Enzymatic Degradation of Lichenan Layers Adsorbed onto Regenerated Cellulose Surfaces

Enzymatic Degradation of Lichenan Layers Adsorbed onto Regenerated Cellulose
Surfaces
Travis DePriest1, Xiao Zhang2 and Alan R. Esker2
1
Department of Physics and 2Department of Chemistry, Virginia Tech
Blacksburg, Virginia, 24061, United States
leeone@vt.edu
Introduction
Generally, secondary plant cell walls are composed of a network of cellulose, hemicelluloses, lignin,
structural proteins, and a few lesser substances such as
pectins and enzymes.1 Hemicelluloses are defined as plant
polysaccharides in the cell wall with β-(1-4)-linked backbones in an equatorial conformation. 2 Hemicelluloses are
grouped into xyloglucans, xylans, mannans, glucomannans, and β-(1→3,1→4)-glucans or mixed linkage glucans
(MLGs).3 Grasses, cereals, and lichens are the main
sources of MLGs which differ in structure and abundance
among sources.
Lichenans are MLGs with a linear chain isolated
from varying species of lichens.3 Over 20,000 species of
lichens are present over a wide range of habitats throughout the world.4 The most abundant source of lichenan is
Cetraria islandica, where lichenan is 42% of the dry mass.3
As such, lichenan could be a successful alternative bioethanol feedstock in the absence of commercialized and centralized lignocellulosic technologies.4
Lichenan is made up of cellulose-like oligosaccharides of D-(1→4) linked glucose monomers that range
from three to nine repeat units, separated by D-(1→3)
linkages.5 The D-(1→3) linkages make the molecules flexible and soluble, while the D-(1→4) linkages are responsible for insolubility in cold water.3 Lichenan is composed of
86.35 % trisaccharides and 2.71% tetrasaccharides, making
the trisaccharide:tetrasaccharide ratio 31.9:1, much higher
than cereal MLGs.5 Pentasaccharides account for 6.82 %
of the mass, while the remaining 4.13 % of lichenan is
attributed to longer segments with degrees of polymerization from 6 to 9.5 Only considering two states, trisaccharides and tetrasaccharides, simplifies the theoretical treatments of lichenan. For these idealized chains the two oligosaccharides are randomly dispersed within the chain, a
feature that might differ in longer segments.6
The gelation mechanism and solution conformation of lichenan are only partially understood. One theory proposes lichenan chains form paracrystalline or sheetlike structures associated through weak intermolecular
bonding when regions of three or more consecutive trisaccharides occur along the chain.7Intermolecular hydrogen
bonds arise between glucose monomers at C6 positions.
Studies of 6% by mass gels in water in the literature supported claims of these regions, or junction zones and concluded an increase in the trisaccharide:tetrasaccharide ratio
increased the density of potential interaction points.5 The
greater the trisaccharide content of the MLG, the more
elastic the gels became. The rheological characteristics of
MLG solutions and gels are dependent upon the distribution of cellulosic oligomers, linkage patterns, molar mass,
temperature, and concentration.8 Fine structure that affects
solution properties are important in drug delivery systems
such as nanocapsules, microcapsules, hydrogels, matrices,
scaffolds, and gels. Variations in fine structure of lichenan
based biopolymer blends9 could allow controlled and sustained drug release of drugs with or without enzyme incorporation for unique degradation-delivery processes.
Lichenase
or
1,3-1,4-β-D-glucanase
(E.C.3.2.1.73) is an enzyme which specifically cleaves β1,4 glycosidic bonds adjacent to D-(1→3) linkages.10 Lichenan subjected to lichenase yields cellulose-like oligosaccharides of 2 to 9 glucose units. Menon et al. found
that subjecting lichenan to lichenase and then to βglucosidase results in complete hydrolysis to glucose.
These hydrolysates can be fermented using thermotolerant
yeast for complete conversion to bioethanol. 4 Thus, lichenan can be used as a decentralized lignocellulosic feedstock for bioethanol production in developing countries
where farming is widely distributed.4 Understanding how
the composition of lichenan, cellulose and other components of the cell wall affect enzyme accessibility is pivotal
in this application, as well as determining optimal conditions for lichenase catalyzed hydrolysis of lichenan.
Previous work by the authors on lichenan using a
quartz crystal microbalance with dissipation monitoring
(QCM-D) addressed lichenan adsorption onto regenerated
cellulose (RC) surfaces and competitive adsorption of lichenan onto RC surfaces coated with hemicelluloses such
as xyloglucan, arabinoxylan, and glucuronoarabinoxylan.
These two studies provided quantitative information about
how lichenan interacts with cellulose and hemicelluloses
common in plant cell walls of Cetraria islandica.
This work addresses the adsorption of lichenan
solutions onto RC surfaces and the subsequent degradation
of these lichenan layers with the enzyme lichenase. Interactions were studied by varying activity and pH of the enzyme, while keeping the surface, temperature and ionic
strength constant.
Experimental
Materials
Lichenan from Cetraria islandica (lot 70901) and
lichenase from Bacillus subtilis (lot 60101a) were purchased from Megazyme. Lichenan had a molar mass of 90
kg·mol-1 and lichenase had a mass of 26.750 kg·mol-1.6
Sample solutions of 0.05% lichenan by mass were prepared in sodium acetate buffers (20 mM, pH 5.5). Sample
solutions of lichenase were prepared in sodium phosphate
buffers (20 mM, pH = 5.5, 6.5 and 7.5). Buffer component
were purchased from Aldrich and dissolved in ultrapure
water (Millipore Gradient A-10, 18.5 ·cm, < 5 ppb
organic impurities).
Preparation of Regenerated Cellulose Surfaces
Regenerated cellulose surfaces were formed on
cleaned sensors (AT-cut quartz crystals covered with 5 nm
of chromium and 100 nm of gold, Q-Sense AB). The sensors were cleaned with a UV/ozone ProCleaner (Bioforce
Nanoscience,Inc.) for 20 min followed immersion into a
heated solution of 10 mL ammonium hydroxide (conc.), 10
mL hydrogen peroxide (30% v/v), and 50 mL of ultrapure
water. The regenerated cellulose films were spincoated
from a solution of trimethylsilyl cellulose (TMSC) in toluene at 2000 rpm for 60 s. Exposure of the TMSC surface
to the vapor of an aqueous solution of hydrochloric acid
(10 wt%) for 5 min yielded a smooth surface. 11
Quartz Crystal Microbalance with Dissipation Monitoring
(QCM-D)
Four sensors coated with regenerated cellulose
were placed into the QCM-D flow cells. Adsorption profiles included scaled frequency changes (Δf/n) and a dissipation changes (ΔD) as a function of time from the intrinsic frequency (4.95 MHz for gold-coated quartz crystals)
and several odd overtones (n = 3 - 13). First, water was
introduced into the flow cells at a rate of 0.200 mL·min-1 at
50 °C (the same rate and temperature was used for all experiments) for several hours until a stable baseline was
obtained. Lichenan solution was then introduced into the
flow cells and Δf/n and ΔD were recorded as a function of
time. The adsorption time for lichenan solution was ~ 1 h
until plateaus in Δf/n and ΔD profiles were obtained. Water was reintroduced into the flow cells for an additional
ten minutes to remove the loosely bound molecules and
determine the amount of irreversibly adsorbed material.
Next, phosphate buffer was introduced into the flow cells
for ten minutes in preparation for enzyme degradation.
Once a stable phosphate buffer baseline was obtained, enzyme solution was introduced for one hour. Finally, phosphate buffer was reintroduced into the enzyme for the removal of reversibly bound material. Experiments were run
in triplicate and reported values indicate the average ± one
standard deviation.
Voigt-based Viscoelastic Modeling
For rigid surface layers (D small), the adsorbed
surface concentration () is directly proportional to f/n
through the Sauerbrey equation.12For soft films (D large),
the Sauerbrey equation is invalid. As this was the case for
adsorbed lichenen layers, a Voigt-based viscoelastic model
was utilized in order to deduce , elasticity, viscosity, and
the density of the absorbed layer.13 The modeling assumed
the regenerated cellulose layer was an extension of the
purely elastic quartz crystal and the surrounding solution
was a purely viscous, semi – infinite Newtonian fluid.
Atomic Force Microscopy (AFM)
Lichenan layers obtained before and after enzyme
degradation were dried in a vacuum oven at 50 °C overnight and then imaged with an MFP-3D-Bio atomic force
microscope (MFP-3D-BIO, Asylum Research) in tapping
mode. AFM images were collected under ambient conditions using a silicon tip (OMCL-AC 160TS, Olympus
Corp.). The reported roughnesses are root-mean-square
(RMS) values determined from a 2 μm × 2 μm scan area.
Results and Discussion
Adsorption of Lichenan onto RC surface
Irreversible adsorption of lichenan onto RC was
observed for adsorption from 0.05% by mass lichenan solutions in sodium acetate buffer (pH = 5.5) at 50 °C. The
adsorption resulted in irreversible Δf/n = -55 ± 1 Hz and
D = (6.9 ± 0.2) x 10-6. Voigt-based viscoelastic modeling
of the lichenan layer irreversibly adsorbed onto RC surfaces using overtones n = 7 - 13 was consistent with a layer
having  = 21 ± 1 mg·m-2, a density of 1050 kg·m-3, an
elastic shear modulus of (0.32 ± 0.04) x 10-5 N·m-2 and a
shear viscosity of (0.98 ± 0.03).The adsorbed lichenen
layers had deduced thicknesses of 19 ± 1 nm, lacked large
aggregates and were only marginally rougher than the RC
surface root-mean-square (rms) roughnesses of ~1.3 vs.
~1.0 nm (Figure 1).
RC
1.0 nm
Lichenan
1.3 nm
Figure 1. 2 m x 2 m AFM images of the RC surface
before and after lichenan adsorption. Numbers on the images represent the rms roughnesses.
Figure 2 shows f/n and D as a function of time
for a lichenan film adsorbed onto RC at pH = 6.5 upon
exposure to enzyme at different concentrations. The initial
rate of hydrolysis increased with concentration until a
limiting value was obtained around 5 U·mL-1. For
solutions with concentrations of 0.5 U·mL-1 and higher,
f/n and D were comparable after ~ 1 h. The increase in
f/n (≈ 40 Hz), was ≈ 15 Hz less than the amount of
lichenan that initially adsorbed onto the surface. This
observation could mean that hydrolysis was incomplete,
some enzyme remained adsorbed on the cellulose, or both.
Figure 3 shows a control experiment in which lichenase
was directly adsorbed onto a RC suface from a 10 U·mL-1
solution. As seen in Figure 3, some enzyme irreversibly
adsorbs onto the RC film, f/n ≈ - 5 Hz after flushing the
system with buffer. However, the amount is too small to
account for all of the ”missing” f/n in Figure 2. As such,
it appears the hydrolysis of lichenan by lichenase is
incomplete.
Figure 4. QCM-D degradation profiles for the 5th overtone for lichenan layers exposed to 5 U·mL-1 lichenase
solutions at different pH.
Conclusions
th
Figure 2. QCM-D degradation profiles from the 5 overtone for lichenan layers exposed to lichenase solutions at
pH = 6.5.
For almost all lichenase concentrations, nearly
complete hydrolysis of the adsorbed lichenan films resulted after about 1 h. Increasing lichenase concentration led
to an increase in the initial rate of hydrolysis of lichenan
films adsorbed onto cellulose until the concentrations exceeded 5 U·mL-1. Comparable degradation occurred for the
entire pH range (5.5 to 7.5) studied.
Acknowledgements
Figure 3. QCM-D adsorption profiles from the 5th overtone for lichenase adsorbed onto RC surfaces from 10
U·mL-1 solutions at pH = 6.5.
In general, enzyme activity is a strong function of
pH and temperature. While the effect of temperature was
not considered in this study, the effect of pH was. Figure 4
shows f/n and D as a function of time for a lichenan
film adsorbed onto RC upon exposure to enzyme at
different pH for lichenase concentration of 1 U·mL-1. As
seen in Figure 4, the variation in enzyme activity over the
pH range of 5.5 to 7.5 is small to insignificant. The optimal pH for this enzyme is 6.5 with an optimal temperature
of 60 °C.14 Another important factor influencing the activity of lichenase is the presence of calcium ions. The enzyme possesses a calcium binding site remote from the
active site. Other studies have shown that calcium ions are
important for the thermal and pH stability of lichenase,
though sodium does bind to the same site with a less stable
geometry.15
The authors are grateful for the financial support
from NSF under Contract DMR-0805179 and the Center
for LignoCellulose Structure and Formation, an Energy
Frontier Research Center funded by the U.S. Department
of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001090.
References
(1) Heredia, A.; Jiménez, A.; Guillén, R. Composition of
plant cell walls. Zeitschrift für Lebensmitteluntersuchung und -Forschung A 1995, 200, 24–31.
(2) Scheller, H. V.; Ulvskov, P. Hemicelluloses. Annual
Review of Plant Biology 2010, 61, 263–289.
(3) Podterob, A. Chemical composition of lichens and
their medical applications. Pharmaceutical Chemistry
Journal 2008, 42, 582–588.
(4) Menon, V.; Divate, R.; Rao, M. Bioethanol production
from renewable polymer lichenan using lichenase
from an alkalothermophilic Thermomonospora sp. and
thermotolerant yeast. Fuel Processing Technology
2011, 92, 401–406.
(5) Tosh, S. M.; Brummer, Y.; Wood, P. J.; Wang, Q.;
Weisz, J. Evaluation of structure in the formation of
gels by structurally diverse (1→3)(1→4)-β-d-glucans
from four cereal and one lichen species. Carbohydrate
Polymers 2004, 57, 249–259.
(6) Staudte, R. G.; Woodward, J. R.; Fincher, G. B.;
Stone, B. A. Water-soluble (1→3), (1→4)-β-dglucans from barley (Hordeum vulgare) endosperm.
III. Distribution of cellotriosyl and cellotetraosyl residues. Carbohydrate Polymers 1983, 3, 299–312.
(7) Tvaroska, I.; Ogawa, K.; Deslandes, Y.; Marchessault,
R. H. Crystalline conformation and structure of lichenan and barley β-glucan. Can. J. Chem. 1983, 61,
1608–1616.
(8) Lazaridou, A.; Biliaderis, C. G. Molecular aspects of
cereal β-glucan functionality: Physical properties,
technological applications and physiological effects.
Journal of Cereal Science 2007, 46, 101–118.
(9) Reijonen, M. WO/2006/125857, December 1, 2006
(10) Malet, C.; Jimenez-Barbero, J.; Bernabe, M.; Brosa,
C.; Planas, A. Stereochemical course and structure of
the products of the enzymic action of endo-1,3-1,4beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis. Biochem J 1993, 296, 753–758.
(11) Kontturi, E.; Thüne, P. C.; Niemantsverdriet, J. W.,
Cellulose model surfaces - Simplified preparation by
spin coating and characterization by X-ray photoelectron spectroscopy, infrared spectroscopy, and atomic
force microscopy. Langmuir2003, 19 (14), 5735-5741.
(12) Sauerbrey, G., The use of quartz oscillators for
weighing thin layers and for microweighing. Z.
Phys1959, 155, 206-222.
(13) Voinova, M.; Rodahl, M.; Jonson, M.; Kasemo, B.,
Viscoelastic acoustic response of layered polymer
films at fluid-solid interfaces: Continuum mechanics
approach. Physica Scripta 1999, 59, 391-396.
(14) McCleary, B. V., Lichenase from Bacillus subtilis.
In Methods in Enzymology, Willis A. Wood, S. T. K.,
Ed. Academic Press: 1988; Vol. Volume 160, pp 572575.
(15) Keitel, T.; Meldgaard, M.; Heinemann, U. Cation
binding to a Bacillus (1,3–1,4)-β-glucanase Geometry,
affinity and effect on protein stability. European
Journal of Biochemistry 1994, 222, 203–214.