
Utilization
Positive
D-glucose(+++), D-fructose (++)
Raffinose (+)
Doubtful
Sucrose
Negative
L(+) arabinose, Cellulose, Galactose,
D-Mannitol, D-xylose, Meso-inositol,
Sucrose, Salicin, Rhamnose
+++: Good growth;
'++: Moderate growth;
+: Poor growth;
Table 3.13 Growth of isolate GAS-4 in the presence of various nitrogen sources
Nitrogen source (0.1% w/v)
Growth response
L-asparagine (positive control)
++
Methionine,
++
Hydroxy proline,
Valine, Threonine & cysteine HC1
Phenylalanine, Serine, Arginine,
-
Histidine, Potassium nitrate
-: No growth;
+: Poor growth
++: Moderate growth;
16S r RNA sequencing of GAS-04
The 16S rDNA gene sequence of the isolate GAS-04 was used as a query to
search for homologous sequence in the nucleotide sequence databases by running
BLASTIN programme. The high scoring similar to 16S rDNA gene sequences were
identified from the BLASTIN result and retrieved from Gene Bank database.
92
Phylogenetic trees were inferred by using the neighbor joining Bootstrap analysis. Isolate
GAS -4 was sent to IMTECH Chandigarh for detrming the biochemical properties and
sequencing the 16s r RNA and its comparison with closely related species by
BLASTINA score and construction of the phylogenetic tree.
The sequence results were trimed and assembled. The assembly of the sequences is
as follows
>GAS-4
ATCCTGGCTCAGGACGAACACTAGCGGCGTGCTTAACACATGCAAGTCGAAC
GATGAACCTCCTTCGGGAGGGGATTAGTGGCGAACGGGTGAGTAACACGTG
GGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCG
GATATGACACGGGATCGCATGGTCTCCGTGTGGAAAGCTCCGGCGGTGAAG
GATGAGCCCGCGCCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCG
ACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACA
CGGCCCAGACTCCTACGGGAGGCAGCAGTGGGAATATTGCACAATGGGCGA
AAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAA
CCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGC
CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTC
CGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCCAGTCGCGTCGGGTGTGA
AAGACCGGGGCTTAACCCCGGTTCCTGCATTCGATACGGGCTGGCTAGAGTG
TGGTAGGGGAGATCGGAATTCCTGGTGTACGGTGAAATGCGCAGATATCAG
GAGGAACAACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAG
GAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCC
GTAAACGGTGGGAACTAGGTGTTGGTCACATTCCACGTGATCGGTGCCGCAG
CTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGAAGGCTAAAACTCAA
AGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGAC
GCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAGCATTAGAG
ATAGTGCCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTGCTCAGCT
CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAGCAGCGCAACCCTTGTCCCG
TGTTGCCAGCAACTCTTCGGAGGTTGGGGACTCACGGGAGACCGCCGGGGTC
AACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTG
GGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGCGAGGT
GGAGCGAATCTCAAAAAACCGCTCTCAGTTCGGATTGGGGTCTGCAACTCGA
CCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCACCCCCCCCTTTCTTGA
ATACGTTCCCGGCCTTGTACAC
93
Table 3.14 Top 9 Sequence Producing Significant Alignments
Mega
Rank
1
2
Name/Title
Streptomyces
indicus
Authors
Luo et.al. (in press)
Strain
IH321(T)
Streptomyces
(Krassilnkov 1941)
LMG
globosus
Waksman 1953
19896(T)
Accession
Pairwise
Diff/Total
BLAS
Similarity
nt
TIN
BLAST
INN
score
score
EF157833
99.120
12/1363
2605
2605
AJ781330
96.741
44/1350
2303
2284
AB184173
96.733
44/1357
2297
2278
AB184349
96.557
46/1336
2268
2240
AY999864
96.557
46/1336
2262
0
AB184280
96.456
47/1326
2250
0
AB184109
96.444
48/1350
2272
2252
AJ781362
96.437
48/1347
2266
2218
AB184652
96.413
48/1338
2264
2230
(Preobrazhenskaya
3
Streptomyces
and Sveshnikoya
NBRC
toxytricini
1957) Pridham et.al.
12823(T)
1958)
4
Streptomyces
xantholitucus
Streptomyces
5
Rubiginosohelvolu
s
6
Streptomyces
Iucensis
(Konev and
Tsyganov 1962)
Pridham 1970)
NBRC
13354(T)
(Kudrina 1957)
IFO
Pridham et.al. 1958
12912(T)
Arcamone et.al. 1957
NBRC
13056(T)
Streptomyces
7
achromogenes
Okami and
NBRC
subsp.
Umezawa 1953
12735(T)
achromogenes
8
9
Streptomyces
tranashiensis
Streptomyces
crystallinus
Hata et.al.1952
Tresner et.al.1961
LMG
20274(T)
NBRC
15401(T)
94
95
Based on the results obtained in these studies our isolate GAS-4
was identified as Streptomyces indicus. It possessed 99.120 pair wise similarity with
Streptomyces indicus 1H32-1(T) reported by Luo et al (2011).
We have carried out a detailed comparison of the biochemical properties of
our isolate GAS-4 with Streptomyces indicus 1H32-1(T)(2011)reportred by L uo et al
(2011).This revealed the following differiences in the biochemical characters between
the two isolates.
Isolate GAS-4 xylose (-), nitrate reduction (-) and mannitol (-).
Isolate 1H32-
1(T) xylose (+), nitrate reduction (+) and mannitol (+).
Based on the differences of these biochemical characters we assign our isolate GAS-4 to
be a new variant and designate it as Streptomyces indicus var.GAS-4.
Moreover our strain has been isolated from terrestrial source whereas Streptomyces
indicus 1H32-1(T) was isolated from a deep sea sediment.
96
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Ichishima, E. (1986). An alkaline proteinase of an alkalophilic Bacillus sp., Curr.
Microbiol. 14:7.
Varley, H., Gowenlock, A. H. and Bell, M(1995). In: practical Clinical Biochemistry.Vol. I,(5th edition), p. 1226, william Heinemann medical Book Ltd, London.
Waksman, S. A.(1958). Proc. Intern. Congr. Biochem., 4th Congr. Vienna.
Waksman, S. A.(1961). The Actinomycetes, Vol. 2, The Williams and Wilkins Co.,
Baltimore, U.S.A.
Williams, S. T. and Cross, T.( 1971). The Actinomycetes, In: Methods in Microbiology,
Vol. 4, p. 295, (ed. Booth, C), Academic Press, London.
Williams, S. T. and Davies, F. L (1967). Use of a Scanning Electron Microscope for the
Examination of Actinomycetes. J. Gen. Microbiol. 48:171-177.
Williams, S. T., Sharpm M. E. and Holt, J. G., Bergey's (1989). Manual of Systematic
Bacteriology, Vol. 4, The Williams and Wilkins Co., Tokyo.
98
CHAPTER-IV
STRAIN IMPROVEMENT STUDIES
STRAIN IMPROVEMENT STUDIES
Strain improvement is an essential part of process development for
fermentation products. Developed strains can reduce the costs with increased
productivity and can possess some specialized desirable characteristics. Such improved
strains can be achieved by inducing genetic variation in the natural strain and subsequent
screening. Thus a major effort of industrial research in producing enzymes is directed
towards the screening programs. Mutation is the primary source of all genetic variation
and has been used extensively in industrial improvement of enzyme production
(Ghisalba,1984; Sidney and Nathan,1975). The use of mutation and selection to improve
the productivity of cultures has been strongly established for over fifty years and is still
recognized as a valuable tool for strain improvement of many enzyme-producing
organisms.
The methods available in applied genetics involve trial and error.
Strain development programs generally involve the following stages: 1) induction of
genetic variation, 2) pre-selection, 3) screening of selected strain in shake flask, followed
by tests in laboratory fermentor, 4) evaluation of selected strain at pilot plant scale and 5)
introduction on a production scale in the main plant.
Mutagenesis
The classical genetic approach to improve the metabolite yield is
to subject the organism to random mutations using various mutagenic agents and then to
screen the survivors after these lethal treatments for colonies that show increased enzyme
production. This process would be repeated until no further increase could be detected.
Choice of mutagen
While selecting mutagen for use in strain improvement program,
one should consider the phenomenon of mutagen specificity, where by a given mutagen
or mutagenic treatment preferentially mutates certain parts of the genome, while
unaffecting the other parts. The industrial geneticist is rarely able to predict exactly what
type of mutation is required to improve the given strain. Hence a series of mutagenic
treatments are carried out to develop a better yielding strain by trial and error.
Various mutagenic agents such as ultraviolet rays (UV), N methylN'- nitro -N-nitrosoguanidine (NTG), X-rays, gamma rays, nitrous acid, ethyl methyl
sulfonate (EMS) etc., are generally used for yield improvement studies. The ultra violet
99
irradiation (UV) is the most convenient of all mutagens to use and it is also very easy to
take effective safety precautions against it. The UV light is the best studied mutagenic
agent in prokaryotic organisms. It gives a high proposition of pyrimidine dimers and
includes all types of base pair substitutions (Meenu et al. 2000).
The EMS is also used for strain improvement as it induces linked
multiple mutations at fairly high frequencies. It promotes base pair substitutions,
primarily GC-AT transitions.
Dosage of mutagen
The optimum concentration of mutagen is that which gives the
highest proportion of desirable mutants in the surviving population. Hopwood et al.
(1985) suggested that 99.9% kill is best suited for strain improvement programs as the
fewer survivors in the treated sample would have undergone repeated or multiple
mutations which may lead to the enhancement in the productivity of the metabolite.
Two important conventional techniques viz., UV irradiation and
HNO2 treatment have been carried out for yield improvement studies in this work..
Experimental
Chemicals
All chemicals and medium constituents used for the present study
were procured from M/S High media, Mumbai.
Microorganisms
Isolated GAS-4(wild strains), that produces protease, was
employed in the present study. These organisms were isolated from soil samples from
Andhra Pradesh State, India. The isolates were grown on starch casein agar slants at
28°C for 96 h, subcultured at monthly intervals and stored in the refrigerator.
Preparation of spore suspension
Each organism grown on starch casein agar slant was scraped off
into sterile water containing tween-80 (1:4000) to give a uniform suspension. The
suspension was transferred into sterile conical flasks (250ml) containing sterile glass
beads and thoroughly shaken for 30 min. on a rotary shaker to break the spore chains.
The spore suspension was then filtered through a thin sterile cotton wad into a sterile
tube, to eliminate vegetative cells from the suspension, so that after plating each spore
was germinated to give a colony. The spore suspension was diluted and used for plating.
100
Shake flask fermentation
Five ml of inoculum (10% v/v) was added to the 45 ml of
production medium in 250 ml Erlenmeyer flask. The flasks were incubated at 28°C on
rotary incubator shaker for 96 h (180 rpm with 5 cm through). At the end of
fermentation, 5ml broth was collected and centrifuged at 3000 rpm for 10 min. and
assayed for protease activity. The composition of production medium is: g/100 ml
Glucose-0.5, yeast extract – 0.25, Tryptone – 0.25, pH-7.0.
Analytical method
The protease activity was determined by Lowry method as described earlier.
Mutation and selection
a) UV Irradiation of parent strain and selection of mutants
Strain improvement
for the selected parent strain were done by
mutation and selection. The wild strain (GAS-4) was subjected to UV irradiation. The
dose survival curve was plotted for selecting the mutants between 10 and 0.1% rate.
Mutation frequency was mentioned to be high when the survival rates were between 10
and 0.1% (Hopwood et al. 1985).
The spore suspension of wild strains was prepared in phosphate
buffer, pH 8.0 and 4 ml quantities were pipetted aseptically into sterile flat bottomed
petridishes of 100 mm dia. The exposure to UV light was carried out in a "Dispensing Cabinet" fitted with TUP 40W Germicidal lamp that has about 90% of its radiation at
2540-2550A. The exposure was carried out at a distance of 26.5 cm away from the center
of the germicidal lamp. The exposure was carried out for 0, 30, 60, 9, 120, 180 and 240
seconds respectively. During the exposure, the lid of the Petridis was removed. Hands
were covered with gloves and the plates were gently rotated so as to get uniform
exposure of the contents of the Petridis. During the treatment, all the other sources of
light were cut off and the exposure was carried out in dark (during night time). The
treated spore suspensions were transferred into sterile test tubes covered with a black
paper and kept in the refrigerator overnight, to avoid photo reactivation.
Each irradiated spore suspension was serially diluted with sterile
phosphate buffer solution (PBS). The spore suspensions after suitably diluting in the
buffer (PBS) with pH -7 were plated onto starch casein agar medium and incubated for
96 h at 28°C. The number of colonies in each plate was counted. It was assumed that
each colony was formed from a single spore. The number of survivals from each
exposure time for GAS-4are represented in Table 4.1. The UV survival curves are
101
plotted (Fig. 4.1). Plates having less than 1% survival rate (60 and 120 sec.) were
selected for the isolation of mutants. The isolates were selected on the basis of
macroscopic differential characteristics. The selected isolates were subjected to
fermentation and tested for their alkaline protease production capacities as described
earlier (Fig. 4.3). The best protease producing UV mutant strain (UV A8) was selected
for HNO2 treatment.
b) Nitrous acid treatment:
The cell suspension of the strain UV A8 was prepared by using
Acetate buffer pH 7.0. To 9mL of the cell suspension in buffer 1mL sterile stock solution
of 0.01 M Sodium Nitrite was added. Samples of 4mL were withdrawn at, 30, 60, 120,
180, 240 seconds respectively. Each of 1mL sample was neutralized with 0.5mL of
0.1M NaOH. Serially diluted and plated on the YEME medium.
Plates having survival rate between 15 and 1% were selected for
the isolation of mutants. The stable mutants UV A1 to UV A13 were selected based on
the consistent expression of the phenotypic characters and maintained on YEME slants.
The plates were incubated at 28⁰C for 5 days.
102
RESULTS AND DISCUSSION:
Genetic improvement is one of the promising approaches for
increased production of enzymes by industrially important microorganisms. Genetic
improvement of the selected GAS-4 strain was carried out by physical and chemical
mutagenesis. In the present investigation, mutations were induced physically by using
UV irradiation and chemically by Nitrous acid treatment.
A. UV irradiation of parent strain GAS-4 and selection of mutants:
The strain was subjected to UV Irradiation. Mutation frequency
was observed to be high when the survival rates were between 27 and 1%..
The dose survival curve was plotted and presented in Table 4.1.
Table. 4.1 Effect of UV Irradiations on Isolate GAS-4
UV Survival Curve:
UV Survival Curve is plotted by taking UV Irradiation time on Xaxis and log survival on Y-axis. (Fig 4.1)
S.No Irradiation
Number
of Percentage
Time
colonies/mL
(secs)
irradiation (x105)
after Kill
(%)
Survival
Log
Percentage
Survival
(%)
1.
0
162
0
100
2.
30
91
43.82
56.18
1.74
3.
60
23
85.80
14.20
1.15
4.
120
93.20
6.80
0.83
5.
180
0.0004
97.53
2.46
0.39
6.
240
0
100
0
0
11
2
103
Fig 4.1 UV Survival Curve of the isolate GAS-4
Protease activity of the UV mutants: A total 13 mutants were
isolated and their Protease production capacity by submerged fermentation was
determined according to the Lowry method (Lowry et al. 1951).
Table. 4.2: Protease activity of UV mutants
S.No
UV Mutants
Protease activity
1
UVA1
90.6
2
UVA2
89.6
3
UVA3
60.5
4
UVA4
73.9
5
UVA5
89.3
6
UVA6
78.2
7
UVA7
76.5
8
UVA8
119.4
9
UVA9
68.7
10
UVA10
75.9
11
UVA11
72.0
12
UVA12
63.2
13
UVA13
65.4
14
Wild Strain
92.0
(U/ml)
104
Fig 4.2 Protease Activity of UV Mutants of the isolate GAS-4

Out of UV 13 mutants
o UVA8 showed a maximum Protease activity of 119.4U/ml while the wild
strain showed 92U/ml.(Table. 4.2) & (Fig. 4.2)
o Production of Protease by UVA8 mutant was 27.4 % higher
than the
parent wild strain (GAS-4).

Hence UVA8 was selected for subsequent Nitrous acid treatment.
B. Nitrous acid treatment of UV-8 mutant:
The selected mutant UVA8 was subjected to nitrous acid
treatment. The dose survival curve was plotted and presented in Table 4.3 and Fig
4.3.The survival curve was plotted by taking Exposure time on X-axis and % survival on
Y-axis. (Fig 4.3)
105
Table. 4.3: Effect of Nitrous acid on UVA8 Mutant
S.No Irradiation
Time
(secs)
Number
of Percentage
colonies/mL
after Kill
5
irradiation (x10 )
1.
0
156
2.
30
3.
(%)
Survival
Log
Percentage
Survival
(%)
0
100
2
87
44.23
55.7
1.74
60
18
88.46
11.54
1.06
4.
120
10
93.58
6.42
0.80
5.
180
0.0005
96.79
3.20
0.50
6.
240
0
100
0
0
106
Fig. 4.3 Survival Curve of UVA8 mutants after Nitrous acid treatment.
Protease activity by various Nitrous acid mutants of UVA8:
A total of 15 mutants were selected and determined for their
Protease production capacities by submerged fermentation and the activity was
determined according to Lowry method (Lowry et al. 1951).
Table. 4.4: Protease Activity by various Nitrous acid mutants of UVA8
S.No
UVA8
Protease activity
(U/ml)
mutants
1
HNB1
129.6
2
HNB2
131.7
3
HNB3
117.2
4
HNB4
109.8
5
HNB5
123.1
6
HNB6
119.6
7
HNB7
132.5
8
HNB8
130.1
9
HNB9
127.2
10
HNB10
117.2
11
HNB11
112.9
12
HNB12
116.2
107
13
HNB13
90.4
14
HNB14
60.7
15
HNB15
78.6
16
Wild Strain
92.0
Fig. 4.4: Protease activity by various Nitrous acid mutants of UVA8
Out of 15 Nitrous Acid mutants
o HNB7 showed maximum Protease activity of 132.5U/ml, which was 12.6 times
more than the mutant strain UVA8 (119.4 U/ml). (Table 4.4) & (Fig. 4.4)
o Compared to the wild strain (80 U/ml) the mutant HNB7 produced 132.5 U/ml.
This represents an increase of 60.6 % compared to the wild strain.
108
REFERENCES:
Ghisalba, O., J. A. L. Auden, T. Schupp, and J. Nuësch. (1984). The rifamycins:
properties, biosynthesis, and fermentation. In E. J. Vandamme (ed.), Biotechnology of
industrial antibiotics. pp. 281-327. Marcel Dekker, Inc. New York.
Hopwood, D. A., M. J. Bibb, K. F. Chater, T. Kieser, C. J. Bruton, H. M. Kieser, D. J.
Lydiate, C. P. Smith, J. M. Ward, and H. Schrempf. (1985). Genetic manipulation of
Streptomyces: a laboratory manual. The John Innes Foundation, Norwich, United
Kingdom.
Lowry O H, Rosebrough N J, Farr A L & Randall R J.(1951). Protein measurement with
the Folin phenol reagent. J. Biol. Chem. 193:265.
Meenu, M., Santhosh, D., Kamia, C. and Randhir, S., Ind. J. Microbiol( 2000).
Production of alkaline protease by a UV-mutant of Bacillus polymyxa .40: 25.
Sidney, P. C. and Nathan, O.K.(1975). Methods In Enzymol.J.Microbiology.3: 26.
109
CHAPTER-V
PURIFICATION AND CHARACTERIZATION STUDIES
PURIFICATION AND CHARACTERIZATION OF ALKALINE PROTEASE
Experimental
Chemicals
Agarose, acrylamide, bis-acrylamide, sodium dodecyl sulphate
(SDS), TEMED, ammonium per sulphate and other chemicals for polyacrylamide gel
electrophoresis (SDS-PAGE) were purchased from Sigma Chemical Co., U.S.A.
Microorganism
A strain of GAS-4 isolated by us in our laboratory was used in the study.
Preparation of cell suspension
The inoculum was prepared as described earlier and used for the fermentation.
Production of alkaline protease in a 2litre fermenter
The alkaline protease production was carried out in a 2 litre
fermenter (B.Braun Biotech International, Micro DCU-200) as shown in Fig. 5.0
containing 1.5 L modified production medium. The composition of modified production
medium was (g/L): Glucose, 10; Soyabean meal, 20; CaCl2, 0.4; MgCl2, 2.0 with pH 10.
A 10% (v/v) level of inoculum was added and the fermenter was run at 37 0C for 48
hours. After the completion of fermentation, the whole fermentation broth was
centrifuged using Sor vall RC 5C centrifuge at 10,000rpm at 40C and the clear
supernatant was separated. The supernatant (crude enzyme) was subjected to recovery
and purification process.
Enzyme recovery and purification procedure
Ammonium sulphate precipitation
A trial was run to determine the optimal concentration
required for the enzyme precipitation with various concentrations of Ammonium
sulphate. For this purpose, the supernatant obtained after centrifugation was subjected to
ammonium sulphate fractionation. Ammonium sulphate was added at different
concentrations ranging from 40 to 80% saturation. The precipitates so obtained were
suspended in cold saline solution (2ml) and tested for protease activity and total protein
content. The salting out concentration of the crude enzyme was established to be 60% on
the basis of enzyme activity. To obtain complete precipitation of the crude enzyme, the
remaining harvest fluid was subjected to ammonium sulphate precipitation at 60%
saturation. For this purpose, solid ammonium sulphate (195g) was added gradually with
110
mechanical stirring to harvest fluid (2x500ml) at40C to a saturation of 60%. The
precipitate so formed was separated by centrifugation (8000g) for 15min., resuspended in
cold saline solution (100ml) and dialyzed in cold against 1L of 0.05M Tris-HCl-0.1M
NaCl (pH 10) for 20 hrs. After dialysis, the solution was centrifuged and supernatant
obtained was designated as fraction –I.
Fig. 5.0 Fermenter (B.Braun Biotech International, Micro DCU-200, Germany
Sephadex G-200 gel filtration chromatography (step III)
The dialyzed enzyme (fraction-II) was centrifuged at 8000g for
15min. and supernatant was chromatographed on a column of Sephadex G-200. The
sample (fraction-II) was loaded on to a column of Sephadex G-200 (1.5cmx24cm)
equilibrated with 0.05M Tris-HCl-0.1M NaCl (pH10). The column was eluted at a slow
rate of 1.0ml/hr with a discontinuous gradient from 0.1M to 0.8M NaCl in the same
buffer. A total of 40 fractions were collected. A typical chromatogram is shown. From
the elution profile it was observed that the protein was eluted as a well resolved peak of
Caseinase activity coinciding with single protein peak at a NaCl concentration of 0.4M.
Fractions (15-18) with high protease activity were pooled together, dialyzed and
concentrated by lyophilization and used for further studies. It was labeled as fractionIII(Deyl.z,1979).
ION-EXCHANGE CHRAMOTOGRAPHY:
The
dialyzed
enzyme
subjected
to
ion
exchange
chromatography containing DEAE-Cellulose .the resin was poured to the column and
111
equilibrated with 10mM Tris –Cl buffer (Ph-7.0).the dialyzed sample was loaded to the
column and its was eluted from column by using gradient elution.
Preparation of elution buffer (gradient elution)
S.NO 1M TRIS –Cl,(10ml)pH-7.0
1M NaCl (10 ml)
1
250
250
2
250
750
3
250
1000
4
250
1250
5
250
1500
6
250
1750
The above are in micro liter .the final volume in each tube was
made to 10 ml with distilled water. All the six tubes were estimated for protein. The tube
containing highest protein was assayed for enzyme
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
After Sephadex G-200 column chromatography, the fractions (1518) showing the highest specific activity was dialyzed, lyophilized and then subjected to
SDS-PAGE. The SDS-PAGE was performed according to Laemmli (1970) using 10%
acrylamide.
The gels were cast by mixing the ingredients as detailed below:
The separating gel mixture was cast in a slab gel apparatus
(Minigel, Genei, Bangalore, India) and overlaid with water. This was left for several
hours to allow for polymerization to occur. Then the water layer was removed and the
stacking gel was cast on top of the separating gel. To run the SDS gel, the
electrophoresis buffer containing 25mM Tris, and 250mM glycine (electrophoresis
grade) with pH 10 was used. The SDS was added to a final concentration of 0.1% to the
electrophoresis buffer. Protein samples containing the five molecular weight markers
Bovine serum albumin (67KD).a crude broth and fractions obtained after dialysis and ion
exchange chromatography were dissolved in sample buffer containing 10mM Tris HCl
(pH 6.8), 4% SDS, 20% glycerol, 0.002% β-mercaptoethanol and 0.002% bromophenol
blue as the tracking dye and boil for 10min. to denature the protein. The treated samples
were loaded in the wells of the slab gel and electrophoresis was started by applying 60
Volts per gel. When the dye front reached the separating gel the voltage was increased to
112
120V and the electrophoresis was continued till the tracking time reached the lower end
of the gel. After the run was over, the gel was soaked overnight (about 16 h) in a fixative
solution containing 50% (v/v) methanol and 12% (v/v) acetic acid. After taking out the
gel from the fixative solution, it was stained by Coomassie Blue R-250 solution. The
results were shown in the Fig. 5.2.
Gel staining
Coomassie blue staining
The gel was stained for 1h with 0.25% Coomassie Blue R-250 in
methanol/water/acetic acid (50:40:10) and the gel was finally destined in a detaining
solution containing water/acetic acid/methanol (87.5:7.5:5).
Native PAGE:
Here the gel electrophoresis is run in the absence of SDS and
DTT. The electrophoretic mobility in SDS-PAGE depends on the molecular mass, while
in native PAGE the mobility depends on both protein’s charge and its hydrodynamic size
(Deyl, Z., 1979). Native PAGE serves as an excellent tool to study conformation, selfassociation or aggregation, and the binding of other proteins or compounds in neutral pH
conditions (Hames, B.D., 1990). Thus it is a powerful technique to study structure and
composition of proteins since both conformation and biological activity remain intact
during the process. (Laemmli., 1970).
PROCEDURE:

Thoroughly clean and dry the glass plates and spacers and insert within bulldog
clips. Fix the chamber in the upright level position.


Prepare 10ml Separating Gel Mixture
40% Acrylamide:Bis Solution (37.5:1)
1ml
4x Separating Gel Buffer
2.5ml
50% Glycerol
2.5ml
Distilled Water
4ml
Degas the solution and then add:
10% Ammonium Persulphate
1
1
TEMED (N,N,N ,N tetramethyl-ethylene diamine)
50ul
10ul
113

Mix gently and use immediately to avoid polymerization reaction. Carefully pour
the solution into the chambers without formation of air bubbles.

Carefully add acrylamide solution with water saturated n-butanol as an overlayer
without mixing to remove oxygen and generate flat surface on the gel.

Polymerize acrylamide layer for an hour.

Prepare 4ml of Stacking gel solution


40% Acrylamide:Bis solution (37.5:1)
0.4ml
4x Stacking gel buffer
1.0ml
Distilled water
2.6ml
Degas the Stacking gel solution and then add:
10% Ammonium Persulphate
20ul
TEMED
5ul
Mix gently and use the mixture immediately. Discard the n-butanol from the
polymerized gel and wash with water to remove the remnants. Fill the voids with
Stacking Gel mixture and insert the comb.

Polymerize the acrylamide for an hour.

After polymerization remove the comb and clips without disturbing plates. Install
the gel in the apparatus.

The apparatus is filled with Reservoir Buffer. Gently remove any air bubbles
from top and underneath the gel using spacers. Use the gel immediately.
SAMPLE PREPARATION:

Dissolve the protein sample in same volume of Sample buffer. The sample
concentration is adjusted such that it gives sufficient amount of protein in a
volume not greater than size of the sample well.

Load the gel with 10-30ul of Protein sample solution by pipette.
114

Electrophoresis is carried out. The bromophenol dye front takes 3hours to reach
the bottom of the gel. Application of greater voltages enhances the speed of
electrophoresis but may generate heat.

Remove the gel from the glass plates.

Stain the gel in Staining Solution for 2-3hours. The composition of Staining
solution is given below:


Coomassie Brilliant Blue R250
0.25g
Methanol
125ml
Glacial Acetic acid
25ml
Deionized H2O
100ml
Remove the dye that is unbound to protein using Destaining Solution.
Methanol
100ml
Glacial Acetic acid
100ml
Deionized H2O
800ml
After 24 hours the gel background becomes colorless and leaves blue, purple or
red colored protein bands. The results were shown in Fig. 5.3.
ZYMOGRAPHY METHOD:
Zymography is the latest method being used to analyze Matrix
Metalloproteinases (MMP) and Tissue Inhibitors of Metalloporteinases (TIMP) in
biological samples. This technique is rather simple, sensitive, accurate and quantifiable.
In zymography, the proteins are separated by gel electrophoresis where separation occurs
in polyacrylamide gel. (Patricia A.M. et al. 2005).
REAGENTS USED IN ZYMOGRAM:

1% Gelatin

1% Casein

SDS-PAGE gel stock without urea

Calcium Chloride

Tris-Acetic acid
115

Tris-Base

Glycine

Triton X-100

30% Acrylamide

0.8% Bis-acrylamide

TEMED

Ammonium persulphate

Sodium Dodecyl sulphate

Coomassie Brilliant Blue R-250
PRINCIPLE OF ZYMOGRAPHY:

Gelatin is retained on the gel during electrophoresis.

The activity of MMP is reversibly inhibited by SDS during electrophoresis.

It also enables the separation of MMP and TIMP complexes. Thus, both
MMP and TIMP can be detected independently.
An advantage of using Zymography is that both proenzymes and active forms of
MMPs can be distinguished on the basis of their molecular weight.
PREPARATION & PROCEDURE:

Samples are prepared in the standard SDS-PAGE treatment buffer without
reducing agent (in order to keep the enzyme in the native state).

A suitable gel is placed in the resolving gel during the preparation of acrylamide
gel.

Electrophoresis is carried out. The SDS is removed from the gel by incubation in
unbuffered Triton-X-100.

The gels are later incubated in digestion buffer for a specified period of time at
370C.
116

The zymogram is subsequently stained using Amido black, Coomassie brilliant
blue dyes.

The areas of digestion appear on clear bands against darkly stained background
where the substrate has been degraded by the enzyme. The results were shown in
the Fig. 5.4.
BIOINFORMATICS MODELING AND INVITER PRODUCTION LABELS
Proteases are the group enzymes involved in hydrolysis of
peptide bonds. Proteases are grouped into four different classes: the cysteine, serine
proteases, metallo and aspartic acid proteases. Based on the structural similarities
alkaline proteases have been grouped into 20 families with six clan subdivisions.
Alkaline proteases hydrolyse the peptide bond containing tyrosine, phenylalanine or
leucine at the carboxyl end of the splitting bond (Vivek Kumar Morya et al. 2011).
The DNA and protein sequence homology have widespread use
now-a-days. The nucleotide and amino acid sequences of number of proteases have been
determined and the results find use in elucidating structure-function relationship.
Availability of genome sequences from several Streptomyces species found their use in
identification of putative secondary metabolism genes and gene clusters which were not
known previously. However, functional analysis is necessary in certain putative
secondary metabolism genes which may not be expressed at a level sufficient to detect
products. This difficulty can be overcome by manipulating structural and regulatory
genes to obtain expression or by experimentation on various strains of the same species
since expression may be strain dependant (Rabbani Syed et al. 2012). The results were
shown in Fig. 5.5.
Analytical methods
Determination of alkaline protease activity
Alkaline protease activity was determined as described earlier.
Protein assay
Protein was measured by the method of Lowry et al. (1951) with
bovine serum albumin (BSA) as the standard. The concentration of protein during
purification studies was calculated from the absorbance at 280 nm.
117
DETERMINATION OF KINETIC PARAMETERS OF THE PURIFIED
ALKALINE PROTEASE FROM THE STRAIN GAS-4.
Effect of substrate concentration on Alkaline protease activity
In order to characterize the alkaline protease produced by the
strain of GAS-4, the enzyme (1mg/ml) was incubated at a time interval of 30 min with
different concentrations of protease. The protease concentration was shown in Table
5.1.1 and Fig.5.7.0. From the graph it was observed that at 15mg concentration, protease
showed the maximum velocity. Further increment of protease concentration did not
enhance the activity significantly.
Characterization of purified enzyme
Effect of pH on purified enzyme activity and stability
Activity of the purified protease was measured at different pH
values to study the effect of different pH values on activity. The pH was adjusted using
the following buffers (0.05 M): phosphate (pH 5.0-7.0), Tris-HCl (pH 8.0) and glycineNaOH (pH 9.0-12.0). Reaction mixtures were incubated at 55°C for 30 min. and the
activity of the enzyme was measured. The results are shown in Fig. 5.6.
To determine the stability of enzyme at different pH values, the
purified enzyme was diluted in different relevant buffers (pH 6.0-12.0) and incubated at
37°C for 2 and 20 h. The relative activity at each exposure was measured as per assay
procedure and the results are shown in Fig. 5.6.
Effect of temperature on enzyme activity and stability
The activity of the purified enzyme was determined by incubating
the reaction mixture at different temperatures ranging from 30 to 100°C for 30 min. in
the absence and presence of
10 mM CaCl2. The results are shown in Fig. 5.7.1.
To determine the enzyme stability with changes in temperature,
purified enzyme was incubated for 30 min. at different temperatures (60, 70, 80 and
90°C) in the presence of 10 mM CaCl2 and relative protease activities were assayed at
standard assay conditions. The results are shown in Fig. 5.8.
118
Effect of protease inhibitors and chelators on enzyme activity
The effect of various protease inhibitors (at 5mM), such as serine
inhibitors [Phenylmethylsulphonyl fluoride (PMSF) and Diisopropyl fluorophosphate
(DFP)], cysteine-inhibitors [p-chloromercuric benzoate (pCMB) and -mercaptoethanol
(-ME), and a chelator of divalent cations [Ethylene diamine tetra acetic acid (EDTA)]
were determined by preincubation with the enzyme solution for 30 min at 37°C before
the addition of substrate. The relative protease activity was measured. The results are
shown in the Fig. 5.9.
Effect of various metal ions on protease activity
The effects of different metal ions viz., Ca2+, Mg2+, Co+2, Cd+2,
Fe+3, Na+, Zn2+ and Cu2+ (10 mM) were investigated by adding them into the reaction
mixture. The mixture was incubated for 30 min. at 37°C and the relative protease
activities were measured. The results are shown in the Fig. 5.10.
Hydrolysis of protein substrates
Protease activity with various protein substrates such as bovine
serum albumin (BSA), casein, egg albumin and gelatin was assayed by mixing 100ng of
the purified enzyme and 200l of assay buffer containing the protein substrates (2
mg/ml). After incubation at 37°C for 30 min, the reaction was stopped by adding 200l
of 10% TCA (w/v) and allowed to stand at room temperature for 10 min. The undigested
protein was removed by centrifugation and the released peptides were assayed. The
specific protease activity towards casein was taken as a control. The results are shown in
the Fig. 5.11.
Enzyme stability in presence of detergents
The compatibility of GAS-4 protease with local laundry detergents
was investigated in the presence of 10 mM CaCl2 and glycine. The following detergents
were used: Nirma (Nirma Chemical, India), Henko (SPIC, India), Surf Excel, Super
Wheel, Rin (Hindustan Lever Ltd, India) and Ariel (Procter and Gamble, India). The
detergents were diluted in distilled water (0.7% w/v), incubated with 0.1 ml of enzyme
(364U/ml) for 4h at 37°C and the residual activity was determined. The enzyme activity
119
of a control sample (without any detergent) was taken as 100%. The results are presented
in the Table 5.2. Our protease showed good stability and compatibility in the presence of
Ariel. As such, the compatibility of our enzyme was studied with Ariel in presence of 10
mM CaCl2 and 1M glycine for different time periods (0.5–3h) at 60°C. The results are
shown in Fig. 5.12.
Washing test with protease preparation
Application of protease as a detergent additive was studied on
white cotton cloth pieces (4  4 cm) stained with blood (0.1ml) and kept aside for 1h.
The stained cloth pieces were taken in separate flasks. The following sets were prepared
and studied:
1. Flask with distilled water (100 ml) + stained cloth (stained with blood).
2. Flask with distilled water (100 ml) + stained cloth (stained with blood) + 1 ml
ariel detergent (7mg/ml).
3. Flask with distilled water (100 ml) + stained cloth (stained with blood) + 1 ml
ariel detergent (7mg/ml) + 2 ml enzyme solution.
The above flasks were incubated at 37°C for 15 min. After
incubation, cloth pieces were taken out, rinsed with water and dried. Visual examination
of various pieces exhibited the effect of enzyme in removal of stains (Fig. 5.13).
Untreated cloth pieces stained with blood were taken as control.
Dehairing of animal skin with protease
The dehairing capacities of the crude broth and purified enzyme
were studied on fresh goat skins. For this purpose, enzyme solutions (20 ml) were
applied as a paste with kaolin (10g) and streptomycin sulphate (100mg) on the flesh side
of freshly slaughtered paired goatskin pieces. The skins were kept aside for 6h. A control
was also kept using water instead of enzyme solution. After 6h contact time, the ease of
dehairing was noted by removing the hairs with a blunt scalpel. The results are shown in
Fig.5.14.
120
RESULTS AND DISCUSSION:
Purification of alkaline protease of Streptomyces indicus GAS-4:
The enzyme production was carried in a 2L fermenter as per the
general procedure. The clear fermentation broth containing the crude enzyme was
subjected to purification.
Sephadex G-200 gel filtration chromatogarphy
The crude broth obtained after fermentation was subjected to
ammonium sulfate precipitation at 60% followed. The pellet obtained was dialyzed in
0.1 M Tris-HC1 buffer.After dialysis, the dialyzed enzyme was subjected to ion
exchange chromatography on a DEAE-Cellulose column .The elution profile shown in
the Fig. 5.2.
It was observed that the protease was eluted as a well resolved
single peak of caseinase activity coinciding with a single protein peak at NaCl
concentrations of 0.4 M. Fractions (15-18) with high protease activities were pooled,
dialyzed and concentrated by lyophilization and used for further studies. The summary of
purification steps involved for alkaline protease is reported in the Table 5.1.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PA GE)
The crude broth, (precipitate obtained after ammonium sulphate
precipitation) and the purified protease along with standard molecular weight markers
were run on SDS-PAGE. Several bands were observed in the case of ammonium
sulphate precipitate (Fig. 5.2) while purified protease showed a single band on SDSPAGE, indicating a homogeneous preparation. The molecular weight of the protease was
determined by comparison of the migration distances of standard marker proteins. The
molecular mass standards used were bovine serum albumin (67 kDa), ovalbumin (45
kDa), carbonic anhydrase (30 kDa), trypsinogen (24 kDa) and – lactalbumin (14 kDa).
The molecular weight was measured by interpolation from a linear semi-logarithmic plot
of relative molecular mass versus the Rf value (relative mobility) (data not shown).
Depending on the relative mobility, the molecular weight of the protein band was
calculated to be around 60 kDa. Thus it was concluded that our alkaline protease enzyme
has a molecular weight of 60 kDa.
121
Many reports had been published on purification of different
microbial proteases using ammonium sulphate precipitate and anion exchange
chromatography method (Yamamato et al. 1987).The molecular weight of purified
enzyme of St.halstedii Salh-12 and St.endus Salh -40 were 60 and 35 kDa.
122
Table 5.1 Summary of purification steps of alkaline protease from S.indicus GAS-4
S.No
Fraction
1.
2.
3.
4.
5.
CRUDE
AMMPPT
DIALYSIS
IEC
GEL
FILTRATION
Total
Total
Activity(IU) Protein
(mg)
306400
1000.2
249930
701.6
200700
400
184048
80
135422
28
Specific
Activity
Fold
Purification
%Yield
306.4
356.2
501.7
2300.6
4836.5
1.162
1.637
7.508
15.78
100
81.56
65.5
60.06
44.1
The purification profile indicated that the enzyme was purified 15.78 fold with an
yield of 44%.
123
Fig. 5.1 Elution profile of alkaline protease from Streptomyces indicus GAS-4.
124
Fig. 5.2 SDS-PAGE of alkaline protease from Streptomyces indicus GAS-4.
Marker
Crude Enzyme Dialysis
IEC
125
Native PAGE
Fig. 5.3 Native PAGE of alkaline protease from Streptomyces indicus
GAS-4.
126
Zymography method:
Fig. 5.4 Zymograph of alkaline protease from Streptomyces indicus GAS-4
`
The arrow mark showing colourless patches is indicative of protease
activity.
127
Fig. 5.5. (A) Production template; (B) 3D Protease respect to template; (C)
Observed structure of protease.
(A)
(B)
(C)
Comparative modeling predicts the 3-D structure of alkaline
protease model as a given protein sequence (target) based on the template. The
hypothetical 3Dstructures of template and the model are given in the above Fig 5.5.
128
Characterization of purified enzyme
pH optimum and pH stability
For the determination of the pH optimum, phosphate (pH 5.0-7.0),
Tris-HCl (pH 8.0) and glycine-NaOH (pH 9.0-12.0) buffers were used. The highest
protease activity was found to be at pH 9.0 using glycine-NaOH buffer. The results are
shown in Fig. 5.6. The stability of the purified protease was also determined by the
preincubation of the enzyme in various buffers of different pH values. In the case of 2 h
preincubation group, the enzyme was stable over a broad range of pH 8-10. On the other
hand, in the case of 20 h preincubation group, the enzyme was stable between pH 8 and
pH 9 (Fig.5.6).
Effect of substrate concentration on protease activity
The substrate profiles of protease activity were shown in Fig. 5.7.0
and Table 5.1.1. From the graph it was observed that at 15mg concentration, protease
showed the maximum velocity.
Temperature optimum and thermal stability
The activity of the purified enzyme was determined at different
temperatures ranging from 30° to 90°C in the absence and presence of 10 mM CaCl 2.
The optimum temperature recorded was at 37°C for protease activity. The enzyme
activity was gradually declined at temperatures beyond 40°C(Fujiwara and Yamamoto,
1987). . The results are shown in Fig.5.7.
The GAS-4 protease had a half-life of 250 min. and less than 50,
min at 70°C and 80°C respectively. The enzyme was almost 100% stable at 37°C even
after 350 min of incubation (Dhandapani and vijayaragavan, 1994). The results are
shown in the Fig. 5.8.
Effect of inhibitors and chelators
Inhibition studies primarily give an insight of the nature of
enzyme, its cofactor requirements and the nature of the active center (Sigma and Mooser,
1975). The effect of different inhibitors on the enzyme activity of the purified protease
129
was studied and the results are presented in the Fig.5.9. Of the inhibitors tested (at 5
mM cone.), PMSF was able to inhibit the protease completely while DFP exhibited 94%
inhibition. In this regard, PMSF sulphonates the essential serine residue in the active site
of the protease and has been reported to result in the complete loss of enzyme activity
(Gold and Fahrney, 1964). Our result were similar to that of Yamagata et al. (1989), who
reported complete in inhibiting protease by PMSF. This indicated that it is a serine
alkaline protease. In the case of other inhibitors, the protease was not inhibited by
EDTA, while a slight inhibition was observed with pCMB and  – ME.
Fig.5.6 Effect of pH on the activity of alkaline protease
Optimum pH: 9.0
130
Table 5.1.1. Effect of substrate concentration on protease production.
Substrate concentration [S],μg
Protease activity (IU/ml)
2
0.16
3
0.2
5
0.24
7
0.28
9
0.31
10
0.33
12
0.38
13
0.44
15
0.46
17
0.48
19
0.51
20
0.52
22
0.53
Fig. 5.7.0 Effect of substrate concentration on protease activity.
.
131
Fig.5.7.1 Effect of temparature on the activity of alkaline protease
Optimum temperature: 370C
132
Fig. 5.8 Effect of temperature on the stability at 370C
133
Fig. 5.9 Effect of protease inhibitors/chelators on enzyme activity.
Effect, of metal ions
Some of the metal ions tested had slight stimulatory effect (Ca2+,
and Na+) or slight inhibitory effect (other ions) on enzyme activity. The results are
presented in the Fig. 5.10. Addition of the metal ions Ca2+, and Na+ increased and
stabilized the protease activity of enzyme probably because of the activation of the
enzyme by these metal ions. These cations also have been reported to increase the
thermal stability of alkaline proteases from Bacillus sp. (Rahman et al. 1994; Paliwal et
al. 1994). Other metal ions such as Zn2+, Cu2+, Co2+, Cd2+, and EDTA did not shown any
appreciable effect on enzyme activity. The residual protease activity was greater than the
control when the enzyme was exposed to Ca++ and Na+ ions and same as the control
when exposed to Mn+2. Similar results were given by Tsuchiya K. et al who reported that
Calcium divalent cation increased pH and Heat stability. This is beneficial when the
enzyme is used industrially.
134
Hydrolysis of protein substrates
When assayed with native proteins as substrates, the protease
showed a high level of hydrolytic activity against casein and moderate hydrolysis of
BSA and egg albumin.The hydrolysis of gelatin was significant and slightly lower
compared to casein .The results are presented in the Fig.5.11.
Compatibility with detergents
Besides pH, a good detergent protease is expected to be stable
in the presence of commercial detergents. The protease from GAS-4 showed
excellent stability and compatibility in the presence of locally available detergents
(Tide, Wheel, Mr. White , Surf Excel, Ariel and Rin). The results are presented in
the Table 5.2.
Fig. 5.10 Effect of various metal ions on alkaline protease activity.
135
Fig. 5.11 Alkaline protease activity against different natural substrates.
136
Table 5.2 Compatibility of alkaline protease activity with commercial detergents in
the presence of CaCl2 and glycine.
Relative residual alkaline protease activity (%)
Time (h)
Control
Ariel
0
100
100
0.5
96
1.0
Surf
Tide
Rin
Mr.White
Wheel
100
100
100
100
100
94
78
85
84
80
90
94
92
73
81
80
78
88
1.5
91
89
70
79
77
73
84
2.0
87
83
65
68
68
69
79
2.5
80
80
68
64
60
66
72
3.0
76
79
60
58
55
52
68
4.0
72
70
59
54
48
46
59
Excel
The protease showed stability and compatibility with the above
commercial detergents at 37°C in the presence of CaCl2 and glycine as stabilizers. Our
protease showed good stability and compatibility in the presence of Ariel detergent
powder. The enzyme retained more than 50% activity with most of the detergents tested
even after 3 hr incubation at 37°C after the supplementation of CaCl 2 and glycine (Table
5.2).
137
Our enzyme was found to be stable in commercial detergents. As
our protease showed good stability and compatibility in presence of ariel detergent
powder, a detailed study was conducted with Ariel in the presence of 10mM CaCl 2 and
1M glycine for different periods (0.5 to 3 h) at 37°C. The results are shown in Fig. 5.12.
The enzyme retained about 60% activity after 1.5 hr in the presence of Ariel at 37°C and
was almost inactivated after 3 hr in the absence of stabilizer. However, the addition of
CaCl2 (10 mM) and glycine (1M), individually and in combination, was very effective in
improving the stability where it retained 50% activity even after 3 h.
As the protease produced by our isolate GAS-4 was stable over a
pH range of 8-10 values and also showed good compatibility with various commercial
detergents tested, it can be used as an additive in detergents. To check the contribution of
the enzyme in improving the washing performance of the detergent, supplementation of
the enzyme preparation with detergent i.e. Ariel significantly improved the removal of
blood stains. The results are shown in Fig. 5.13.
Dehairing of animal skin with protease
The dehairing capacities of the crude and purified enzyme were
studied. Freshly slaughtered paired goat skin pieces were treated with crude enzyme and
purified enzyme. The skins were kept aside for 6h. A control was also kept using water
instead of enzyme solution. After 6h contact time, the ease of dehairing was determined
by removing the hairs with a blunt scalpel. It was observed that the purified enzyme
could dehair with greater ease than the crude enzyme. The results are shown in Fig.5.14.
138
Fig. 5.12 Compatibility of alkaline protease with Ariel in the presence of CaCl2 and
Glycine
Enzyme+Detergent+Glycine
Enzyme+ Detergent
Enzyme+Detergent+Calcium+Glycine
Control
Enzyme+Detergent+Calcium.
Fig. 5.13. Washing performance of alkaline protease from Streptomyces indicus
GAS-4 in the presence of detergent (Ariel). (A, Cloth stained with blood; B, blood
stained cloth washed with detergent only; and C, blood stained cloth washed with
detergent and enzyme.)
A
B
C
139
The isolate GAS-4 producing an alkaline phosphate was identified
as Streptomyces indicus var GAS-4. The purified enzyme was studied in pH range 8-10
with maximum activity at pH
9. The enzyme was completely inhibited by
Phenylmethylsulphonyl fluoride (PMSF) indicate that it is a serine alkaline protease .ca 2+
and Na+ metal ions increased and stabilized the protease activity. Stability and activity
in the presence of these metal ions is industrially very useful where high concentration of
these metal ions are present. The protease strain GAS-4 showed excellent stability and
compatibility in the presence of locally available detergents. The blood strain removing
and dehairing experiments showed that the enzyme improved the washing capability of
the detergent.
Fig. 5.14 Comparision of proteolytic activities on dehairing of goat skin before (A)
and after (B) addition of the enzyme
A
B
140
REFERENCES
Deyl,
Z.(1979).Electrophoresis.A
survey
of
techniques
and
applications.Part
A:Techniques. Elesevier,Amsterdam.
Dhandapani, R. and R. Vijayaragavan. 1994. Production of thermophilic extracellular alkaline protease by Bacillus stearothermophilus. Ap-4. World J. Microbiol.
Biotechnol. 10: 33-35
Fujiwara, N., and Yamamoto, K. 1987. Decomposition of gelatin layers on X-ray film by
the alkaline protease of Bacillus sp.B21. J. Ferment. Technol. 65: 531–534.
Gold, A. M. and Fahrney,D.(1964). Sulfonyl Fluorides as Inhibitors of Esterases. II.
Formation and Reactions of Phenylmethanesulfonyl α-Chymotrypsin*) .Biochem. 3: 783.
Laemmli, U.K (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4 .Nature. 227: 680.
Lowry O H, Rosebrough N J, Farr A L & Randall R J.(1951). Protein measurement with
the Folin phenol reagent. J. Biol. Chem. 193:265.
Paliwal, N., S.D. Singh and S.K. Garg,( 1994). Cation: induced thermal stability of an
alkaline protease from a Bacillus sp. Biores Technol. 50: 209-211.
Patricia A.M., Snoek-van Beurden and Johannes W. Von den Hoff .(2005).Zymographic
techniques for the analysis of matrix metalloproteinases and their inhibitors.
Biotechniques. 38:73-83.
Rabbani Syed., Roja Rani., Sabeena., Tariq Ahmed Masoodi., Gowher Shafi., Khalid
Alharbi (2012). Boinformation- A Discovery at the interface of physical and biological
science. 8:232.
Rahman RNZA, Razak CN, Ampon K, Basri M, Yunus WMZW, Salleh AB .(1994)
Purification and characterization of a heat stable alkaline protease from Bacillus
stearothermophilus F1. Appl Microbiol Biotechnol. 40:822-827
Sigma DS, Mooser G.(1975).Chemical studies of enzyme active
sites. Annu Rev
Biochem.44:889-931.
Tsuchiya, K., Y. Nakamura, H. Sakashita and T. Kimura.(1992). Purification and
character-rization of a thermostable alkaline protease from alkaliphilic Thermoactinomyces
sp. HS682. Biosci. Biotechnol. Biochem.56: 246-250
141
Vivek Kumar Morya., Sangeeta Yadav., Eun-Ki-Kim., Dinesh Yadav .(2011). In Silico
Characterization of Alkaline Proteases from Different Species of Aspergillus . Appl
Biochem Biotechnol.166: 243-257
Yamagata, Y. and Ichishima, E . (1989) .A new alkaline proteinase with pI 2.8 from
alkalophilicBacillus sp., Curr. Microbiol. 19: 259.-264.
142
CHAPTER-VI
OPTIMIZATION STUDIES BY SmF
OPTIMIZATION OF BIOPARAMETERS FOR ALKALINE PROTEASE
PRODUCTION USING STREPTOMYCES INDICUS GAS-4 BY SMF
Bioprocessing in its many forms involves a multitude of complex
enzyme-catalysed reactions within specific microorganisms and these reactions are
critically dependent on the physical and chemical conditions that exist in their immediate
environment. Successful bioprocessing will occur only when all the essential factors are
brought together. To understand and control a fermentation process, it is necessary to
know how the organism responds to a set of measurable environmental conditions.
Medium development (formulation) is an essential prerequisite to
get higher productivity using any microbial strain. The strain production potential not
only depends on the genetic nature, but also on nutrients supply and cultural conditions.
So it is important to know the suitable nutrients and cultural conditions required to
achieve higher productivity (Singh et al. 1982). Several authors have reported the
production of alkaline protease from alkalophilic and neutrophilic Bacillus sp. in
complex media (Gessesse and Gashe, 1997; Margesin and Schinner, 1994; Takii et al,
1990; Takami et al. 1989). However, there are almost no reports on production of
alkaline protease by using Streptomyces sp. in synthetic medium.
In the preliminary stage, it was planned to develop/formulate a
suitable production medium for alkaline protease production. It was proposed to study
the of effect of various cultural factors on alkaline protease production followed by
optimization of nutritional parameters by Plackett-Buman design (PBD).
143
The ideal production medium must meet as many criteria as
possible as referred by Stanbury et al. (1997). Therefore, it is proposed to study the
following major nutritional and cultural parameters for the production of alkaline
proteases.
1. Effect of various incubation temperatures
2. Effect of various initial pH values
3. Effect of various incubation periods
4. Effect of level of inoculum
5. Effect of age of inoculum
6. Effect of various vitamins, amino acids, trace elements, metabolic inhibitors,
surfactants and antibiotics
7. Optimization of various nutrient sources by Plackett-Burman design .
Experimental:
Chemicals
All chemicals used in this study were of analytical grade.
Microorganism
A mutant strain of Streptomyces indicus GAS-4, was used in the present study.
This culture was maintained on starch casein agar slants at 4°C and subcultured every 4
weeks.
144
Inoculum preparation
Five ml of sterile water was added to 24 h old slant of S.indicus
GAS-4. The cells were scrapped from the slant into sterile water and the resultant cell
suspension was transferred at 10% level, aseptically into 250ml Erlenmeyer flasks
containing 45 ml of sterile inoculum medium. The composition of the inoculum medium
is (g/L): Soluble starch, 10; casein, 3; KNO3, 2; NaCl, 2; K2HPO4, 2; MgSO4 7H2O, 0.05;
CaCO3 0.02; FeSO4.7H2O, 0.01. The flasks were kept on a rotary shaker (180 rpm) at
28°C for 48 h. The contents of the flasks were centrifuged at 3000 rpm for 10 min and
the supernatant was decanted. The cell pellets were washed thoroughly with sterile saline
followed by sterile distilled water. Finally the cell mass was suspended in sterile saline
and used as inoculum for subsequent experiments.
Shake flask fermentations
Five ml of cell suspension (equivalent to 0.03 g dry cell weight)
was inoculated into 45 ml of basal production medium [(g/L): glucose, 10; soya bean
meal, 20; CaCl2, 0.4 and MgCl2, 2) contained in 250 ml EM flask and incubated at 28°C
on incubator shaker for 96 h (180 rpm). At the end of fermentation, 5 ml broth was
centrifuged at 3000 rpm for 10 minutes and assayed for en2yme activity. The
experiments were carried out in triplicate throughout the studies and the average values
are-presented.
Optimization of cultural parameters
Effect of various temperatures on alkaline protease production.
Incubation temperature was shown to affect protease production.
To study the effect of incubation temperature for maximum protease production, the
flasks with the production medium were inoculated and incubated at various
temperatures such as 25°C, 26°C, 28°C, 30°C, 32°C, 35°C and 37°C for 96 h. The
general procedure mentioned earlier was followed for protease production and assay. The
results are presented in Fig. 6.1. The optimal temperature 37°C) obtained was used for
further studies.
Effect of various initial pH values on alkaline protease production.
The initial pH of the medium was shown to affect protease
production. The effect of initial pH on alkaline protease production was studied. The
production medium was adjusted at various levels of pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0,
10.0, and 11.0). General procedure mentioned earlier was followed for protease
145
production and samples were assayed as described earlier. The results obtained are given
in Fig. 6.2. The optimal pH (9.0) obtained was used in all subsequent experiments.
Effect of various incubation periods on alkaline protease production.
The duration of incubation plays an important role in the
production of a microbial metabolite. To study the optimal incubation period for
maximum protease production, the flasks with the production medium (pH 9.0) were
inoculated and incubated at 37°C. Samples were withdrawn periodically at every 12 h up
to 96 h and assayed for protease activity as described earlier. The results are presented in
Fig. 6.3. The optimal incubation period (96 h) thus obtained was used in the subsequent
experiments.
Effect of level of inoculum on alkaline protease production
The effect of level of inoculum was studied for optimal alkaline
protease production. Experiments were carried out using 2.0%, 3.0%, 5.0% and 7.0%
inoculum volume each containing 6.24xl06 spores/ml. The flasks with the production
medium (pH 9.0) were inoculated as above and incubated at 28°C for 96 h. The general
procedure mentioned earlier was followed for protease production and assay. The results
are presented in Fig. 6.4.
Effect of age of inoculum
Fermentation experiments were carried out using cultures of
different age (24, 36, 48, 60, and 72 h old culture as inoculum). The flasks with the
production medium were inoculated using cultures of different age at 10% level and
incubated at 28°C for 96 h. The general procedure mentioned earlier was followed for
protease production and assay. The results are presented in Fig. 6.5.
146
Effect of amino acids on protease production
Effect of amino acids on protease production
A large number of amino acids are known to be effective
nutritional sources for fungi and bacteria. They were assimilated as such and were found
in both free and bound form in fungal mycelium .Their rate of assimilation from the
nutrient medium varied with the nature of microorganism and other constituents present
in the medium. However, no reports are available on their effect on growth and
metabolism of actinomycetes.
In the present work, the effect of different amino acids viz., Dalanine,
DL-alanine, L-alanine, L-arginine, L-asparagine, L-cysteine, L-cysteine HC1, glycine,
L-glutamic acid, L-glutamic acid sodium salt, L-leucine, L-Iysine, L-histidine,
L-tryptophan and L-tyrosine on protease production was studied.
The solutions of individual amino acids were prepared in distilled
water, sterilized by filtration and added aseptically to the sterile basal medium at a
concentration of 0.5%. A control was run using water. Protease activities were
determined as described earlier. The results were given in Fig. 6.7. Effect of trace
elements on protease production.
147
RESULTS AND DISCUSSIONS:
The results of the effect of various amino acids, trace elements,
metabolic inhibitors and surfactants on protease production by S.indicus GAS-4 were
given in Fig 6.1 to 6.5.
Effect of incubation temperature
To study the effect of various temperatures on the growth and
alkaline protease production, different temperature ranges (25, 26, 28, 30, 32, 35 and
37°C) were used. The fermentations and assays were carried out in triplicate as described
earlier. The results are shown in Fig. 6.1.
Fig. 6.1 Effect of initial temperature on enzyme production.
The results indicated that the organism grew over a wide range of
temperatures (250 to 370C). At 37, 45 and 450C, the protease production was 109, 148
and 204U/ml respectively. The maximum alkaline protease production (222U/ml) was
observed at 37°C at 96 h. Increase in incubation temperature to 60°C decreased the yield
to 186 U/ml. Hence the optimum incubation temperature for protease production by this
organism is 37°C.
Effect of initial pH
148
The effect of initial medium pH on protease yield was studied in
shake flask. Different initial pH values (3.0-11.0) were used to study their effect on the
protease production. The fermentations and assays were carried out in triplicate as per
the general procedure and the results are shown in Fig. 6.2. The organism has produced
reasonable amounts of protease in acidic and highly alkaline conditions with the highest
yield (226.2 U/ml) at pH 10.0 where it had maximum growth. So the optimum pH for
protease production was found to be 9.0. It is clear from Fig. 6.2 that the organism grew
well at a wide range of pH 3.0-11.0.
Fig. 6.2 Effect of initial pH on enzyme production
Effect of incubation time
To study the optimal incubation time for maximum protease
production, the fermentation samples were withdrawn periodically and assayed. The
results are shown in Fig. 6.3. The results indicate that the organism grew well in the
medium and maximum protease production (224 U/ml) was achieved at 96 h. After that
the protease production decreased gradually with increased incubation periods.
149
Fig. 6.3 Effect of age of inoculum on enzyme production.
Effect of age of inoculum
Fermentation experiments were carried out using inoculua cultures
of different age (24, 36, 48, 72, 96 and 120 h). The results from Fig. 6.3 indicate that
culture of 96 h age had maximum protease producing ability (72.8 U/ml)
Effect of level of inoculum
Initial microbial load to a medium does affect the growth and in
turn metabolite production. To study the effect of inoculums level the experiments were
conducted using 2.0%, 3.0%, 5.0% and 7.0% inoculum volume. The results in Fig. 6.4
indicate that protease production was increased with increase in level of inoculums upto
10% level (230 U/ml) and further increase in inoculums level did not increase the
protease production.
150
Fig. 6.4 Effect of inoculum level on enzyme production.
Effect of Amino acids on protease production
The Fig. 6.5 indicates that many of the amino acids (at 5µg/ml
concentration) tested were shown stimulating effect on protease production. Among
them, L- cysteine had shown maximum (198%) increase in protease production.
Stimulating effect of amino acids are in the order of L-cysteine > L-tyrosine, Ltryptophan (176% increase) > L-lysine HCl, L-cysteine HCl (48% increase) > L-alanine
(25% increase) > DL-alanine (18% increase) > L-arginine, L-aspargine (16% increase).
Other amino acids had an inhibitory effect on the protease production with L-glutamic
acid having maximum inhibitory effect.
151
Fig.6.5 Effect of amino acids on protease production.
Optimization Of Process Variables Using Placket Burman Design (P.B.D) For
The Strain Gas-4.
Designing of an appropriate fermentation medium is of critical
importance as medium composition influences product concentration yield and
volumetric productivity (Akhnazarova and Kafarov, 1982; Mabrouk et al. 1999; Rao et
al. 2006). The commonly used optimization method is one at a time method (Prakasham
et al. 2006). But in one at a time method determination interactions among the different
parameters is not possible. However, it was impractical to optimize all parameters and to
establish the best possible conditions by inter-relating all parameters as this involves
numerous experiments to be carried out with all the possible combinations (Prakasham et
al. 2005a; Sreenivas Rao et al. 2004). Experimental designs based on statistical tools are
known to provide economic and practical solutions in such cases (Prakasham et al.
2005a; Ravichandra et al. 2007). Statistical experimental designs have been used for
many decades and can be adopted on to several steps of an optimization strategy, such as
for screening experiments or searching for the optimal conditions of a targeted response
(Kim et al. 2005; Nawani and Kapanis, 2005; Senthilkumar et al. 2005).
152
Statistical methods show better performance than one at a time method even
though it has some limitations
Central composite design (CCD)
Central composite design (CCD) is one of the response surface
methodologies used after identifying the components affecting the product yield
significantly. A CCD was adopted to optimize the major variables, which were selected
through Plackett Burman design or conventional methods (Chakravarthi and Sahai,
2002).
Optimization of process parameters for protease production in
submerged fermentation was carried out a statistical approach.Coded factor levels
provide a uniform framework to investigate the effects of a factor in any experimental
context, while the actual factor level depends on a particular factor to be studied;
factorial design is usually given in the form of coded factor levels. One can assign each
actual factor level to the corresponding coded factor level of a factorial design when
using it. The analysis and the model-fitting for a factorial design can be performed based
on either the coded factor level analysis or the actual factor levels. However, in almost
all situations, the coded factor level analysis is preferable, because in a coded factor level
analysis, the model coefficients are dimensionless and thus directly comparable, which
make it very effective to determine the relative size of factor effects.
153
EXPERIMENTAL DESIGN AND OPTIMIZATION OF PROTEASE
PRODUCTION BY PLACKET BURMAN DESIGN( P.B.D)
Inoculum preparation:
As described earlier in the Chapter-III.
Estimation of alkaline protease activity:
Alkaline protease activity was assayed by following the method of Lowry et al. (1951)
which was described earlier in Chapter-III.
After doing some basic experimental work regarding the output profile vs input variables
and their levels with conventional experimentation, it was thought of going for
optimization of the process by applying DOE ( Design of Experiments) statistical
techniques. Statistical techniques will offer a clear understanding of the process variables
and their quantitative effect on output.
The process involved 9 variables. They are: pH, Inoculum, Temperature, rpm, Age of
inoculum, Incubation period, Glucose, Yeast extract and Tryptone.
Sno
Nam e of variable
Lower level
Higher level
1
pH
6
7
2
Inoculum
3
5
3
Temperature
280C
320C
4
RPM
180
200
5
Age of inoculum
36h
48 h
6
Incubation period
48 h
96 h
7
Glucose
1%
2%
8
Yeast extract
0.5%
1%
9
Tryptone
0.5%
1%
154
To optimize the process with 9 variables by conventional methods
, it takes a long time and consumes large resources as the number of experiments will run
into several hundred and it will not be economic. Hence by using statistical methods for
DOE( designing of experiment),the minimum the minimum no of experiments which
enable the statistical analysis to find out the most effective variables that govern the
process can be determined. As per Paretos Law, when there are many variables, it is the
vital few significant factors that affect the process rather than many trivial factors. This
process is called screening. Therefore Placket Burmann a statistical technique has been
used. The advantage of this technique is that it offers a plan with minimum no of
experiments to be conducted. For 9 variables only 12 experiments need to be conducted.
For this purpose software SigmaTech has been used for statistical planning and analysis
to screen the effective process variables.
Table 6.1.Placket Burmann Design plan and observations of experiments
Inference it can be seen from the observations that when ever
temperature is lower at 28C , the enzyme is the highest.
S.N
p
Inoculu
temperat
RP
Age of
Incubat
Gluco Yeast Trypto Concentrat
o
H
m
ure
M
inoculu
ion
se
m (h)
period
extra
ne
ct
tion
unknown.
(h)
1
7
5
28
200
48
96
1.0
0.5
0.5
136.0
2
7
3
32
200
48
48
1.0
0.5
1.0
40.9
3
6
5
32
200
36
48
1.0
1.0
0.5
30.1
4
7
5
32
180
36
48
2.0
0.5
1.0
42.0
5
7
5
28
180
36
96
1.0
1.0
1.0
144.8
6
7
3
28
180
48
48
2.0
1.0
0.5
115.7
7
6
3
28
200
36
96
2.0
0.5
1.0
120.0.
8
6
3
32
180
48
96
1.0
1.0
1.0
38.3
9
6
5
28
200
48
48
2.0
1.0
1.0
106.4.
10
7
3
32
200
36
96
2.0
1.0
0.5
39.7
11
6
5
32
180
48
96
2.0
0.5
0.5
34.0
12
6
3
28
180
36
48
1.0
0.5
0.5
110.2
The above data was used for statistical analysis by ANOVA
155
Statistical analysis: ANOVA.
S.No
Variable
Coefficient
SS Ratio
1
pH
66.9167
2.3586
2
Inoculum
23.9167
0.3013
3
Temperature
-423.5833
94.5055
4
RPM
-9.75
0.0501
5
Age of inoculum
-12.75
0.0856
6
Incubation period
56.4167
1.6765
7
Glucose
-35.5833
0.6669
8
Yeast extract
-6.9167
0.0252
9
Tryptone
22.0833
0.2569
10
Dummy
5.25
0.0145
11
Dummy
10.5833
0.059
The analysis as given above is explained in table-2 with comments
Table 6.2. Statistical analysis : ANOVA
Sno
1
Variable
pH
Coefficient/
% SS
effect
(contribution)
66.92
2.36
Measures to be taken
Optimization with higher
pH if possible.
2
Inoculumn
23.92
0.30
Keep it as constant in
optimization at higher level ie
5.
3
Temperature
-423.58
94.51
Optimization with further
reduction in temperature.
4
RPM
-9.75
0.05
Keep it as constant in
optimization at lower level
ie180.
5
Age of inoculum
-12.75
0.09
Keep it as constant in
optimization at lower level
ie36 hrs.
156
6
Incubation
56.42
1.68
period
7
Glucose
Optimization with higher
levels of incubation period.
-35.58
0.67
Keep it as constant in
optimization at lower level
ie1.0.
8
Yeast extract
-6.92
0.03
Keep it as constant in
optimization at lower level ie
0.5.
9
Tryptone
22.08
0.26
Keep it as constant in
optimization at higher level ie
1.0 .
Analysis of the above results:
1) At the out set it can be said that only temperature is contributing to the output
increase very significantly since its contribution is 94.51% in terms of SS%
where as all others are of small contributing ones. Lower temperature is preferred
since its coefficient is negative.
2) However some of the variables are of positive by contributing though they are
very small in their contribution like pH, Inoculum, Incubation period and tryptone.
If One percent and above contributing variables are selected for optimization of
the process, only pH, temperature & Incubation period can be only three
parameters for optimization.
3) All other variables having negative coefficient can be kept as constant at lower
levels where as variables of positive coefficient can be kept as constant at higher
level so that some effect of these variable though small can be available for the
process.
157
Histogram:
Histogram indicates that only temperature, inoculum, and pH are
only contributing factors .thus out of 9 process variables, only three are the variables that
have significant effect on the process output and hence they only are selected for
optimization.
Pie Diagram
158
In both the pictures only temperature is one single contributing
factor. But in order to see the further scope finally 3 variables have been selected for
optimization with Factorial Design of Experiments a part of RSM.
In fact next optimization can be done only changing the temperature to a lower level.
However since the experimentation is to be done to verify to increase the out put, we can
as well try with three variables as shown below.
Levels of variables to be considered for experimentation:
Sno
Designate
Variable
Units
Lower level
Higher
level
1
X1
Temperature
C
24
28
2
X2
pH
No
7
8
3
X3
Incubation
Hrs
48
96
period
A software SigmaTech has been used for statistical planning and analysis.
Factorial Design of Experimental Plan.
Sno
Variables
X1
X2
X3
Y actual out put
Incubation
Enzyme activity
combination
Temperature pH
period
1
I
24
7
48
2
X1
28
7
48
3
X2
24
8
48
4
X1X2
28
8
48
5
X3
24
7
96
6
X1X3
28
7
96
7
X2X3
24
8
96
8
X1X2X3
28
8
96
9
Mid points
26
7.5
72
10
Mid points
26
7.5
72
11
Mid points
26
7.5
72
12
Mid points
26
7.5
72
159
As per this the experiments were conducted and the outputs are recorded in the following
table.
Actual experimental observations with DOE plan:
Sno
Variables combination
X1
X2
X3
Y actual out
put
Temperature pH
Incubation Enzyme
period
activity
1
I
24
7
48
25.4
2
X1
28
7
48
28.0
3
X2
24
8
48
18.5
4
X1X2
28
8
48
120.0
5
X3
24
7
96
16.8
6
X1X3
28
7
96
129
7
X2X3
24
8
96
20.1
8
X1X2X3
28
8
96
153.2
9
Mid points
26
7.5
72
100.0
10
Mid points
26
7.5
72
110.0
11
Mid points
26
7.5
72
104.31
12
Mid points
26
7.5
72
101.01
Statistical analysis of the above data by ANOVA
Sno
Combination of
Coefficient = b
SS%
variables
1
I
63.875
2
X1= temperature
43.675
62.72
3
X2=pH
14.075
6.51
4
X1X2
14.975
7.37
5
X3
15.900
8.31
6
X1X3
17.650
10.24
7
X2X3
-7.200
1.70
8
X1X2X3
-9.750
3.13
160
Where
Coefficient means the incremental output per unit of input variable.
SS% of each variable and interactions is expressed as a percentage of individual SS to
the total SS ( Sum of squares) .This is also called contribution of each variable on the
output.
While sign of coefficient is a direction (increase or decrease), the SS % is a relative
weightage of each factor over the output.
Graphical display:
Histogram:
Fig: 6.6 . Histogram showing the Coefficient of each Variable
161
Pie diagram:
Fig 6.7 Pie Diagram showing the SS% of each variable and interactions
Inference:
1. Temperature X1 has the highest positive coefficient and also highest SS%
contribution .however the optimum temperature determined was 280c with further
increasing temperature protease yield decreased
2. All variables have positive coefficient and therefore it calls for increase in
variables to boost up output.
3. The observations indicate that at higher temperature , increase in pH or
Incubation period will offer higher output.
4. Therefore the process model has been simulated.
The process model:
Y^= b0 +b1X1 +b2X2 +b3 X3+ b12 X1X2 +b13X1X3 + b23 X2X3 + b123 X1X2X3
By substituting the coefficients from the above table in place of bs the process model can
be described as follows.
Y^= 63.875 +43.675 X1 + 14.075 X2 + 15.9 X3 +14.975 X1X2 + 17.65 X1X3 - 7.2
X2X3 – 9.75 X1X2X3
Where,
Y^ = estimated value of output
X1= Temperature.
X2= pH
X3= Incubation period in hrs
162
Verification of model accuracy:
By substituting the above planned variables in this model, we get the Y^ ie estimated
output
Combinations X1
X2
X3
Y actual
Y^
estimated
Temperature
pH
C
Incubation
Enzyme
Enzyme
period hrs
concentration
concentration
I
24
7
48
25.4
25.4
X1
28
7
48
28.0
28.0
X2
24
8
48
18.5
18.5
X1X2
28
8
48
120.0
120.0
X3
24
7
96
16.8
16.8
X1X3
28
7
96
129
129
X2X3
24
8
96
20.1
20.1
X1X2X3
28
8
96
153.2
153.2
1. It can be seen that the estimated values match with actual observations by which
we can
infer that it is perfect model.

The highest output is 153.2 (U/ml) which is most satisfactory. Thus just 12
experiments with Plackett Burman method and 8 experiments as per Factorial
with Design of Experiments we could obtain give a maximum output of 153.2
U/ml of protease activity.
163
RESULTS AND DISCUSSION:
The promising isolate GAS-4 was used for protease production.
Initially the culture was grown in media containing glucose, yeast extract and tryptone.
Maximum yield of protease (80.1 U/ml)protease was obtained in a medium containing
Glucose 1%, Yeast extract 0.5% and Tryptone 0.5%. Later protease production was
carried out in four modified production media. Among them the highest protease (90.0
U/ml) was produced in the medium containing Glucose 1%, Yeast extract 0.5% and
Tryptone 0.5% as observed earlier.
After selecting the medium constituents and their concentrations
we employed statistical methods to optimize the process variables. Nine variables were
selected and protease production at two levels (Lower level, higher level) were selected
for protease production by Placket-Burmann design in 12 experimental runs. It was
observed that maximum yield of protease 144.8 U/ml was obtained in run no. 5.
Statistical analysis by ANOVA revealed temperature is contributing 94.5% in terms of
SS% where as all other variables are contributing less. pH, Inoculum and temperature are
the other variables whose contribution is greater than 1% and hence were selected for
further optimization.
Later a factorial design of the experimental plan was made for
these three variables (temperature, pH, incubation period) at three levels (Lower level,
midpoint and higher level). Highest protease 153.2U/ml was produced in run no. 8 with
X1, X2 and X3 being 280C, pH 9.0 and incubation period 96 h respectively. Statistical
analysis of the above data revealed that temperature has highest positive coefficient and
highest SS% contribution. The process model was simulated and the observed yields of
protease matched the estimated yields. From this it was inferred that it was a perfect
model. Finally after Strain improvement by mutation
optimization of medium
constituents and bioprocess variables the yield of alkaline protease produced by isolate
GAS-4 increased from 80.1U/ml wild strain to 153.2 U/ml . Which represents an
increase of 87.5 % this shows that the strain has the potential to be used industrially.
164
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Chakravarti, R. & Sahai, V.(2002). Optimization of compactin production in chemically
defined production medium by Penicillium citrinum using statistical methods. Process
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Gessesse, A. & Gashe, B. A. (1997). Production of alkaline protease by an alkaliphilic
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Kim,H.O.,Lim,J.M.,Joo,J.H.,Kim,S.W.,Hwang,H.J.,Choi,J.W.,Yun,J.W.(2005).
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Nawani,N.N.,Kapadins,B.P.(2005). Optimization of chitinase production using statistics
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,R.S.,Subbarao,Ch.,
Sarma,P.N.,Sreenivas
Rao,R.,Rajesham,
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R.S., Subba
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Ch., Sreenivas
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R. and Sarma,
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Prakasham, R.S., Subba Rao, Ch., Sreenivas Rao, R., Rajesham, S. and Sarma,
P.N. (2005b) Optimization of alkaline protease production by Bacillus sp. using Taguchi
methodology. Appl Biochem Biotechnol .120, 133–144.
Ravichandra, P., Sudhakar,Ch.and Annapurna Jetty .( 2007,)alkaline
protease production by submerged fermentation in stirred tank reactor using Bacillus
licheniformis NCIM-2042: Effect of aeration and agitation regimes Biochem Eng .
34:113-120.
Lowry O H, Rosebrough N J, Farr A L & Randall R J.(1951). Protein measurement with
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Rao,
R.S., Jyothi,
C.P., Prakasham,
R.S., Rao,
Ch.S., Sarma,
P.N. and Rao,
L.V. (2006) Strain improvement of Candida tropicalis for the production of xylitol:
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biochemical and physiological characteristics of wild-type and mutant strain CTOMV5. J. Microbiol.44:113–120.
Senthilkumar S.R., Ashok kumar , B.,Raj , K., Chandra , Gunasekhran , P.(2005).
Optimization
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P.N. and Venkateswar Rao, L. (2004). Xylitol production by Candida sp.: parameter
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Stanbury, P.F., Whitaker, A. and Hall, S.J. (1997). Principles of Fermentation
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166
CHAPTER-VII
SUMMARY AND CONCLUSION
SUMMARY AND CONCLUSIONS
Actinomycetes are a large group of Gram positive eubacteria
present in a wide range of terrestrial and marine environments. They are prolific
producers of antibiotics and other secondary metabolites. Actinomycetes are reported to
produce a number of enzymes among which proteases occupy a significant position.
In the present study an attempt has been made to isolate protease
producing actinomycetes from different soil and water samples. A promising isolate
GAS-4 was obtained from a soil sample collected from Sangam diary, Jagarlamudi in
Guntur district and it initially produced 80U/ml of protease. Initially protease was
produced in submerged fermentation in media reported in literature and later in modified
production media when the yield of protease increased to 92.3U/ml. The yield of
protease was increased to 119.4U/ml and 132.5U/ml after mutation by UV and HNO 2
respectively.
The yield of protease was improved by employing statistical
methods like PBD. For this 9 variables were selected and an experimental plan with
twelve runs was designed. A yield of 144.8U/ml was obtained with the following
conditions: pH 7, Inoculum 5%, temperature 280C, rpm 180, Age of inoculum 36 h,
Incubation period 96 h, Glucose 1%, Yeast extract 1% and tryptone 1%. The analysis of
the data revealed that only temperature is contributing very significantly to protease
production. pH and Incubation period are the other variables whose contribution is 2.36
and 1.68 respectively.
Subsequently a plan was designed for these three variables
viz.Temperature, pH and incubation period with high level, midpoint and low levels.
Maximum yield of protease 153.2 U/ml was observed in the eighth run with X1, X2, X3
being at temperature 280C, pH 9.0 and 96 h incubation period. The yield of protease
increased from 80Units (Parent strain) to 153.2 U/ml which represents an increase of
87.5%. Isolate GAS-4 was identified as S.indicus after evaluation of biochemical
properties and 16S rRNA sequencing. We observed some differences in biochemical
properties between the reference strain 1H32-1(T) reported by Luo et al and our isolate.
The differences were observed in utilization of Xylose, Nitrate reduction and Mannitol.
Hence we designated our isolate as S.indicus var. GAS-4.
167
The enzyme was purified from 2 ltr fermentation broth by
Ammonium sulphate precipitation, dialysis, ion exchange chromatography and gel
filtration. The molecular weight of the enzyme determined by Native PAGE was 60KDa.
The enzyme activity was assessed by Zymography method. The 3D structure of the
enzyme was determined by ROSOMAL method. The enzyme had an optimum pH 9.0
and it was completely inhibited by PMSF (Phenyl Methyl Sulphonyl Fluoride) indicating
that it was a serine alkaline protease. The Km of the enzyme was 6.4mg which is
equivalent to 1.06x10-4M and the Vmax was 0.54IU.
The purified enzyme was used in dehairing goat skin and
considerable loosening of hair was observed by applying moderate force. Washing
performance of the alkaline protease in the presence of detergent was assessed by the
removal of blood from a stained cloth. In the presence of the enzyme and detergent the
stain was almost completely removed. The compatibility of the enzyme with different
detergents was evaluated and it was found that Aerial was the best detergent. The
purified enzyme exhibited enhanced activity in the presence of Ca++ and Na+ ions. This
is advantageous when the enzyme is used industrially. In this study a serine alkaline
protease was produced by a variant of Streptomyces indicus GAS-4. The yield of enzyme
after optimization of nutritional and cultural conditions increased from 80U/ml (parent
strain) to 153.2 U/ml which represents an increasing 87.5%. The yield of alkaline
protease is better or comparable to those reported in the literature. The results obtained in
this study indicate the scope for utilization of this enzyme in industry after pilot plant
ans scale up studies are conducted.
168
APPENDIX
APPENDIX-I
All media were sterilized at 1210C for 20 minutes unless otherwise stated. Antibiotics &
B-Vitamins were sterilized by filtration.
1. Yeast Extract Malt Extract agar (YEME) medium:
Yeast Extract
: 4.0g
Malt Extract
: 10.0g
Dextrose
: 4.0g
Agar
: 20.0g
Distilled Water
: 1000ml
pH
: 7.3
2. Glucose-aspargine agar medium:
Glucose
: 10.0g
L-aspargine
: 0.5g
K2HPO4
: 0.5g
Agar
: 20.0g
Distilled Water
: 1000ml
3. Nutrient Agar medium (NAM):
Peptone
: 5.0g
Beef extract
: 3.0g
NaCl
: 5.0g
Agar
: 20.0g
Distilled Water
: 1000ml
pH
: 7.0-7.5
4. Half-strength nutrient agar medium:
Peptone
: 2.5g
Beef Extract
: 1.5g
NaCl
: 2.5g
Agar
: 10.0g
Distilled Water
: 1000ml
169
5. Gelatin Agar medium:
Peptone
: 5.0g
Beef Extract
: 3.0g
Gelatin
: 5.0g
Agar
: 20.0g
Distilled Water
: 1.0L
pH
: 7.0
6. Potato Dextrose Agar medium (PDA):
Peeled Potatoes
: 20.0-30.0g
Dextrose
: 20.0g
Agar
: 20.0g
Distilled Water
: 1000ml
pH
: 7.2-7.4
Peeled potatoes are steamed for 20mins. Glucose was dissolved in the extract and water
was then added to make up the volume.
7. Oat meal agar medium:
Oat meal
: 20.0g
Agar
: 18.0g
20g Oatmeal was cooked or steamed in 1000ml of distilled water for 20 minutes filtered
through cheese cloth. Distilled water was added to restore volume of filtrate to 1000ml.
Trace salts solution
: 1000ml
Agar
: 18.0g
pH
: 7.2
8. Trace salts solution:
FeSO4.7H2O
: 0.1g
MnCl2.5H2O
: 0.1g
ZnSO4.7H2O
: 0.1g
Distilled Water
: 1000ml
9. L.C Agar medium:
Tryptone
: 10.0g
Yeast Extract
: 5.0g
NaCl
: 5.0g
Agar
: 20.0g
Distilled Water
: 900ml
170
pH
: 7.3
After sterilization, 10% pasteurized milk was added.
10. Starch Agar medium:
Beef Extract
: 3.0g
Starch
: 2.0g
Agar
: 20.0g
Distilled Water
: 1000ml
11. Inorganic Salts- Starch Agar (ISP- medium 4):
Solution I: 1.0 gm of soluble starch was made into a paste with a small amount of cold
water and then the volume was made up to 50ml with distilled water.
Solution II:
K2HPO4
: 0.1g
MgSO4.7H2O
: 0.1g
NaCl
: 0.1g
(NH4)2SO4
: 0.2g
CaCO3
: 0.2g
Distilled Water
: 50ml
Trace salt solution
: 0.1ml
pH
: 7.0-7.4
Solutions I and II were mixed and 2.0g of agar was added.
12. Starch Casein Agar medium:
Soluble Starch
: 10.0g
Casein
: 3.0g
KNO3
: 2.0g
NaCl
: 2.0g
K2HPO4
: 2.0g
MgSO4.7H2O
: 0.05g
CaCO3
: 0.02g
FeSO4.7H2O
: 0.01g
Agar
: 20.0g
Distilled Water
: 1000ml
171
13. Skimmed milk agar medium:
Skim milk powder
: 1.0g
Glucose monohydrate
: 1.0g
Casein enzymic hydrolysate : 5.0g
Yeast extract
: 2.5g
Distilled Water
: 1000ml
pH
: 7.0
14. Glycerol- Aspargine agar medium:
L-aspargine
: 1.0g
Glycerol
: 10.0g
K2HPO4
: 1.0g
Trace salts solution
: 1.0g
Agar
: 20.0g
Distilled Water
: 1000ml
pH
: 7.0-7.4
15. Jowar Starch agar medium:
Beef Extract
: 3.0g
Tryptone
: 2.0g
Jowar Starch
: 2.0g
Agar
: 20.0g
Distilled Water
: 1000ml
pH
: 7.6
16. Peptone-yeast extract-iron agar medium:
Bacto-Peptone iron agar, dehydrated (Difco) : 36.0g
Bacto-Yeast extract (Difco)
: 1.0g
Distilled Water
: 1000ml
pH
: 7.0-7.2
Bacto-Peptone-iron agar dehydrated contained the following ingredients. When
reconstituted as 36.58 g/L of water:
Bacto-Peptone
: 15.0g
Protease Peptone (Difco)
: 5.0g
Ferric ammonium citrate
: 0.5g
Di Potassium phosphate
: 1.0g
Sodium thiosulphate
: 0.08g
172
Bacto-Agar
: 12.0g
17. Milk casein agar medium:
Peptone
: 1.0g
Agar
: 20.0g
Sterile skimmed milk (10%) : 100ml
Distilled Water
: 1000ml
pH
: 7.6
18. Bennett’s agar medium:
Yeast Extract
: 1.0g
Beef extract
: 1.0g
Nz-amine A
: 2.0g
Agar
: 15.0g
Glucose
: 10.0g
Distilled Water
: 1000ml
pH
: 7.3
19. Carbon utilization medium:
A) Sterile Carbon source
B) Pridham and Gottlieb trace salts (only 1ml of this solution was used per liter of final
medium)
CuSO4.7H2O
: 0.64g
FeSO4.7H2O
: 0.11g
MnCl2.4H2O
: 0.79g
ZnSO4.7H2O
: 0.15g
Distilled Water
: 1000ml
C) Basal mineral salts agar (analytical reagent grade chemicals were used)
(NH4)2SO4
: 2.64g
KH2PO4 (anhydrous)
: 2.38g
K2HPO4.3H2O
: 5.65g
MgSO4.7H2O
: 1.0g
Pridham and Gottlieb trace salts (B) : 1ml
Distilled Water
: 1000ml
pH
: 6.8-7.0
Agar (Difco)
: 15.0g
173
D) Complete medium
The sterile basal agar medium (C) was cooled to 600C and sterile carbon source
was aseptically added to give a concentration of approximately 1%.
174
ACRONYMS AND ABBREVIATIONS
ng
: Nanogram
µg
: Microgram
mg
: Milligram
g
: Gram
kg
: kilogram
µL
: Microliter
mL
: Milliliter
L
: Liter
g/L
: Gram per liter
nm
: Nanometer
0
: Temperature in Celsius degrees
C
M
:
Molar
rpm
:
Revolution per minute
sec
:
min
: Minutes
h
:
Hour
IU/mL
:
International Units per milliliter
U/g
:
Units per gram
Fig
:
Figure
%
:
Percentage
kDa
:
Kilodalton
SDS
:
Sodium dodecyl sulphate
BSA
: Bovine serum albumin
Tris
: Tri (hydoxymethyl) methylamine
v/v
: Volume per unit volume
w/v
: Weight per unit volume
w/w
: Weight per unit weight
BOD
: Biological Oxygen Demand
Β
: Beta
COD
: Chemical Oxygen Demand
γ
: Gamma
Second
175
SA
: Specific activity
SmF
: Submerged fermentation
OD
: Optical density
TCA
: Trichloro acetic acid
mM
: Millimolar
Vs
: Versus
sp
: Species
sub sp
: Sub Species
var
: Variety
176