Enzyme-polysaccharide interaction and its influence on enzyme activity and stability

Article abstract of DOI:10.1016/j.carbpol.2010.04.045

An attempt was made to probe and elucidate the influence of three kinds of polysaccharides including the negatively charged sodium carboxymethyl cellulose (CMC), the uncharged methyl cellulose (MC) and the positively charged sodium carboxymethyl chitosan (CMCS), on the catalytic activity and stability of the model enzyme, β-d-glucuronidase (GUS). DSC analysis showed that the denaturing temperature of GUS was increased by 7 °C in the presence of CMC, but decreased in the presence of MC or CMCS by 5 and 3 °C, respectively. This variation was in good accordance with changes in the enzyme's catalytic activity. Circular dichroism was employed to characterize the conformational changes of GUS before and after the addition of the polysaccharide. It suggested that charged polysaccharides, CMC and CMCS, were favorable for improving the pH stability and the storage stability of GUS, whereas uncharged MC did not show such a stabilizing effect. At an elevated temperature up to 70 °C, GUS in CMC solution remained 78% activity and displayed the highest thermal stability among the three enzyme-polysaccharide pairs. The electrostatic interaction between enzyme and polysaccharides was closely relevant to the enzyme conformation, activity and stability.

Full text of DOI:10.1016/j.carbpol.2010.04.045

Carbohydrate Polymers 82 (2010) 160–166
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Enzyme–polysaccharide interaction and its influence on enzyme activity
and stability
Jian Lia, Zhongyi Jianga,b, Hong Wua,∗, Yanpeng Lianga, Yufei Zhanga, Jiaxian Liua
a Key Laboratory for Green Chemical Technology of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China
b State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
An attempt was made to probe and elucidate the influence of three kinds of polysaccharides including
the negatively charged sodium carboxymethyl cellulose (CMC), the uncharged methyl cellulose (MC) and
the positively charged sodium carboxymethyl chitosan (CMCS), on the catalytic activity and stability of
the model enzyme, ␤-d-glucuronidase (GUS). DSC analysis showed that the denaturing temperature of
GUS was increased by 7 ◦C in the presence of CMC, but decreased in the presence of MC or CMCS by
5 and 3 ◦C, respectively. This variation was in good accordance with changes in the enzyme’s catalytic
activity. Circular dichroism was employed to characterize the conformational changes of GUS before and
after the addition of the polysaccharide. It suggested that charged polysaccharides, CMC and CMCS, were
favorable for improving the pH stability and the storage stability of GUS, whereas uncharged MC did not
show such a stabilizing effect. At an elevated temperature up to 70 ◦C, GUS in CMC solution remained
78% activity and displayed the highest thermal stability among the three enzyme-polysaccharide pairs.
The electrostatic interaction between enzyme and polysaccharides was closely relevant to the enzyme
conformation, activity and stability.
Received 19 December 2009
Received in revised form 15 March 2010
Accepted 20 April 2010
Available online 29 April 2010
Keywords:
Polysaccharides
␤-d-Glucuronidase
Microenvironment
Enzyme activity
Enzyme stability
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
1998; Zhang, Foegeding, & Hardin, 2004). In our recent work,
polysaccharides were chosen as the liquid core to prepare enzyme-
Recently, much attention has been given to maintenance of bio-
catalytic functions of enzyme including its activity and stability.
Enzymes in nature are present in the cytoplasm or organelle of liv-
ing cells wherein the cytosol is actually a complex biocolloid which
is rich in proteins, nucleic acids, lipids and mono/polysaccharides
(Miyoshi & Sugimoto, 2008). The interactions between the enzyme
and these ambient biomacromolecules play crucial roles in sta-
bilizing the structure and function of enzymes. For example,
␤-d-glucuronidase (GUS), a hydrolase which catalyzes the cleav-
age of terminal glucuronic acid, exists in the lysosome. Lysosome
is a membrane-bounded vesicle providing an acidic (pH 4–5)
containing microcapsule with a cell-like or organelle-like structure
(Zhang et al., 2008; Zhang et al., 2009). The compatibility and toler-
ance against harsh condition of these capsules were both improved.
Thorough understanding and elucidation of enzyme-additive inter-
action will enable the control and optimization of the enzyme
microenvironment and an enhancement in the enzyme activity as
well as stability.
It is believed that there are at least two factors pertaining to
the effect of polysaccharides on enzyme thermal stability. First,
the molecular crowding or molecular confinement effect caused by
addition of polysaccharides not only maintains the native structure
of enzyme but also inhibits enzyme aggregation (Allison et al., 2000;
Eggers & Valentine, 2001). Second, the presence of hydrophilic
polysaccharides enhances the preferential hydration of enzyme to
build up a more stable hydrate water layer around the enzyme
surface and the hydrophobic interactions among the non-polar
amino acid residues on the enzyme are simultaneously strength-
ened (Athes` & Combes, 1998; Back, Oakenfull, & Smith, 1979; Cui et
al., 2008; Sathish, Kumar, & Prakash, 2007). However, little research
on the electrostatic interaction between enzyme and polysaccha-
rides has been reported.
colloid microenvironment with
a high density of biomacro-
molecules (Bechet, Tassa, Taillandier, Comaret, & Attaix, 2005).
Enzyme molecules have nonspecific interactions with neighboring
biomacromolecules in the extracellular liquid microenvironment,
exerting a notable influence on enzyme activity (Chen, Xu, & Wang,
2007; Cui, Du, Zhang, & Chen, 2008). It has been demonstrated
that polysaccharides are very important biomacromolecule com-
ponents for enhancing the stability of free enzyme against pressure
and temperature fluctuation (Allison et al., 2000; Athes` & Combes,
Herein, three polysaccharides with similar molecular structure
but different charges were chosen to alter the microenvironment of
the model enzyme GUS. The enzyme GUS possesses an isoelectric
∗
Corresponding author. Tel.: +86 22 23500086; fax: +86 22 23500086.
E-mail address: wuhong2000@gmail.com (H. Wu).
0144-8617/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2010.04.045

J. Li et al. / Carbohydrate Polymers 82 (2010) 160–166
161
point (pI) of 4.8–5.0 (Witcher et al., 1998) and is thus nega-
tively charged under a neutral pH condition. Different electrostatic
interactions would be generated between GUS and the negatively
charged sodium carboxymethyl cellulose (CMC), uncharged methyl
cellulose (MC) and positively charged sodium carboxymethyl chi-
tosan (CMCS). Conformational changes were monitored by CD to
tentatively analyze how the polysaccharide-enzyme electrostatic
interactions affected the enzyme activity and stability.
diluted with methanol and analyzed by HPLC (HP1100, Agilent,
equipped with Agilent ZORBAX SB-C18 column) to determine the
amount of baicalein produced. A mixture of methanol/H2O/H3PO4
(60/40/0.2) was used as the mobile phase at a flow rate of 1 mL/min.
The detection wavelength was set at 274 nm. The relative enzyme
activity was calculated by comparing the activity in the presence
of polysaccharides with that without any polysaccharides.
2.4. Thermal stability
2. Experimental
GUS and the polysaccharides were dissolved separately in
30 mM Tris–HCl buffer (pH 7.0) to get a GUS solution with a con-
centration of 0.5 mg/mL, a CMC solution with a concentration of
0.2% (w/v), a MC solution with a concentration of 1.0% (w/v) and
a CMCS solution with a concentration of 1.0% (w/v). 0.4 mL of GUS
solution was mixed with 10 mL polysaccharide solution and the
mixture was incubated at different temperatures (30–70 ◦C) for 1 h
and then heated up or cooled down to 37 ◦C. The residual activity
of GUS after incubation was measured at 37 ◦C, pH 7.0. The relative
activity of GUS after incubation was determined by comparing the
activity with that without thermal incubation.
2.1. Materials
␤-Glucuronidase (GUS) (EC 3.2.1.31) from Escherichia coli
(type IX-A, lyophilized powder, 1,000,000–5,000,000 units/g pro-
tein) was purchased from Sigma Chemical Co. Methyl cellulose
(MC), sodium carboxymethyl chitosan (CMCS) and sodium car-
boxymethyl cellulose (CMC) were obtained from Tianjin Reagent
Chemicals Co. Ltd. Baicalin and baicalein standards for analysis
were obtained from the National Institute for the Control of Phar-
maceutical and Biological Products of China. Baicalin (purity >98%),
used as the substrate, was purchased from Sichuan Xieli Pharma-
ceutical Co. Ltd. All the other chemicals were of analytical reagent
grade.
2.5. pH stability
The GUS and the polysaccharides were dissolved in 30 mM
Tris–HCl buffer at different pH 4.0–8.0. The concentrations of
GUS and polysaccharides of each sample were the same as
those described in the above Section 2.4. The 0.4 mL GUS/10 mL
polysaccharide mixture was incubated at different pH conditions
(pH4.0–8.0) for 1 h. Then the residual enzyme activity was mea-
sured at 37 ◦C, pH 7.0. The relative activities of GUS after incubation
under different pH conditions were determined by comparing the
activity with that without incubation.
2.2. Characterizations
The thermodynamic property of GUS in the presence or absence
of various polysaccharides was determined using a PerkinElmer
Pyris Diamond differential scanning calorimeter (DSC). GUS and
polysaccharide were dissolved in 30 mM sodium phosphate buffer
(pH 7.0) to get a final concentration of 23 mg/mL for GUS and 0.5%
(w/v) for polysaccharide. 40 mL of the above solution was sampled,
hermetically sealed in a stainless steel pan and weighed. A sealed
empty pan was used as a reference. Three replicates of each sam-
ple suspension were scanned at a heating rate of 10 ◦C/min in the
temperature range of 20–150 ◦C.
2.6. Storage stability
The GUS/polysaccharide mixture was prepared according to the
above procedure at room temperature and pH 7.0 and stored at 4 ◦C.
The residual activity of GUS was monitored with time. The initial
GUS activity was regarded as a standard to calculate the remaining
activity.
Changes in the secondary structure of GUS in the presence or
absence of polysaccharides at room temperature were determined
by circular dichroism spectroscopy (CD) on a JASCO J715 spectropo-
larimeter (JASCO, Japan). The enzyme and the polysaccharide were
dissolved in 5 mM Tris–HCl buffer (pH 7.0). The final enzyme con-
centration was 62.5 ␮g/mL and the polysaccharide concentrations
were, respectively, 0.025% (w/v) for CMC, 0.125% (w/v) for MC and
0.125% (w/v) for CMCS. Six scans were conducted and averaged for
each sample from 200 nm to 260 nm at a 0.5 nm interval with a rate
of 50 nm/min and a response time of 8 s. The optical path was 1 cm.
3. Results and discussion
3.1. Effect of polysaccharides on the enzyme activity
The influence of the three polysaccharides on the bioconversion
reaction could be intuitively observed by the color change of the
solutions (Fig. 1). The initial substrate solutions were bright yel-
low. As the reaction proceeded, the solutions containing CMCS and
MC turned brown and yellow-brown, respectively. In contrast, little
change in color was observed for the CMC-containing solution. The
notable color change for CMCS system was due to the reduced sta-
bility of baicalin in the presence of CMCS. This unfavorable effect on
the substrate usually caused a decrease in production of baicalein.
The enzyme activities for various polysaccharide-containing
systems were presented in Table 1. The addition of negatively
charged CMC and uncharged MC led to an increase in the enzyme
activity by 12% and 5%, respectively, compared with the blank one
without any polysaccharides. A loss of 4% in enzyme activity was
found in the presence of positively charged CMCS. The influence
of polysaccharides on the enzyme activity was likely attributed to
the following two aspects. On one hand, the interaction between
polysaccharide and enzyme result in some conformational changes
of the enzyme molecules. This aspect could be further studied by
2.3. Enzyme activity
Bioconversion of bailcalin to baicalein catalyzed by GUS in the
presence or absence of polysaccharide was carried out. The enzyme
activity was determined by measuring the amounts of baicalein
produced with time. All the chemicals used were dissolved in
30 mM Tris–HCl buffer (pH7.0). A certain amount of baicalin and
Na2SO3 were added into the polysaccharide solution followed by
preheating at 37 ◦C for 15 min. Na2SO3 was used as an antioxidant.
Then, the GUS solution (37 ◦C) was added into the above mix-
ture to induce the reaction. The total volume of reacting solution
was 20 mL containing baicalin (0.4 mg/mL), GUS (0.01 mg/mL) and
Na2SO3 (0.2%, w/v). To get an equal viscosity for the three react-
ing systems, the concentrations of the three polysaccharides were
kept at 0.1% (w/v) for CMC, 0.5% (w/v) for MC and 0.5% (w/v) for
CMCS, respectively. The difference in diffusion resistance caused by
the different viscosity property of various polysaccharides could be
ignored. 100 ␮L of the reacting solution was sampled every 15 min,

162
J. Li et al. / Carbohydrate Polymers 82 (2010) 160–166
Fig. 1. Effect of polysaccharides on the color of the reaction liquid.
On the other hand, the electrostatic interaction between
polysaccharide and substrate was another reason that influenced
the enzyme activity by affecting the combination of enzyme
and substrate. Since baicalin molecules with dissociable carboxyl
groups are negatively charged under neutral pH condition, the
electrostatic repulsion between baicalin and CMC (also negatively
charged) enhanced the molecular movement of baicalin. As a result,
the enzymatic reaction rate was accelerated because the proba-
bility of collision between substrate and enzyme increased. The
electrostatic attraction between CMCS and baicalin, on the con-
trary, inhibited the molecular movement of baicalin. In addition,
the active center of GUS might be shielded by CMCS due to the
electrostatic attraction between CMCS and GUS. The above two
factors weakened the effective combination between the substrate
and enzyme, resulting in a decrease in enzyme activity.
3.2. Thermal stability
Fig. 2. Effect of polysaccharides on the CD spectra of GUS.
DSC analysis was made to test the thermal stability in terms of
denaturation temperatures (data listed in Table 1). The denatura-
tion temperature of native GUS was 103 ◦C, while in the presence of
CMC, MC and CMCS, the denaturation temperatures were, respec-
tively, increased by 7 ◦C, decreased by 5 and 3 ◦C. The denaturation
temperature indicates the destruction of secondary structure, but
the destruction of secondary structure is not the only reason for
enzyme deactivation. Hydrate layer destroying, enzyme aggre-
gation and some other unfavorable factors could also lead to a
notable decrease and even a total loss of enzyme activity even when
temperature was much lower than its denaturation temperature.
Therefore, the effect of polysaccharides on the enzyme deactivity
was further investigated by examing the activity after incubation
at temperatures range from 30 to 70 ◦C (Fig. 3). For native GUS, no
loss in activity was found after incubation at 30 ◦C for 1 h and only
3% loss after incubation at 40 ◦C. However, when the incubation
temperature increased to 50 ◦C or higher, the enzyme lost activity
remarkably, 43% loss at 50 ◦C and nearly 100% loss at ≥60 ◦C. In the
presence of the negatively charged CMC, 78% of original activity
could be preserved after incubation at 50 ◦C. A negative effect was
found for the uncharged MC and positively charged CMCS, the rela-
tive activity being even lower than native enzyme. This variation in
deactivity temperatures regarding the three polysaccharides was
in accordance with that found in the denaturation temperature.
The secondary conformation of GUS after thermal incubation
in the presence of polysaccharides was studied by CD. Comparing
Fig. 4a with Fig. 2, little change in CD spectra for GUS blank sample
was found before and after 30 ◦C incubation. In contrast, after incu-
bation at 70 ◦C, the peaks attributed to ␣-helix at 208 and 222 nm
completely disappeared and transformed into a random-coil struc-
the analysis of CD spectra. GUS molecule is composed of four sub-
units and its secondary structure is mainly in ␣-helix (Jain et al.,
1996). As shown in Fig. 2, the GUS in blank Tris–HCl buffer pre-
sented three negative peaks at 208, 216 and 222 nm in the CD
spectrum. The introduction of CMC and MC exerted little effect
on the CD spectrum of GUS, indicating that the original secondary
conformation of GUS was well retained. In contrast, the CD spec-
trum of GUS changed remarkably after mixing with CMCS. The
negative peaks at 208 and 216 nm disappeared, the mole elliptic-
ity decreased and the negative peak at 222 nm was red-shifted to
226 nm. This change in secondary conformation was likely due to
the electrostatic attraction between the negatively charged GUS
and the positively charged CMCS that destroyed the secondary
bonds in GUS molecule.
Table 1
Effect of polysaccharides on the relative activity, denaturation temperature and
storage stability of GUS.
Blank
CMC
MC
CMCS
Enzyme activitya (relative activity, %)
100
103
112
110
105
98
96
100
Denaturation temperatureb (◦C)
Storage stabilityc (remaining activity, %)
0 day
10 days
26 days
100
74
4
100
115
41
100
57
7
100
97
94
a
The enzyme activity was expressed in terms of relative activity calculated by
setting the activity in buffer without polysaccharide as 100%.
b
Denaturation temperature was determined by DSC.
Storage stability was shown and compared by setting the fresh (0 day) activity
c
as 100% for each polysaccharide.

J. Li et al. / Carbohydrate Polymers 82 (2010) 160–166
163
ture. The enzyme lost all its activity after 70 ◦C incubation although
part of its secondary structure retained. In the presence of CMC, the
CD spectra of GUS did not change after thermal incubation, only the
mole ellipticity decreased slightly (Fig. 4b). This suggested a stabi-
lizing effect of CMC on the conformation of GUS, which resulted in
an enhancement in enzyme thermal stability. Because of the heat-
sensitive sol–gel transformation of MC at 40–50 ◦C, the secondary
structure of GUS with addition of MC underwent complex changes
with increase of temperature, while the results was depicted in
Fig. 4c. The molar ellipticity began to decrease at 40 ◦C and the
␣-helix peak showed a red shift when the temperature increased
to 50 ◦C. Correspondingly, the activity of GUS decreased rapidly.
Regarding the positively charged CMCS, the original ordered struc-
ture of GUS was destroyed during thermal incubation (Fig. 4d),
resulting in a significant reduction in activity.
With the increase of temperature the hydration layer would
be gradually destroyed and thus resulting in enzyme aggrega-
tion, conformational change and deactivation. In this study, only
the negatively charged CMC was found to be able to improve the
thermal stability of GUS. This result implied that the electrostatic
Fig. 3. Effect of polysaccharides on the thermal stability of GUS.
Fig. 4. Effect of polysaccharides on the CD spectra of GUS after thermal incubation.

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J. Li et al. / Carbohydrate Polymers 82 (2010) 160–166
interaction was a very important factor for enzyme stability. For
GUS/CMCS mixture, the net charge of GUS decreased because of
the electrostatic attraction between the positively charged CMCS
and negatively charged GUS, the hydration layer on enzyme sur-
face was easier to destroy hence, weakening the thermal stability.
In regard to gelation of MC via physical cross-linking at 40–50 ◦C
(Desbrier`es, Hirrien, & Ross-Murphy, 2000; Lin, 2002), the orig-
inal hydration layer on the enzyme surface was destroyed by
the interaction with hydroxyl groups of MC through hydrogen
bonds, resulting in a decrease in thermal activity. The addition of
negatively charged CMC could inhibit conformational changes as
proved by CD study. Under neutral pH conditions, the electrostatic
repulsion between CMC and GUS prevented the polysaccharide
molecules from approaching the enzyme, thus retaining the hydra-
tion layer on the enzyme surface. As this protective effect on the
hydration layer inhibited the aggregation of enzyme molecules, the
steric confinement effect of the polysaccharides further restricted
enzyme unfolding. Coincidentally, the native microenvironment
for GUS provided by lysosome is also a negatively charged one
created by negatively charged neighboring macromolecules and
the inner lysosome membrane surface. It could be concluded that
charge property is crucial in generating a suitable microenviron-
ment mimicking their natural existence for enzymes.
3.3. pH stability
Enzymes will generally undergo reversible or irreversible
conformational changes under extreme pH conditions. The con-
formational changes of GUS under different pH conditions with or
without polysaccharides were characterized by CD (Fig. 5). The con-
formation stability in ␣-helixes and ␤-sheets of enzyme mainly
depends on the hydrogen bonds formed between the hydrogen
atoms and the oxygen atoms on neighboring amino acid residues
(Federici, Masulli, Gianni, Ilio, & Allocati, 2009). These hydrogen
bonds as well as the net charge of the enzyme surface would
be greatly influenced by pH variation (Boscolo, Leal, Salgueiro,
Ghibaudi, & Gomes, 2009). A significant change in GUS conforma-
tion under acidic or alkaline conditions was observed compared
Fig. 5. Effect of polysaccharides on the CD spectra of GUS under different pH conditions.

J. Li et al. / Carbohydrate Polymers 82 (2010) 160–166
165
Table 2
Effect of polysaccharides on the pH value of 30 mmol/L Tris–HCl buffer solution.
pH value
Tris–HCl blank
CMC
MC
4.0
6.5
4.0
7.2
5.0
6.7
5.0
7.5
6.0
6.7
6.0
7.5
7.0
7.0
7.0
7.5
8.0
7.5
8.0
7.5
CMCS
alkaline conditions, thus regulating the pH condition in microenvi-
ronment.
The enzyme activity of GUS after incubation under different
pH (4.0–8.0) conditions was measured to test the influence of
various polysaccharides on the pH stability of GUS. After mix-
ing with MC solution, a remarkable reduction in the pH stability
of GUS was found with a total lost of activity in acidic environ-
ments (pH ≤ 6.0). This could be explained by the incapability of
the uncharged MC to prevent the hydrogen bonds inside GUS from
being destroyed under the pH fluctuation. In contrast, the pres-
ence of polysaccharides with buffering function (CMC and CMCS)
remarkably improved pH stability of GUS due to the buffering effect,
especially under acidic conditions. At pH 4.0, GUS retained nearly
80% activity in presence of CMC or CMCS. It could be deduced from
the above results that electrostatic interactions played an impor-
tant role in maintaining pH stability of GUS.
Fig. 6. Effect of polysaccharides on the pH stability of GUS.
with that at neutral pH. Correspondingly, as shown in Fig. 6, a sharp
decrease was found after blank GUS was incubated under either
acidic or alkaline condition and complete deactivation appeared at
pH 4.0.
In native microenvironment of enzyme, the pH change was
regulated through the buffering function of some weak acids
and their conjugate bases, such as –COOH/–COO−, –NH3+/–NH2,
3.4. Storage stability
2−
H2PO4−/HPO4 and H2CO3/HCO3−. The buffering function of the
The storage stabilities of GUS with and without polysaccha-
rides were compared in Table 1. The activity of GUS in blank
solution decreased sharply by 26% after 10-day storage and con-
tinued to decrease thereafter. On the 26th day of storage, only 4%
of its initial activity was found. Storage stability of GUS could be
significantly improved by adding CMC. No loss but even a slight
increase in activity was found during the first 10 days, and 41%
of activity could be retained in 26 days. GUS with CMCS pre-
polysaccharides used herein was tested by measuring the change
of pH value before and after addition of polysaccharides (Table 2).
MC with no dissociable groups showed no buffering ability. Both
the anionic CMC and amphoteric CMCS showed buffering capacity,
being able to buffer the solution of pH 4.0–8.0 to around neutral
pH (6.5–7.5). The –COOH/–COO− and –NH3+/–NH2 pairs on CMC
and CMCS consume H+ under acidic conditions or release H+ under
Fig. 7. Effect of polysaccharides on the CD spectra of GUS during storage.

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J. Li et al. / Carbohydrate Polymers 82 (2010) 160–166
sented the highest storage stability and 94% of activity was retained
after 26 days. However, MC showed little effect on the storage
stability of GUS. The above results suggested that charged polysac-
charides (CMC, CMCS) had a more favorable effect on the storage
stability of the model enzyme GUS than the uncharged polysac-
charide (MC). The electrostatic interaction (attractive or repulsive)
between polysaccharide and enzyme was supposed to be a crucial
factor determining enzyme storage stability. As for the different
influence extent of CMC and CMCS on storage stability, it may
be tentatively explained as follows. The model enzyme GUS (pI
4.8) was negatively charged in the neutral storage environment
and it seemed that the attractive electrostatic interaction between
CMCS and GUS exerted a more favorable effect on storage sta-
bility than the repulsive electrostatic interaction between CMC
and GUS.
Acknowledgements
The authors thank the financial support from the National
High-Tech Research and Development Plan (No. 2007AA10Z305),
the Natural Science Foundation of Tianjin (No. 09JCYBJC06700),
the National High-Tech Research and Development Plan (No.
2007AA10Z305), the National Science Foundation of China (No.
20976127), the program for Changjiang Scholars and Innovative
Research Team in University (PCSIRT) and the State Key Laboratory
of Fine Chemicals, Dalian University of Technology (No. KF0605).
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4. Conclusions
The presence of polysaccharides significantly influenced the
enzyme activity and stability by the electrostatic interaction
between polysaccharide and enzyme. Since the enzyme GUS was
negatively charged at neutral pH, the positively charged CMCS
reduced catalytic activity of GUS, while the negatively charged CMC
and uncharged MC enhanced catalytic activity of GUS. The electro-
static repulsion between CMC and GUS reinforced the hydration
layer on the enzyme surface, thus improving the enzyme ther-
mal stability. The buffering effect of charged polysaccharides CMC
and CMCS enhanced the pH stability and storage stability of GUS.
Considering all the influences on enzyme activity and stability in
the presence of the three variously charged polysaccharides under
study, the negatively charged CMC was the most favored one. This
result was in accordance with the natural existence microenviron-
ment of GUS.
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