Research Paper
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Acta Biochim Biophys Sin
2005,37:702-708 |
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doi:10.1111/j.1745-7270.2005.00099.x |
Purification and
Characterization of Two Endo-b-1,4-glucanases from Mollusca,
Ampullaria crossean
Yan-Hong LI1#, Rui GUO3#, Qiu-Yu YIN1, Ming DING1, Si-Liang ZHANG3, Gen-Jun XU1,2, and Fu-Kun ZHAO1,2*
1 Key
Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences,
Graduate
School of the Chinese Academy of Sciences, Chinese Academy of Sciences,
Shanghai 200031, China;
2 College
of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China;
3 Bioengineering
Institute, East China University of Science and Technology, Shanghai 200233,
China
Received: May 31,
2005
Accepted: July 16,
2005
This work was
supported by the grants from the National Natural Science Foundation of China
(No. 30370336), the Major State Basic Research Development Program of China
(No. 2003CB716006 and No. 2004CB719702) and the Creation Foundation from
Shanghai Institutes for Life Sciences
# These authors
contributed equally to this work
*Corresponding
author: Tel, 86-21-54921155; Fax, 86-21-54921011, E-mail, [email protected]
Abstract Two novel endo-b-1,4-glucanases, EG45 and EG27, were
isolated from the gastric juice of mollusca, Ampullaria crossean, by
anion exchange, hydrophobic interaction, gel filtration and a second round of
anion exchange chromatography. The purified proteins EG45 and EG27 appeared as
a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with
a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC
activity was 5.5 for EG45 and 4.4-4.8 for
EG27. The optimum temperature range for EG27 was broad, between 50 ºC and 60
ºC; for EG45 it was 50 ºC. The analysis on the stability of these two endo-b-1,4-glucanases showed that EG27 was
acceptably stable at pH 3.0-11.0 even when the incubation
time was prolonged to 24 h at 30 ºC, whereas EG45 remained relatively stable at
pH 5.0-8.0. About 85% of the activity
of EG27 could be retained upon incubation at 60 ºC for 24 h. However, less than
10% residual activity of EG45 was detected at 50 ºC. Among different kinds of
substrates, both enzymes showed a high preference for carboxymethyl cellulose.
EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of
146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat
spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-b-D-cellobioside, salicin and
starch.
Key words cellulase; endo-b-1,4-glucanase; Ampullaria crossean;
purification; substrate specificity
Today, cellulases and
hemicellulases are commonly used in many industrial applications, especially in
textile, food, brewing and wine-making as well as in the pulp and paper
industries [1,2]. With the shortage of fossil fuels, the emission of
greenhouse gases and air pollution by incomplete combustion of fossil fuel,
there has been increasing worldwide interest in the production of bioethanol
from lignocellulosic biomass [3,4]. To utilize those materials and to avoid
waste pollution, one of the most important approaches is to find applicable
cellulases and hemicellulases to hydrolyze the lignocellulosic biomass [5,6].
However, the cost of enzymes involved in the production of bioethanol might be
an obstacle [3]. Therefore, there is a perpetual interest in finding new,
stable and highly efficient cellulases and hemicellulases to meet industrial
requirements.
Many microorganisms have been
reported as good sources for the production of cellulases and hemicellulases.
The soft rot fungus Trichoderma reesei has been studied in detail due to
its ability to secrete large amounts of enzymes (up to 35 g per liter) [7]. In
recent years, owing to the unique characteristics of animal cellulases, a
number of laboratories have paid great attention to finding animal sources for
cellulases, for example, root-knot nematode Meloidogyne incognita [8],
crayfish [9], blue mussel [10], abalone [11], beetle [12], and A. crossean [13].
We have reported a multifunctional cellulase EGX from the gastric juice of A. crossean. The
cellulolytic and hemicellulolytic enzymes in mollusca, A. crossean, have
not been investigated systematically. It is worthwhile identifying new enzymes
from this species.
Cellulases are responsible for
the hydrolysis of the b-1,4-glucosidic bonds in
cellulose. Ordinarily it was accepted that effective biological hydrolysis of
cellulose to glucose requires synergistic collaboration of three different
kinds of enzymes: endo-b-1,4-glucanase (EC 3.2.1.4,
EG) which randomly cleaves internal linkages in cellulose chains;
cellobiohydrolase (EC 3.2.1.91, CBH) which specifically cleaves cellobiosyl
units from non-reducing ends of cellulose chains; and b-D-glucosidase
(EC 3.2.1.21) which cleaves glucosyl units from cellooligo-saccharides [14].
This study focused on the
purification of two novel cellulases from the gastric juice of A. crossean.
In addition, different substrates were used to analyze the substrate
specificity of the purified enzymes. Other biochemical properties of these two
enzymes were also studied.
Materials and Methods
Materials
A. crossean was purchased locally
(Xiamen, China). Carboxymethyl cellulose (CMC, medium viscosity), Sigmacell
101, xylan, starch and p-nitrophenyl-b-D-cellobioside
(pNPC) were purchased from Sigma Chemical (St. Louis, USA), Avicel pH101 was
purchased from Fluka (Buchs, Switzerland), DEAE-Sepharose CL-6B,
phenyl-Sepharose CL-4B and DEAE-Sepharose fast flow were bought from Pharmacia
AB (Uppsala, Sweden). Bio-gel P-100 was from Bio-Rad Laboratory (Hercules,
USA). The other chemicals used were of reagent grade and from Shanghai Chemical
Industries (Shanghai, China).
Enzyme activity assay
Endo-b-1,4-glucanase
activity is determined by the hydrolysis of 200 ml
1% CMC in 100 mM sodium acetate buffer containing 100 mM NaCl (pH 5.2) at 50 ºC
for 10 min. Dinitrosalicylic acid (0.5 ml) was added to stop the reaction by boiling
in a water bath for 5 min and quick cooling to room temperature. The absorbance
at 540 nm was measured (as standard assay). One unit of endo-b-1,4-glucanase activity is defined as the
amount of enzyme that yields 1 mmol glucose in 1 min at 50 ºC.
Determination of protein
concentration
Protein concentration was
determined by Lowry's method [15], using bovine serum albumin as a standard.
Purification of EG45
The purification of EG45 was
carried out at 4 ºC. The A. crossean stomachs (18 g) were cut into small
pieces, blended in 24 ml (1:1.5, w/v)
of buffer A (Table 1), and centrifuged at 12,000 rpm for 15 min. The
supernatant was adjusted to 60% saturation and stood overnight. The precipitate
was centrifuged at 12,000 rpm for 15 min and dissolved in buffer A and dialyzed
against buffer A, then applied onto a DEAE-Sepharose fast flow column (2.6 cm´16 cm) pre-equilibrated with
buffer A and eluted with buffer A. the
eluted fractions with CMC activity were collected and changed to buffer C (Table
1), then applied onto a phenyl-Sepharose CL-4B column (2.0 cm´20 cm) pre-equilibrated with
buffer C. The column was eluted with a linear gradient from 170 ml buffer C to
170 ml buffer B (Table 1), and subsequently with a linear gradient from
buffer B to buffer A, each 200 ml, then eluted with buffer A. The small peak
with CMC activity appeared in the first gradient, which corresponds to EG27.
The large peak with cellulase activity appeared in the second gradient, which
was concentrated with Ultrafilter PM10 (Millipore Corporation, Bedford, USA)
and applied to a Bio-gel P-100 column (2.7 cm´92 cm) pre-equilibrated with
buffer A. The enzyme was eluted with buffer A. The large peak with enzyme
activity was concentrated and dialyzed with buffer D (Table 1). The
enzyme was applied to a DEAE-Sepharose fast flow column (1.0 cm6.0 cm)
pre-equilibrated with buffer D and eluted by a gradient of NaCl from 0 M to 0.2
M in buffer D. EG45 was concentrated with Ultrafilter PM10.
Purification of EG27
The purification procedure for
EG27 was similar to that for EG45: anion exchange, hydrophobic interaction, gel
filtration and a second round of anion exchange chromatography, consecutively,
except that different buffers were used. Buffer E (Table 1) was used
for extraction and first chromatography (DEAE-Sepharose CL-6B), and buffer F (Table
1) was used in the next step. The enzyme was further purified by Bio-gel
P-100 column with buffer G (Table 1). Buffer H (Table 1) was used
for the final purification.
Electrophoresis and gel
diffusion assay
Sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by
Laemmli [16], and native PAGE was carried out according to routine procedures
with 10% or 15% gel at 4 ºC. Proteins were stained with Coomassie brilliant
blue R-250 or silver nitrate, and cellulase activity was measured by the gel
diffusion method. After native PAGE, the gel was transferred onto an agarose
plate containing 0.1% CMC. After being incubated at 45 ºC for 1 h, the plate
was stained with 0.1% Congo red for 30 min. The stained gel was finally washed
with 1 M NaCl to detect enzyme activity [17].
Optimum pH and temperature for
activity and stability
Aliquots of appropriate
amounts of enzymes were incubated with 200 ml
1% CMC at 50 ºC and different pH levels: 100 mM citric acid-Na2HPO4 of pH 3.0-8.0, for EG45; 100 mM citric acid buffer
of pH 3.0, 100 mM acetate buffer of pH 4.0-6.0,
and 100 mM phosphate buffer of pH 7.0-8.0, for
EG27). To establish the optimum temperature for CMC activity, the enzymes were
incubated with 1% CMC for 10 min at different temperatures in the range of 25
ºC to 70 ºC. The reaction was stopped by 0.5 ml dinitrosalicylic acid.
The pH stability was studied
by incubating the enzyme at different pH levels: 100 mM KCl-HCl, pH 1.0-2.0; 100 mM Tris-HCl, pH 9.0; 100 mM Na2CO3-NaHCO3, pH 10.0; 100 mM Na2HPO4-NaOH, pH 11.0; 100 mM
KCl-NaOH, pH 12.0-13.0; and pH 3.0-8.0 buffer, the same as the above buffer
used for EG27, at 30 ºC for 24 h. The temperature stability measurement was
performed by incubating the enzyme at different temperatures in 100 mM acetate
buffer (pH 4.8 for EG27; pH 5.2 for EG45) for 24 h. The enzyme activity was
measured as described above.
Substrate specificity
Substrate specificity of the
purified glucanases was determined by measuring the sugars reduced from CMC
(1%), Sigmacell 101 (1%), oat spelt xylan (1%), birchwood xylan (0.5%), potato
starch (0.5%), salicin (0.5%) or the p-nitrophenol released from pNPC (0.9 mM)
under standard conditions.
Results
Purification of enzymes
EG45 and EG27 were purified
from the gastric juice of mollusca, A. crossean, by ammonium sulfate fractionation,
anion exchange, hydrophobic interaction, gel filtration and a second round of
anion exchange chromatography, consecutively (Fig. 1). CMC hydrolytic
activity was assayed at each step. The final purified enzymes all appeared as
a single band on SDS-PAGE. EG45 was purified 38.7-fold with a specific
activity of 146.5 IU/mg against CMC and recovery was 1.48% (Table 2).
EG27 was purified 12.2-fold in a rather low yield. Its specific activity
against CMC was 11.0 IU/mg (Table 2). The molecular mass was 45 kDa for
EG45 and 27 kDa for EG27 (Fig. 2). Both enzymes exhibited activities on
the native PAGE gels by staining with 0.1% Congo red (data not shown).
Determination of optimum pH
and temperature
Both of the enzymes showed the
highest hydrolytic activity against CMC under acidic conditions. EG45 exhibited
maximum activity at pH 5.5, while the activity of EG27 reached its maximum at
pH 4.4-4.8. There was a comparatively
great decrease in the activity of two enzymes, respectively. At pH 4.0, EG27
retained over 70% activity while EG45 was almost inactive. Under alkaline
conditions, both EG45 and EG27 had approximately 20% activity at pH 7.0 and
were inactive at pH 8.0 (Fig. 3).
EG45 had an unusually sharp
curve in optimum temperature. At 50 ºC, the enzyme catalyzed the hydrolysis of
CMC efficiently. Above 50 ºC and below 45 ºC, the activity of the enzyme
declined rapidly. A unique characteristic of EG27 was its broad optimum
activity temperature range, between 50 ºC and 60 ºC, and about 90% of its
maximum activity was obtained at 45 ºC (Fig. 4).
Effect of pH and temperature
on enzyme stability
The pH stability was
determined by measuring the residual activity after incubation of the enzyme
at 30 ºC for 24 h in buffers with differing pH. EG27 was stable in a broad
range of pH 3.0-11.0, and more than 80% maximum
activity of EG45 was observed at pH 5.0-8.0.
In addition, EG27 showed an unusual, almost acidophilic and alkaliphilic
feature in that it retained more than 70% of its maximum activity at pH 12.0
and more than 40% at pH 1.0 after incubation at 30 ºC for 24 h. However, EG45
expressed only about 20% activity at pH 12.0 and was inactive below pH 3.0 (Fig.
5).
In a separate temperature
stability study (the remaining activity was measured with CMC as the
substrate), we found that EG27 exhibited high thermostability. About 85% of the
enzyme activity was retained when the incubation time was even prolonged to 24
h at 60 ºC. As far as EG45 was concerned, less than 10% activity was retained
at 50 ºC. Both enzymes were completely inactive after incubation at 70 ºC for
24 h (Fig. 6).
Substrate specificity
Various substrates, such as
CMC, Sigmacell 101, xylan, starch, salicin and pNPC, were used to analyze the substrate
specificity of EG45 and EG27, and results are shown in Table 3. EG45 had
much higher specific activity against CMC than EG27. The CMC hydrolytic
activity of EG45 was 146.5 IU/mg and EG27 was 11.0 IU/mg. Apart from CMC, EG45
and EG27 were found to hydrolyze oat spelt xylan (8.1 IU/mg and 0.5 IU/mg,
respectively) and showed low activity towards microcrystalline cellulose
Sigmacell 101 (0.8 IU/mg and 0.2 IU/mg, respectively). EG45 also had lower
hydrolytic activity towards xylan from birch wood; EG27 showed none. No
activity was found with pNPC, starch or salicin for either of these two
enzymes. These results suggested that both EG45 and EG27 were typical endo-b-1,4-glucanases.
Discussion
A. crossean is a kind of herbivorous mollusca.
The activities of exo-b-1,4-glucanase, endo-b-1,4-glucanase and b-glucosidase have been detected in the
gastric juice of A. crossean [18]. In a previous paper, we reported a
cellulase named EGX which belongs to the glycosyl hydrolase family 10 (GHF10)
and is a multifunctional cellulase with exo-b-1,4-glucanase,
endo-b-1,4-glucanase and endo-b-1,4-xylanase [18]. Furthermore, the
endogenous origins of the EGX gene [13] and a kind of endo-b-1,4-glucanase gene (EG65) (data
not shown) were confirmed by PCR amplification from the ovary genomic DNA of
the mollusca. Here two novel endo-b-1,4-glucanases,
EG45 and EG27, were purified from the gastric juice of the mollusca. Although
the origins of EG45 and EG27 remain to be confirmed, genes encoding cellulase
and xylanase at least existed in the mollusc itself. These results indicated
that A. crossean might have its own intact cellulase system.
Although optimal pH values for
cellulase activity vary from acidic to alkaline [19-22],
all animal cellulases reported until now have optimal activity under weak
acidic conditions [23-25]. The optimal pH for
purified EG45 and EG27 from the gastric juice of the mollusca, A. crossean,
against CMC was also 5.5 and 4.4-4.8, respectively.
This is in accordance with the acidic environmental conditions of the stomach.
In some biotechnological applications, some processes need acidic enzymes to
hydrolyze the cellulose materials. Currently, a combination of pectinases,
cellulases and hemicellulases, collectively called macerating enzymes, are
used in the extraction and clarification of fruit and vegetable juices [26,27].
a-amylase and amyloglucosidase were active
at acidic pH and used to process starch-containing fruits, especially apples harvested
early in order to prevent haze formation [27,28].
In addition to acidophilic
property, EG45 and EG27 had their own unique characteristics. EG45 had high
activity against CMC-Na. The specific activity reaches 146.5 IU/mg protein
which is much higher than some cellulases from other species [11,25,29] and is
similar to that reported for purified endoglucanase from the yellow-spotted longicorn
beetle, Psacothea hilaris [12]. Stability is an important criterion of
enzymes, especially for cellulases and hemicellulases with their enormous
potential application in biotechnology and industry [1]. The thermostable analysis
of EG27 showed that the enzyme exhibited high thermostability. About 85% of the
enzyme activity was retained when the incubation time was prolonged to 24 h at
60 ºC. Furthermore, EG27 is stable at a broad pH range (pH 3.0-11.0), and more than 70% of its activity
remained after incubation at pH 12.0 for 24 h, at 30 ºC. It is comparable to
the endoglucanase from Bacillus pumilus that 75% of the activity
remained after incubation at pH 12.0 for 20 h, at 30 ºC [30]. Hence, these two
novel enzymes would attract active research and commercial interest due to
their potential extensive applied value.
Cellulases have been
extensively studied on cellulolytic microorganisms [31] and studies of
cellulolytic enzymes at the molecular level have revealed some of the features
that contribute to their activity. However, the structure and catalytic
mechanisms of animal cellulases are poorly understood. Until now, no crystal
structure of any animal cellulase has been solved because of glycosylation or
other post-translational modifications. Without glycosylation and other
post-translational modifications, the low molecular mass of EG27 would be an
ideal system easily expressed in Escherichia coli and obtained in large
quantities for structure and function studies at the molecular level.
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