Biotechnology Letters (2005) 27: 1369–1373  Springer 2005

DOI 10.1007/s10529-005-3683-8

Expression, purification and characterization of a recombinant

b-glucosidase from Volvariella volvacea

Xun Li1,2, Jianjun Pei1, Guogan Wu1 & Weilan Shao1,2,*

1
The Key Laboratory of Microbial Engineering, Nanjing Normal University, 210097, Nanjing, China
2
The Key Laboratory of Industrial Biotechnology under Ministry of Education, Southern Yangtze University,
214036, Wuxi, China
*Author for correspondence (Fax: +086-025-83598838; E-mail: wlshao@jsmail.com.cn)

Received 7 December 2004; Revisions requested 4 January 2005 and 18 April 2005; Revisions received 8 April 2005 and 27 June 2005;
Accepted 30 June 2005

Key words: Escherichia coli, expression, b-glucosidase, purification, Volvariella volvacea

Abstract
For the first time, a b-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has
been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on
SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The
maximum activity was at pH 6.4 and 50
C over a 5 min assay. The purified enzyme was stable from pH
5.6–8.0, had a half life of 1 h at 45
C. The b-glucosidase had a Km of 0.2 mM for p-nitrophenyl-b-D-
glucopyranoside.

Introduction and the cleavage of glucosylated flavonoids in

Cultivation of edible mushrooms on cellulosic
wastes represents a prime example of the direct
conversion of low-grade residues into a value-ad-
ded food source and also as a source of commer-
cially important metabolites (Buswell & Chang
1993). The edible straw mushroom, Volvariella
volvacea, is grown on an industrial scale in many
tropical and subtropical regions and currently
ranks fifth among the world’s most important
commercially cultivated species (Chang 1996). It
grows faster at a higher temperature than other
mesophilic filamentous fungi when using straw as
substrate. These characteristics indicate that
V. volvacea has a whole series of cellulolytic
enzymes which may have better stability than
those from other mesophiles.
b-Glucosidase catalyzes one of the major rate-
limiting reactions in the saccharification of cellu-
lose (Grohmann 1993, Philippidis et al. 1993). It
also plays a key role in the degradation of cellu-
lose and glycolipids in mammalian lysosomes,
plants (Dan et al. 2000). Cai et al. (1998) purified
and partially characterized two b-glucosidases
from V. volvacea V14. The sequence of a gene
encoding b-glucosidase from V. volvacea has
been submitted to GenBank, but the b-glucosi-
dase from V. volvacea has not been expressed in
a heterologous host and characterized in compar-
ison with the purified natural enzymes. In this
work, the b-glucosidase gene was cloned from
cDNA library of V. volvacea, and over-expressed
in E. coli, and the recombinant enzyme was puri-
fied and characterized.
Materials and methods
Microbial strains and growth media
Volvariella volvacea V1-1 (ACCC50447) from
China General Microbiological Culture Collection
Center (Beijing, China) was grown on solid med-
ium containing (g/l): 0.2% carboxymethyl cellu-
lose (CMC); 0.2% xylan; 0.2% KH2PO4; 0.2%
1370
(NH4)2SO4; 0.1% MgSO4; 2.5 mg thiamine/l;
1.5% (w/v) agar. E. coli JM109 and JM109 (DE3)
(Promega Corp., Madison, WI, USA) were used
as hosts for plasmid pTrc99A (Amersham Phar-
macia Biotech, Sunnyvale, CA, USA) and pET-
20b(+) (Novagen, Darmstadt, Germany) to clone
and over-express the b-glucosidase gene (bgl).
They were grown at 30 or 37
C in Luria–Bertani
(LB) medium containing ampicillin (100 mg/ml).
All chemicals, unless specially referenced in the
paper, were purchased from Sigma.
Cloning of the b-glucosidase gene
Total RNA was isolated from 5-day-old mycelia
as described by Sambrook & Russell (2001).
Poly(A)+ mRNA was purified from total RNA
by using a PolyATtract mRNA Isolation Systems
(Promega). The cDNA was synthesized by using
BD Powerscript Reverse Transcriptase (Clontech
Lab. Inc. Palo Alto, CA) under manufacture’s
instructions and was kept at )70
C as a collec-
tion of desired genes. For amplification of the
bgl, a pair of primers was designed on the nucleo-
tide sequence of the V. volvacea strain 14 bgl
cDNA (GenBank: AF329731). The bgl was
flanked by EcoRI and HindIII restriction sites at
the 5¢ and 3¢ ends, respectively, and was amplified
by PCR using Probest DNA polymerase (Takara
Biotech, Co., Ltd., Dalian, China) with primers
as follows: 5¢-CCCGAATTCATGCCTCCCTCT-
GATTTTGCA-3¢ and 5¢-CCCAAGCTTTCAAA-
TGCCTCTCCATTCGAAC-3¢. After digestion,
the fragments were ligated at the EcoRI/HindIII
sites of the expression vectors pTrc99A and pET-
20b(+) to yield the constructs, pTrc99A-BGL
and pET-BGL.
Gel electrophoresis of the recombinant
b-glucosidase
The molecular mass of subunits was determined
by SDS–PAGE according to the method of Lae-
mmli. The molecular mass of active b-glucosidase
was determined by native gradient gels of 3–25%
polyacrylamide as described by Bollag (1996).
Enzyme and protein assays
Quantitative assays were performed using
p-nitrophenyl-b-D-glucopyranoside (pNPbG) as a
substrate. The reaction mixture comprised 1 mM
pNPbG, 50 mM citric acid/sodium phosphate
buffer, and appropriately diluted enzyme solution
in a total volume of 200 ll, and was performed
at 50
C for 5 min at pH 6.4 and was terminated
by the addition of 600 ll 1 M Na2CO3, and the
absorbance at 410 nm was measured. One unit
(U) of enzyme activity was defined as the
amount of the enzyme to produce one lmol
p-nitrophenol per min, and the specific activity
was defined as the number of units per mg pro-
tein. Protein concentrations were determined by
the Bradford method using bovine serum
albumin as the standard.
Results
Cloning and expression of the b-glucosidase gene
(bgl)
A bgl gene was amplified and sequenced from
cDNA library of V. volvacea V1-1. It showed
99.8% homology with the gene of V. volvacea
V14. When the E. coli strain harboring pET-BGL
was cultivated for expression at 30 and 37
C,
b-glucosidase activities of 0.04 and 0.01 U/mg
protein were detected in disrupted cells. But the
strain harboring pTrc99A-BGL produced 6 U/mg
protein at 30
C. As the total protein was 138 mg
from the cells harvested from 167 ml cultures, the
b-glucosidase was expressed at about 5000 U/l.
At 37
C, this strain produced about the same
amount of recombinant protein but with only
0.01 U/mg activity; most of the recombinant pro-
tein was insoluble (Figure 1), and no inclusion
bodies were obtained through the procedures for
inclusion body isolation.
Purification of the recombinant b-glucosidase
The recombinant b-glucosidase was purified to
homogeneity from E. coli by a simple two-step
procedure involving DEAE-Sephacel and gel fil-
tration chromatography (Table 1).
Characterization of the recombinant b-glucosidase
The purified active b-glucosidase showed on nat-
ive gradient gels a single band corresponding to
molecular mass of 380 kDa, and its subunits
 1371

Km was 0.2 mM, and the Vmax was 93 U/ mg.

The enzyme was also active towards p-nitrophe-
nyl-b-D-cellobioside (pNPC), p-nitrophenyl-a-D-
xylopyranoside (pNPaX), and cellobioside
(Table 2). No hydrolytic activity was detected
towards CMC, oat spelt xylan, p-nitrophenyl-a-
D-arabinofuranoside (pNPA), and p-nitrophenyl-
b-D-xylopyranoside (pNPbX). The activity of the
enzyme was unaffected by Mn2+, Mg2+, Co2+,
EDTA and Tween-20, but completely inhibited
by Cu2+, Hg2+ and SDS, and partial inhibitions
of 84 and 27% were also observed with Zn2+
and Ni2+. Triton X-100 activated the b-glucosi-
dase under the assay conditions (Table 3).

Fig. 1. SDS–PAGE analysis of expressed b-glucosidase. SDS- Discussion

PAGE of protein was performed in a 10% (w/v) acrylamide
gel. Broad range protein molecular weight markers (Promega
Corp.) were used as the standard of proteins for the determi-
nation of molecular weights. Lane 1: Protein marker; lane 2:
Crude extract of JM109 transformed with pTrc99A-BGL in-
duced at 30
C (approx. 5 lg proteins); lane 3: Crude extract
of JM109 transformed with pTrc99A-BGL induced at 37
C
(approx. 5 lg proteins); lane 4: Cell free extract of JM109
transformed with pTrc99A-BGL induced at 30
C (approx.
7 lg proteins); lane 5: Cell free extract of JM109 transformed
with pTrc99A-BGL induced at 37
C (approx. 4 lg proteins);
lane 6: Purified recombinant BGL (approx. 3 lg proteins).
showed a molecular mass of 97 kDa on 10%
SDS-PAGE. The b-glucosidase exhibited optimal
activity at 50
C and pH 6.4. The purified
enzyme was stable from pH 5.6 to 8.0, and had a
half life of 1 h at 45
C (Figure 2). The purified
recombinant b-glucosidase had a specific activity
of 96 U/mg against pNPbG. Determined by Ea-
die–Hofstee plots of reaction rate on pNPbG, the
The b-glucosidase gene from V. volvacea V1-1
and that from V. volvacea V14 have almost the
same sequence. The recombinant b-glucosidase
was expressed at about three times higher than
that of native b-glucosidase (1.9 U/mg protein or
1260 U/l) produced by V. volvacea V14 in cellu-
lose-grown cultures (Cai et al. 1998). b-Glucosi-
dase genes from other fungi were successfully
expressed in yeasts, Pichia pastoris and Saccharo-
myces cerevisiae, or in filamentous fungi (Cum-
mings & Fowler 1996, Skory et al. 1996,
Takashima et al. 1999, Dan et al. 2000). None of
these genes, however, have been successfully ex-
pressed in E. coli. Very low activity was detected
when bglI of Aspergillus niger was expressed in
E. coli (Dan et al. 2000), and expression of bglB
from Candida wickerhamii was unstable in E. coli
(Skory & Freer 1995). Usually, the host cells of

Table 1. Purification of the recombinant b-glucosidase from E. coli.

Step Total protein (mg) Total activity (U) Specific activity (U/ mg) Recovery (%) Purification (fold)

Crude extracta 138 819 6 100 1

DEAE-SepharoseCL-6Bb 14 617 46 75 7.7
HW-55c 3 285 96 35 16
a
The recombinant strain was grown in LB medium with 100 lg amplicillin/ml at 30
C to OD600 0.6 and was incubated further with
isopropyl-b-thiogalactopyranoside (IPTG) for 8 h. The cells of 167 ml cultures were harvested by centrifugation at 10,000  g for
10 min at 4
C, resuspended in 20 ml imidazole buffer (50 mM imidazole, pH 6.7), then disrupted by sonication.
b
The resulting supernants were loaded on to an DEAE-Sephacel column (2.5  30 cm) equilibrated with the imidazole buffer (50 mM
imidazole, pH 6.7), and the bound proteins were eluted with a linear gradient of 0–1 M NaCl in the same buffer. The fractions
exhibiting enzyme activity were pooled, and protein was concentrated by (NH4)2SO4 precipitation and was dissolved in the imidazole
buffer.
c
The concentrated sample was put on a HW-55F-Toyopearl column (2.5  90 cm, SUPELCO) pre-equilibrated with the imidazole
buffer containing 0.2 M NaCl, and the protein was eluted with the same buffer.
1372

enzyme at 30
C but an inactive form at 37
C,

Fig. 2. Effect of pH and temperature on the b-glucosidase
activity. (a) The optimal pH was determined at 50
C for
10 min in the 0.1 M citric acid/sodium phosphate buffer from
pH 4.4 to 8.0 (100% relative values of data was 89 U/mg).
(b) The optimal temperature for the enzyme activity was
determined by standard assay ranging from 35 to 65
C in
0.1 M citric acid/sodium phosphate buffer, pH 6.4. The spe-
cific activity was 94 U/mg, defined as 100%. (c) The pH sta-
bility of the recombinant enzyme was determined by
measuring the remaining activity after incubating 0.07 lg
purified enzyme at 16
C for 1 h in 0.2 ml 0.1 M citric acid/
sodium phosphate buffer from pH 5.2 to 8.0. Full activity
was determined at each pH value. (d) The thermostability. A
0.07 lg purified enzyme in 0.2 ml of 0.1 M citric acid/sodium
phosphate buffer (pH 6.4) was pre-incubated for various
times at 30
C (¤); 37
C ( n ); 42
C (m); 45
C () and
50
C (*) in the absence of the substrate. The activity of the
enzyme without pre-incubation was defined as 100%
(activity=94 U/mg).
yeast and filamentous fungi are cultivated at 28
or 30
C, while E. coli cells are grown for heter-
ologous gene expression at 37
C. In this work,
the construct, pTrc99A-BGL, gave an active
indicating that temperature was an important
factor affecting the activities of the enzyme ex-
pressed from mesophilic fungus genes in E. coli.
At 30
C, the construct pET-BGL was expressed
about 100 times less enzyme than pTrc99A-BGL,
indicating that expression vector was also an
important factor relating to the bgl expression.
The purified recombinant b-glucosidase had a
molecular mass of 380 kDa; two b-glucosidases
purified from V. volvacea V14 had molecular
masses of 158 kDa (BGL-I) and 256 kDa (BGL-
II) (Cai et al. 1998), and the purified re-
combinant b-glucosidase also had a specific
activity of 1.5–2-fold higher than levels observed
with native enzymes from V. volvacea V14 (62 U/
mg protein; 44 U/mg protein) (Cai et al. 1998).
The recombinant enzyme differed from the native
enzymes in substrate specificity as well (Table 2).
The recombinant b-glucosidase did not exhibit
activities on pNPaA and pNPbX, but it did have
activity on pNPbC, and higher activity on cello-
biose than on pNPbX.
The recombinant b-glucosidase was com-
pletely inhibited by 1 mM Cu2+, while BGL-I and
BGL-II activities were inhibited 69 and 39% by
1 mM Cu2+, respectively. A low level inhibition
of BGL-II (7%) was observed with Zn2+ (5 mM),
while the high level inhibition of the recombinant
b-glucosidase (84%) was observed with Zn2+
(1 mM). The recombinant b-glucosidase only kept
about 40% of original activity after 2 h incuba-
tion at 37
C. Most of enzymes were denatured
after incubated for 12 h at 37
C in the induction
medium. This is not consistent with the character-

Table 2. Relative initial rates of hydrolysis of various substrates by the purified b-glucosidase.

Substrate Recombinant BGLa Native BGL-Ib Native BGL-IIb

p-Nitrophenyl-b-D-glucopyranoside 100 100 100

p-Nitrophenyl-a-D-xylopyranoside 18 nd nd
p-Nitrophenyl-b-D-cellobioside 46 0 0
Cellobioside (0.5 mM) 164 65 32
p-Nitrophenyl-b-D-xylopyranoside 0 29 0
p-Nitrophenyl-a-D-arabinofuranoside 0 18 12
a
Various substrates were used at 1 mM or as shown. Activity on xylan, CMC and cellobiose was determined at 50
C for 10 min by
measuring the release of glucose or xylose (410 nm) and on aryl-glycosides by measuring the release of p-nitrophenol (410 nm). A
0.07 lg purified b-glucosidase was added to each substrate in 0.2 ml of 0.1 M citric acid/sodium phosphate buffer (pH 6.4) to initiate
the enzyme reaction. The absolute rate of hydrolysis of pNPbG by the recombinant BGL was 94 U/mg protein. Values shown are the
mean of duplicate experiments with each substrate, and the variation about the mean was below 5%.
b
Data reported by Cai et al. (1998).

Table 3. Effect of metal ions and surfactants on the recom-
binant b-glucosidase activity.
Metal ions and b-Glucosidase
surfactants (relative activity, %)
Control 100
Zn2+ 16
Cu2+ 0 Buswell JA, Chang ST (1993) Edible mushrooms: Attributes
Ni2+ 73
Hg2+ 0 eds. Genetics and Breeding of Edible Mushrooms, Philadel-
Triton-X100 (0.05%, w/v) 208
SDS (0.1%, w/v) 0 nents of the cellulolytic system of the edible straw mush-
Various reagents were used at 1 mM or as shown. A 0.07 lg
purified b-glucosidase was incubated with each reagent for
8 min before the addition of pNPbG to initiate the enzyme
reaction. The activity without a reagent was taken as 100%
(activity=94 U/mg). Values shown are the mean of duplicate
experiments, and the variation about the mean was below 5%.
istics of the native BGL-I or BGL-II from V. volv-
acea V14 which were more stable than the recom-
binant b-glucosidase to exposure of 50
C and
above (Cai et al. 1998).
Our results showed that the recombinant
b-glucosidase of V. volvacea V1-1 was successfully
over-expressed in E. coli. The expression level of
the bgl in pTrc99A was about 100 times higher
than that in pET-20(b); and most recombinant
enzyme was inactive when expressed at 37
C. The
recombinant enzyme was purified by a two-step
chromatography procedure, and its properties dif-
fered from the native enzymes purified from V.
volvacea V14. It would be of great benefit to real-
ize commercial use, and to generate strains with
improved capability for cellulose degradation.
Acknowledgments
This work was supported by grants from the
National Natural Science Foundation of China
1373
(30370034), the 211 Foundation of Ministry of
Light Industry of China (2001).
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