L-ASPG86 - Journal of Microbiology and Biotechnology

J. Microbiol. Biotechnol. (2016), 26(6), 1115–1123
http://dx.doi.org/10.4014/jmb.1510.10092
Review
Research Article
jmb
A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from
the Marine Bacterium Mesoflavibacter zeaxanthinifaciens
Su-Jin Lee1, Youngdeuk Lee1, Gun-Hoo Park1, Navaneethaiyer Umasuthan2, Soo-Jin Heo1, Mahanama De Zoysa3,
Won-Kyo Jung4, Dae-Won Lee5, Hanjun Kim5, Do-Hyung Kang1*, and Chulhong Oh1*
1
Jeju International Marine Science Research & Education Center, Korea Institute of Ocean Science & Technology, Jeju Special Self-Governing
Province 63349, Republic of Korea
2
Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Special Self-Governing Province
63243, Republic of Korea
3
College of Veterinary Medicine, Chungnam National University, Daejeon 34134, Republic of Korea
4
Department of Biomedical Engineering and Centre for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National
University, Busan 48513, Republic of Korea
5
Marine Ecosystem and Biological Research Center, Korea Institute of Ocean Science & Technology, Ansan 15627, Republic of Korea
Received: October 28, 2015
Revised: February 7, 2016
Accepted: March 8, 2016
First published online
March 14, 2016
*Corresponding authors
C.O.
Phone: +82-64-798-6102;
Fax: +82-64-798-6039;
E-mail: och0101@kiost.ac.kr
D.H.K.
Phone: +82-64-798-6100;
Fax: +82-64-798-6039;
E-mail: dohkang@kiost.ac.kr
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2016 by
The Korean Society for Microbiology
and Biotechnology
L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the
potential to effect leukemic cells and various other cancer cells. We identified the Lasparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open
reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino
acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective
homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned
into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells.
Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40oC, pH 9.
Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its
enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and
magnesium (3.35-fold) compared with the untreated control. The specific activity of r- LASPG86 was 687.1 units/mg under optimum conditions (37°C, pH 9, and 5 mM MnSO4).
Keywords: L-Asparaginase, Mesoflavibacter, cloning, expression, manganese, glutaminase-free
Introduction
L-Asparaginase
is a member of the homologous
amidohydrolase family that catalyzes the hydrolysis of
L-asparagine to L-aspartic acid and ammonia [7]. This
enzyme, which is distributed in diverse sources such as
animals [28], plants [42], algae [37], fungi [40], and bacteria
[38], exists in two types: I and II. Type I asparaginase is a
low-affinity enzyme found in the cytoplasm, whereas type
II asparaginase is a high-affinity enzyme [36] identified
within the periplasmic space [9].
L-Asparaginase was the first enzyme discovered with
antileukemic activity [41]. The antitumor activity of
asparaginase was first observed in guinea pig serum [21]
and elucidated the asparagine depletion by asparaginase
[7]. Asparagine is an amino acid that is required for protein
synthesis and survival. Most normal cells have asparagine
synthetase for the synthesis of asparagine from aspartate
and glutamine. However, some tumor cells are dependent
on an exogenous source of asparagine for protein synthesis,
because tumor cells lack or have very low levels of
asparagine synthetase [8]. Therefore, depletion of asparagine
by L-asparaginase leads to the selective death of tumor
cells. Currently, L-asparaginase from Escherichia coli and
Erwinia chrysanthemi is being used for the clinical treatment
of acute lymphoblastic leukemia (ALL) and non-Hodgkin’s
lymphoma [25, 31]. Many countries use asparaginase from
one of these sources as an antileukemic drug [1]. However,
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Lee et al.
the glutaminase activity of L-asparaginase causes side
effects, such as liver dysfunction, pancreatitis, leucopenia,
neurological seizures, and coagulation abnormalities [11].
Asparaginase is also used in the food industry during food
processing to reduce acrylamide [22]. Acrylamide, which is
formed from asparagine when starchy foods such as breads
and cookies are baked or fried, is known to have a toxic
effect on the nervous system and fertility [44]. Acrylamide,
a potential carcinogen, results from the reaction between
asparagine and reducing sugars found in carbohydraterich foods when they are processed above 180oC [32].
Pretreatment with L-asparaginase prevents acrylamide
formation in carbohydrate-rich foods during the frying or
baking process [16, 35].
In this study, we identified the L-asparaginase (L-ASPG86)
gene from Mesoflavibacter zeaxanthinifaciens and produced
the recombinant protein to characterize its enzymatic
properties.
Materials and Methods
Analysis of L-ASPG86 Gene
M. zeaxanthinifaciens strain S86 was isolated from the seawater
of Chuuk State in Micronesia and the genome sequenced in our
previous study [34]. The L-ASPG86 gene was identified from the
genome of M. zeaxanthinifaciens, and its homology and domain
structure were examined using the protein BLAST of the NCBI
(http://blast.ncbi.nlm.nih.gov). The molecular mass and isoelectronic
point (pI) were predicted by the Compute pI/Mw tool (http://
web.expasy.org/compute_pi/). Multiple sequence alignment of
the L-asparaginase sequences was performed using ClustalW [45].
A phylogenetic tree was constructed for L-asparaginase amino
acid sequences using the neighbor-joining (NJ) method from
MEGA 6.0 [43]. The bootstrap values were replicated 1,000 times
to obtain the confidence values for a robust analysis. Homology
search in the PDB database using BLAST and PSIBLAST was
performed to find a template structure. The 3D model of the
L-ASPG86 was predicted using the Swiss Model Server [4], using
as template E. coli (PDB code 2P2N). The 3D structural models
were visualized using PyMOL [10].
Cloning of L-ASPG86 Coding Sequence
M. zeaxanthinifaciens S86 was cultivated after growing in marine
broth (BD Biosciences, USA) at 20oC for 2 days. Genomic DNA
was extracted with the AccuPrep Genomic DNA Extraction Kit
(Bioneer, Korea). PCR was performed to amplify the full-length
ORF of L-ASPG86 from genomic DNA of M. zeaxanthinifaciens S86
using a pair of primers (forward primer 5’-gagacatATGGG
TAAAAGAGCTAGAATATTA-3’ with NdeI site, and reverse
primer 5’-gagactcgagttaATTTTGGGACATCTCCCC-3’ with XhoI
site and stop codon). The PCR conditions were as follows: initial
J. Microbiol. Biotechnol.
denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec,
annealing at 48°C for 30 sec, and extension at 72°C for 60 sec for 30
cycles; and a final extension at 72°C for 5 min. The product was
purified by the AccuPrep Gel Purification Kit. The purified PCR
product and pET-16b vector (Novagen, USA) were digested with
NdeI (Takara, Japan) and XhoI (Takara) restriction enzymes, and
ligated with T4 DNA ligase (Takara). The ligation product was
transformed into E. coli DH5α cells and purified with the
AccuPrep Nano-Plus Plasmid Mini Extraction Kit (Bioneer). The
recombinant plasmids were confirmed by sequencing.
Overexpression and Purification of r-L-ASPG86
The plasmid was transformed into E. coli BL21 (DE3) and a
positive colony was inoculated onto 4 ml of LB broth containing
ampicillin (100 µg/ml) and incubated overnight at 37°C in a
shaking incubator. The overnight culture was transferred to
250 ml of LB broth containing ampicillin and incubated until the
optical density at 600 nm reached 0.6. The r-L-ASPG86 expression was
induced by addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside
(IPTG) for 24 h at 30°C with shaking at 200 rpm. Thereafter, cells
were collected by centrifugation at 8,000 rpm for 10 min at 4°C.
The cell pellets were resuspended in binding buffer and sonicated
by a Q700 sonicator (Qsonica, USA). The lysate was centrifuged at
13,000 rpm for 30 min at 4°C, and the supernatant was collected.
The r-L-ASPG86 was purified using a His-Bind kit (Novagen,
USA) according to the manufacturer’s instruction. The purified
r-L-ASPG86 was keep at -20°C with 20% glycerol until use. Purified
r-L-ASPG86 was examined by 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) with standard
marker proteins of known molecular weight. Protein concentration
was determined using a BCA protein assay kit (Pierce, USA).
L-Asparaginase
Activity Assay for r-L-ASPG86
The L-asparaginase enzyme assay was performed by using
Nessler’s reaction [17]. Briefly, 1 ml of the total reaction mixture
consisted of 1 µg of purified enzyme solution, 50 mM Tris-HCl
(pH 8.6), and 10 mM L-asparagine and was incubated at 37°C for
10 min. The reaction was stopped by adding 50 µl of 1.5 M
trichloroacetic acid. After centrifugation at 10,000 rpm for 5 min,
0.2 ml of the supernatant was mixed with 3.8 ml of distilled water
and 0.5 ml of Nessler`s reagent. The absorbance was measured at
450 nm using a spectrophotometer. A standard curve of ammonium
sulfate was used for calculating the ammonia concentrations. One
unit of the L-asparaginase activity was defined as the amount of
enzyme that liberates 1 µmol of ammonia per minute. Glutaminase
activity was assayed by the same method using L-glutamine as
substrate.
Biochemical Characterization of Purified r-L-ASPG86
The optimum temperature for the enzyme activity was determined
by incubating the assay mixture for 10 min at temperatures ranging
from 30ºC to 80ºC, including 37ºC, in Tris-HCl buffer (pH 8.6).
Effects of pH on enzyme activity were evaluated by measuring the
L-ASPG86
from Mesoflavibacter zeaxanthinifaciens
1117
activity for 10 min at 37ºC with different buffers as follows;
citrate-phosphate (pH 3.0-7.0), sodium-phosphate (pH 6.0-8.0),
Tris-HCl (pH 8.0-9.0), and glycine-NaOH (9.0-10). To determine
the effect of metal ions and chelating agent on the activity of r-LASPG86, the enzymatic activity was assayed in the presence of
various metal ions and chelating agents (CaCl2, CuSO4, FeSO4,
KCl, MgSO4, MnSO4, NaCl, ZnSO4, and EDTA) with 1 or 5 mM
final concentrations. The thermostability of r-L-ASPG86 was
investigated by pre-incubating at 40ºC or 50ºC for 1 h (in 10 min
intervals). The specific activity was determined at optimal conditions.
Results
Sequence Characterization of L-ASPG86
An asparaginase homolog was identified from
M. zeaxanthinifaciens S86 and named L-ASPG86 (GenBank
Accession No. KP705084). The nucleotide and amino acid
sequences are shown in Fig. 1. The L-ASPG86 gene contains
a 1,035 bp open reading frame (ORF) and encodes a putative
protein of 344 amino acid (aa) residues. The predicted
molecular mass and isoelectric point (pI) are 38 kDa and
6.6, respectively. A conserved asparaginase type I domain
was found between aa 6 and 342. Active site residues (aa
14-15, 60-61, 90-92, 117, 168 aa) and homodimer interface
sites (15, 25-27, 56, 59-65, 67-68, 92-93, 96, 118-119, 168-173,
217-227, 229-230, 233, 236-237, 247, 249-251, 255, 279, 282286, 290, 309, 313, and 317 aa) were also predicted. To
determine the relationship between L-ASPG86 and other
known asparaginase members from various bacteria, a
phylogenetic tree was constructed (Fig. 2). The phylogenetic
tree consisted of two main clades: glutaminases (E.C.
3.2.1.2) and asparaginases (E.C. 3.2.1.1) with glutaminaseasparaginase (E.C. 3.2.1.38). The asparaginasee clade was
further divided into two subclades, namely asparaginases
and glutaminase-asparaginases, the first of which was again
divided into two groups: type I and type II asparaginases.
The L-ASPG86 grouped within the type I asparaginase clade
with respective homologs in E. coli and Y. pseudotuberculosis.
L-ASPG86 was most closely related, with 41.4% identity, to
E. coli type I L-asparaginase. The three-dimensional (3D)
modeling results indicate that L-ASPG86 exists as a
homotetramer. Each of the five active sites is located at the
N-terminal domains (Fig. 3).
Expression and Purification of r-L-ASPG86
IPTG-induced overexpression of the His-tagged L-ASPG86
fusion protein was verified by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (Fig. 4). A strong single
band similar in mass to the predicted molecular mass of r-
Fig. 1. Nucleotide and deduced amino acid sequences of Lasparaginase from M. zeaxanthinifaciens S86.
Large box, L-asparaginase type I domain; dot boxes, active site;
underlines, residues at the homodimer interface.
L-ASPG86
(41 kDa, including the 6-His tag) was detected,
corresponding to the purified recombinant protein. This
confirmed successful overexpression of r-L-ASPG86 along
with sufficient purity and integrity of the eluted fusion
protein.
Biochemical Characterization of Purified r-L-ASPG86
We investigated the relative activity of r-L-ASPG86 over
a range of temperatures (between 30ºC and 80ºC with 10ºC
intervals, including 37ºC). r-L-ASPG86 showed the highest
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Lee et al.
Fig. 2. Phylogenetic analysis of amino acid sequences of L-ASPG86 with other known characterized asparaginase and glutaminase
counterparts.
Phylogenetic analysis was conducted by the NJ method using MEGA 6.0, based on sequence alignment using ClustalW. The numbers indicate the
bootstrap confidence values of 1,000 replicates. The accession numbers of L-asparaginase homologs are given in parentheses. Bar, 0.2 substitutions
per amino acid position.
Fig. 3. Three-dimensional models of L-ASPG86.
(A) L-ASPG86 tetramer with identical subunits colored cyan, yellow, green, and pink. (B, C) A representation of the L-ASPG86 monomer. α-Helices
are shown in red and β-strands in yellow. The active site residues are shown in blue.
J. Microbiol. Biotechnol.
L-ASPG86
Fig. 4. SDS-PAGE analysis of r-L-ASPG86.
Protein samples were separated on 12% SDS-PAGE and stained with
Coomassie brilliant blue. M, molecular mass marker (Takara, Japan);
lane 1, total cellular extract from E. coli BL21 (DE3) before IPTGinduction; lane 2, total cellular extract after induction; lane 3, purified
from Mesoflavibacter zeaxanthinifaciens
1119
enzymatic activity at 40ºC, and similar activity was detected
at 37ºC (Fig. 5A). Its activity decreased gradually from
50ºC. In the optimum pH assay, the highest activity was
observed at pH 9, and more than 60% of the relative
activity was retained within the pH range of 7-10, as
shown in Fig. 5B. The effects of metal ions and EDTA on rL-ASPG86 activity are shown in Fig. 5C at two different
concentrations (1 and 5 mM). r-L-ASPG86 was strongly
inhibited by both the zinc ion and EDTA. However, r-LASPG86 demonstrated increased activity upon addition of
5 mM manganese (3.97-fold) and 5 mM magnesium (3.35fold) ions compared with the untreated control. The
thermal stability of r-L-ASPG86 was examined following
pre-incubation with the enzyme at 40ºC or 50ºC for 60 min
(Fig. 5D). At 40°C, the activity of r-L-ASPG86 was reduced
by approximately 50% after 20 min and then decreased
over time. However, r-L-ASPG86 was completely inactivated
after a 10-min incubation at 50ºC. Specific activity was tested
under optimal conditions (40ºC, pH 9, and 5 mM manganese);
r-L-ASPG86 had a specific activity of 687.1 units/mg.
Glutaminase activity of r-L-ASPG86 was not detected under
the tested conditions (data not shown).
r-L-ASPG86.
Fig. 5. Biochemical properties of purified r-L-ASPG86.
(A) Effects of temperature on r-L-ASPG86 activity at temperature in the range of 30-80°C. (B) Effects of pH on r-L-ASPG86 activity at pH in the
range of 3-10. (C) Effects of metal ions and chelating agents on r-L-ASPG86 activity at 1 and 5 mM, measured after incubation at 37°C for 10 min.
(D) Effects of thermostability on remaining r-L-ASPG86 activity at 40°C and 50°C for 0-60 min.
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Lee et al.
those from Erwinia carotovora [19], Staphylococcus sp. [15],
Pectobacterium carotovorum [26], Penicillium brevicompactum
[13], Pseudomonas aeruginosa [12], and Pseudomonas stutzeri
[30], have exhibited optimal activity between 30oC and
40oC. The r-L-ASPG86 produced here also demonstrated its
highest activity at 37-40oC, which is similar to those of
other bacteria (Table 1). r-L-ASPG86 showed optimal activity
at pH 9, which is similar to homologs from Rhizobium etli
[3], Staphylococcus sp. [15], Pseudomonas aeruginosa [12],
Pseudomonas stutzeri [30], and Pyrococcus furiosus [5] (Table 1).
It also retained more than 60% of its activity at a pH range
between 7 and 10 (Fig. 5B). Upon addition of metal ions
and the chelate effect, r-L-ASPG86 activity was highly
enhanced in the presence of Mn2+ and Mg2+ (Fig. 5C).
The activity of bacterial asparaginases is dramatically
increased in the presence of cations. For instance, Na+ and
K+ increase the asparaginase activity in Bacillus coagulans
[27], Pseudomonas stutzeri [30], and Erwinia carotovora [46].
Meanwhile, Mg2+ enhances the asparaginase activity in
bacterial species, such as Acinetobacter calcoaceticus [18] and
Pseudomonas stutzeri MB-405 [30]. Similarly, the activity of
r-L-ASPG86 was strongly enhanced by Mg2+; however, its
activity was inhibited by 5 mM Na+. r-L-ASPG86 was not
significantly affected by the K+ ion. Interestingly, r-L-ASPG86
Discussion
In this study, we identified an asparaginase gene (LASPG86) from the M. zeaxanthinifaciens S86 genome. The
identified gene was cloned, expressed, and characterized.
This is the first enzymatic characterization of an asparaginase
isolated from the family Flavobacteriaceae. In our study,
we found that the amino acid sequence of L-ASPG86
contains a highly conserved L-asparaginase type I domain
with a homodimer interface. Phylogenetic analysis grouped
L-ASPG86 into a type I asparaginase with E. coli and
Y. pseudotuberculosis (Fig. 2). These results suggest that
L-ASPG86 belongs to a type I asparaginase and is localized
in the cytoplasm. Furthermore, the presence of a homodimer
interface suggests that L-ASPG86 may be found as a
homodimer upon a protein BLAST search on the NCBI
website (Fig. 1). However, the 3D structure of L-ASPG86
(Fig. 3) was predicted as a homotetramer that was most
closely related to E. coli type I L-asparaginase, with 41.4%
similarity. The structure of the E. coli type I L-asparaginase
analyzed by X-ray crystallography suggested a dimer of
two intimate dimers [48]. It is also possible that L-ASPG86
is a homotetramer as an E. coli type I L-asparaginase.
Most of the reported bacterial asparaginases, such as
Table 1. Biochemical properties of L-asparaginase from microbial sources.
Types
Specific activity
(µmol/min/mg)
Optimum
temp. (ºC)
Optimum
pH
Monomer
molecular
mass (kDa)
Relative
glutaminase
activity (%)a
Oligomer
Ref.
NTb
3.1
35
8.6
33.5
NT
NT
[19]
Erwinia chrysanthemi
II
118.7
NT
NT
37.2
Less than 0.2%
Homotetramer
[2, 23]
Escherichia coli
I
173
NT
NT
39.2
Less than 0.2%
Homotetramer
(dimer of two
intimate dimers)
[48]
Escherichia coli
II
188-200
43
NT
37
4%
Homotetramer
[20, 24, 33]
Rhizobium etli
II
20
50
9
47
NDc
Homodimer
[3]
Species
Erwinia carotovora
Staphylococcus sp.
NT
113
37
9-10
37.5
58.2%
Homodimer
[15]
Pectobacterium carotovorum
NT
2,020.9
40
8.5
36.5
ND
Homotetramer
[26]
Penicillium brevicompactum
NT
574.2
37
8
94
ND
NT
[13]
Pseudomonas aeruginosa
NT
1900
37
9
160
NT
NT
[12]
Pseudomonas stutzeri
NT
732.3
37
9
33
ND
NT
[30]
Pyrococcus furiosus
NT
500
85
9
37.8
ND
Homodimer
[5]
Pyrococcus horikoshii
Yersinia pseudotuberculosis
Mesoflavibacter
zeaxanthinifaciens
a
I
NT
NT
NT
NT
NT
Homodimer
[47]
NT
62.7
60
8
36.4
5.7%
NT
[39]
I
687.1
37-40
9
38
ND
NT
This study
Relative glutaminase activity: compared against L-asparaginase activity.
b
c
NT: not tested.
ND: not detected.
J. Microbiol. Biotechnol.
L-ASPG86
showed the highest asparaginase activity in the presence of
5 mM Mn2+. In previous studies, only the activity of an
asparaginase from Streptomyces sp. [6] was reported to be
enhanced by Mn2+. In our analogous study to clone and
characterize the endo-β-1,3-glucanase from M. zeaxanthinifaciens
S86, we found that its activity was strongly enhanced by
5 mM Mn2+. This observation suggests that Mn2+ may be
an important metal that affects enzymatic functions in
M. zeaxanthinifaciens.
L-Asparaginase can be used for the treatment of ALL [14,
29]. However, cancer treatment with asparaginases has
been hampered by their potent glutaminase activity, which
can cause liver dysfunction, pancreatitis, leucopenia,
neurological seizures, and coagulation abnormalities,
leading to intracranial thrombosis or hemorrhage [11].
Therefore, glutaminase-free asparaginase is necessary for
medical treatment in humans. In this study, the assay for
determining the substrate-specific activity of r-L-ASPG86
showed that it possesses only L-asparaginase activity and
not glutaminase activity. Taken together, the biochemical
properties of r-L-ASPG86 are similar to those of previously
reported L-asparaginases that are used as therapeutic agents
in the treatment of ALL. Generally, type I L-asparaginases
display high Km values toward L-asparagine and show
catalytic activity toward L-glutamine, whereas type II
L-asparaginases exhibit affinity to L-asparagine and low-tonegative activity toward L-glutamine [49]. Interestingly,
r-L-ASPG86 showed no activity toward L-glutamine, which
is similar to previously reported type II asparaginases,
although it showed the typical molecular features of a type I
L-asparaginase.
In summary, L-ASPG86 contains type I asparaginase
domain features with no signal peptide, which are typical
molecular characteristics of type I L-asparaginases. Purified
r-L-ASPG86 showed high catalytic activity against L-asparagine,
but no glutaminase activity, which is comparable to other
commercially available asparaginases used as anticancer
agents. These results suggest that r-L-ASPG86 from
M. zeaxanthinifaciens S86 could be a potential candidate
enzyme to use in the treatment of ALL. Further studies are
necessary to confirm its use as an anticancer therapeutic
enzyme.
Acknowledgments
This research was supported by research grants (PM59122,
PE99411, PO0125C) from the Korea Institute of Ocean Science
and Technology (KIOST) and the Marine Biotechnology
Program funded by the Ministry of Oceans and Fisheries.
from Mesoflavibacter zeaxanthinifaciens
1121
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