Identification of a metagenome-derived β-glucosidase from bioreactor contents

Article abstract of DOI:10.1016/j.molcatb.2009.11.009

A novel β-glucosidase gene designated as bgl1T was cloned by function-based screening of a metagenomic library from uncultured microorganisms in contents of a bioreactor. The gene has an open reading frame of 1860 base pairs and encodes a 620 amino acid polypeptide with a predicted molecular mass of about 65 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Bgl1T and other putative β-glucoside-specific II ABC subunit components, were closely related. Functional characterization with a high performance liquid chromatography method demonstrated that the recombinant Bgl1T protein hydrolyzed d-(+)-cellobiose to glucose. The maximum activity for Bgl1T protein occurred at pH 7.0 and 37 °C using p-nitrophenyl-β-d-glucoside as the substrate. The putative β-glucosidase had an apparent K_m value of 1.45 mM, a V_max value of 20.5 U/mg, a k_cat value of 1370/min and a k_cat/K_m value of 943/mM/min. The biochemical characterization of Bgl1T protein indicated its potential applications for better industrial production of glucose or ethanol by biological processes under moderate conditions. Crown Copyright

Full text of DOI:10.1016/j.molcatb.2009.11.009

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 11–16
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Identification of a metagenome-derived ␤-glucosidase from
bioreactor contents
Chengjian Jianga,∗, Zhen-Yu Haoa, Ke Jina, Shuang-Xi Lia, Zhi-Qun Chea,
Ge-Fei Maa, Bo Wua,b,∗∗
a Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and
Plant Genetic Engineering, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, People’s Republic of China
b College of Chemistry and Ecology Engineering, Guangxi University for Nationalities, 188 Daxue East Road, Nanning, Guangxi, 530006, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel ␤-glucosidase gene designated as bgl1T was cloned by function-based screening of a metagenomic
library from uncultured microorganisms in contents of a bioreactor. The gene has an open reading frame
of 1860 base pairs and encodes a 620 amino acid polypeptide with a predicted molecular mass of about
65 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Bgl1T and
other putative ␤-glucoside-specific II ABC subunit components, were closely related. Functional charac-
terization with a high performance liquid chromatography method demonstrated that the recombinant
Bgl1T protein hydrolyzed d-(+)-cellobiose to glucose. The maximum activity for Bgl1T protein occurred
at pH 7.0 and 37 ◦C using p-nitrophenyl-␤-d-glucoside as the substrate. The putative ␤-glucosidase had
an apparent Km value of 1.45 mM, a Vmax value of 20.5 U/mg, a kcat value of 1370/min and a kcat/Km
value of 943/mM/min. The biochemical characterization of Bgl1T protein indicated its potential appli-
cations for better industrial production of glucose or ethanol by biological processes under moderate
conditions.
Received 6 September 2009
Received in revised form
22 November 2009
Accepted 23 November 2009
Available online 27 November 2009
Keywords:
Metagenomic library
Uncultured sludge microorganisms
␤-Glucosidase
Functional characterization
Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction
[4]. However, at present, its use in industrial cellulose hydroly-
sis for ethanol production is probably its primary biotechnological
␤-Glucosidases (␤-d-glucoside glucohydrolases, EC 3.2.1.21)
catalyze the hydrolysis of ␤-glucosidic linkages of various oligosac-
charides and glycosides to form glucose and a shorter/debranched
(oligo)saccharide or aglycone [1]. For most bioconversion pro-
importance [5].
␤-Glucosidases are commonly found in both prokaryotic and
eukaryotic organisms. Based on amino acid sequence similarities,
␤-glucosidases have been classified in family 1 and family 3 of the
glycoside hydrolase families [2,6]. At present, many ␤-glucosidases
have been purified and characterized from cultured bacteria and
fungi [2–8]. Recently, some ␤-glucosidases have been identified
from uncultured samples including wetlands [9], alkaline polluted
soils [10], contents of rabbit cecum [11], the cow rumen [12], and
the large bowel of mice [13]. Nonetheless, detailed biochemical
characterizations of these metagenome-derived ␤-glucosidases are
currently lacking.
In the present study, a cosmid library was constructed from
a metagenome derived from sludge samples from a bioreactor.
Through enzyme function-based screening of a DNA library, a
gene encoding a novel ␤-glucosidase (named Bgl1T) was isolated.
The deduced amino acid sequence had moderate similarity to
the known ␤-glucosidases in the Genbank database. Biochemi-
cal analysis of the overexpressed recombinant protein revealed
that Bgl1T catalyzed hydrolysis of d-(+)-cellobiose to form glucose.
To our knowledge, this is the first report of a metagenome-
derived ␤-glucosidase isolated from the sludge content of a
bioreactor.
cesses, ␤-glucosidase is
a key rate-limiting enzyme for the
cellulose-hydrolyzing system in bacteria and fungi [2]. It is one
of the three enzymes in the complete cellulase system composed
of ␤-1,4-endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91),
and ␤-glucosidase [3]. Thus, ␤-glucosidase plays an important
role in the utilization of biomass. It has also attracted consider-
able attention in the food industry for its potential applications
in various biotechnological processes, such as hydrolysis of bitter
compounds during fruit-juice extraction, release of aromatic com-
pounds from flavorless precursor glucosides [3], and removal of
bitter components of citrus products, and detoxification of cassava
∗
Corresponding author. Tel.: +86 771 3239283; fax: +86 771 3239403.
Corresponding author at: Guangxi University for Nationalities, 188 Daxue East
∗∗
Road, Nanning, Guangxi, People’s Republic of China. Tel.: +86 771 3239283;
fax: +86 771 3239403.
E-mail addresses: jiangcj0520@gmail.com (C. Jiang), wubogxu@yahoo.com.cn
(B. Wu).
1381-1177/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.11.009

12
C. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 11–16
2. Materials and methods
(5ꢀ-TGTGAAATTTGTGATGCTATTGCT-3ꢀ) provided by the manufac-
turer. Complete coverage of the sequences for the target gene was
obtained by primer-walking sequencing from both strands.
2.1. DNA manipulation and protein analysis
All DNA manipulations, including cloning and subcloning,
transformation of Escherichia coli cells, and PCR were per-
formed according to standard techniques [14] or following the
manufacturer’s instructions, unless indicated otherwise. Protein
preparation and analysis, including protein extraction from E. coli,
protein quantification, and sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE), were performed as described in the
standard protocols [15].
2.5. Overexpression and purification of the recombinant
ˇ-glucosidase
The sequence of bgl1T, excluding the sequence encoding the
N-terminal signal peptide, was amplified by polymerase chain
reaction (PCR) using pGXN10 as the template, using the fol-
lowing primers: sense primer 5ꢀ-CCGGAATTCTATGGAATATCAA-
GCACTGGC-3ꢀ (containing an EcoRI site at the 5ꢀ end) and antisense
primer 5ꢀ-AACTGCAGCTGATGGCTGACGCAAAGTA-3ꢀ (containing a
PstI site at the 5ꢀ end). Amplified DNA was digested with EcoRI and
PstI prior to its ligation into the vector pETBlue-2 (Qiagen, Valen-
cia, CA, USA) digested with the same enzymes, resulting in the
plasmid pGXN10A. The recombinant plasmid, pGXN10A, then was
transformed into E. coli Tuner (DE3) pLacI (Qiagen, Valencia, CA,
USA). His-tagged Bgl1T was expressed and purified using nickel-
nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen, Valencia, CA,
USA), according to the manufacturer’s instructions.
2.2. Sludge metagenomic DNA preparation and purification
Sludge samples were collected from a biogas reactor located
in WuMing county of Guangxi Province, South China (23◦10ꢀN,
108◦17ꢀE). This particular biogas reactor has been running success-
fully for over 30 years. The metagenomic DNA was extracted from
the sludge samples following the direct lysis method of Roh et al.
described [16], with some modifications. Briefly, the DNA pellet
from 5 g of sludge sample was washed twice with 75% ethanol,
air dried, and dissolved in 1× TE buffer (10 mM Tris–HCl pH 8.0
and 1 mM Na2EDTA). To remove contaminants, the crude metage-
nomic DNA extract was loaded onto a column (200 mm × 10 mm)
containing Sephadex G200 and acid-washed polyvinylpolypyrroli-
done (PVPP) at a ratio of 50:1. Finally, DNA was eluted from the
column with TE buffer and further purified by electroelution, as
described [14].
2.6. Identification of the hydrolysis product
The hydrolysis product of the putative ␤-glucosidase was
identified by high performance liquid chromatography (HPLC). d-
(+)-Cellobiose (Sigma, St. Louis, MO, USA) was used as the substrate
for characterization of the ␤-glucosidase. The enzymatic reaction
mixture contained 10 mM d-(+)-cellobiose, 0.2 M Na2HPO4-citric
acid buffer, pH 7.0, 1.0 mM MgCl2, and approximately 20 ␮g puri-
fied recombinant protein. The reaction was conducted in a total
volume of 2.0 ml at 35 ◦C for 15 min. Upon termination of the reac-
tion with 2.0 ml 1 M Na2CO3, the residual protein was removed
by centrifugation through a membrane (Vivaspin 500, Vivascience,
Littleton, MA, USA). The filtered reaction sample was separated
on a Fast Carbohydrate Cartridge Column (150 mm × 4.6 mm,
Waters, USA) eluted with double distilled H2O and acetonitrile
(15:85, v/v) as the mobile phase at a flow rate of 1.0 ml/min
and detected using a Waters R-401 differential refractometer
[10,18].
2.3. Construction and screening of the cosmid library for
ˇ-glucosidase genes
A metagenomic library was constructed using the pWEB::TNC
Cosmid Cloning Kit (Epicentre, Madison, WI, USA) according to
the manufacturer’s instructions. Briefly, the purified DNA was
end repaired with T4 DNA polymerase and T4 polynucleotide
kinase to generate blunt ends. End-repaired DNA was separated
on a low-melting-point agarose gel (FMC Corporation, Philadel-
phia, PA, USA). The DNA fragments between 40 and 50 kb were
recovered and ligated into the cosmid vector pWEB::TNC that
had been linearized at the unique SmaI site and dephosphory-
lated. The ligated products were packaged and infected into E. coli
EPI100. After overnight growth on LB agar plates containing ampi-
cillin (50 ␮g/ml) and chloramphenicol (25 ␮g/ml), white colonies
harboring plasmids bearing inserts were collected and used to con-
struct the metagenomic library. The library was stored at −80 ◦C
until screening.
Colonies of the library were replica plated onto Luria–Bertani
(LB) agar plates supplemented with ampicillin (50 ␮g/ml) and
chloramphenicol (25 ␮g/ml). Esculin hydrate and ferric ammo-
nium citrate (Sigma, St. Louis, MO, USA) were used to detect
␤-glucosidase activity according to the method described by Kwon
et al. [17]. The plasmids of the positive clones were retransformed
into E. coli DH5␣ to confirm that the activities were due to the
cloned DNA.
2.7. Physico-chemical characterization of recombinant
ˇ-glucosidase protein
Unless otherwise specified, ␤-glucosidase activity was assayed
at 40 ◦C by incubating an enzymatic reaction mixtures (1 ml)
containing 20 ␮g of suitably diluted enzyme with 2 mM p-
nitrophenyl-␤-d-glucoside (Sigma, St. Louis, MO, USA) in 50 mM
Na-phosphate buffer (pH 7.0) for 15 min. The reaction was stopped
by adding 600 ␮l of 0.4 M Na2CO3 and the p-nitrophenol (pNP)
released was determined by reading the absorbance at 420 nm.
One unit of ␤-glucosidase activity (U) is defined as the amount of
enzyme that will hydrolyze 1 ␮mol pNP-␤-d-glucoside per minute
under the experimental conditions described above [19]. The pro-
files of activity versus pH and activity versus temperature were
determined using standard assay methods. To measure the effect of
pH on the activity of Bgl1T protein, the enzyme activity was assayed
in the range of pH 4.5–9.0 (50 mM Na-phosphate buffer) and pH
8.6–10.0 (0.1 M glycine-NaOH buffer). For the effect of temperature
on activity of Bgl1T protein, the enzyme was assayed at various
temperatures (20–60 ◦C) for 15 min, using pNP-␤-d-glucoside as
the substrate in 50 mM Na-phosphate buffer (pH 8.0). Various metal
compounds (NaCl, KCl, LiCl, MgCl2, ZnCl2, CuCl2, CaCl2, MnCl2,
FeCl2, FeCl3, KNO3), chelating agents (ethylenediaminetetraacetic
acid, EDTA), and surfactant (SDS) were added to optimal reaction
systems to investigate their effects on enzyme activity. The con-
2.4. Identification of ˇ-glucosidase genes
To identify the ␤-glucosidase genes carried on recombinant
plasmid pGXN10, the target plasmids were nested-deleted from
one end by in vitro Tn5 transposition (pWEB::TNC Deletion Cosmid
Transposition Kit; Epicentre, Madison, WI, USA) according to the
manufacturer’s protocol. The plasmid with the smallest insertion
expressing ␤-glucosidase was sequenced from priming sites on the
vector adjacent to the deletion start sites using the following primer

C. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 11–16
13
centrations of metal ions, EDTA, and SDS used were 1 mM, 2 mM,
and 1% (v/v), respectively.
(GenBank accession no. YP 001177456) from Enterobacter sp. 638.
Others ␤-glucosidases include a cellobiose-specific PTS permease
(GenBank accession no. AAB51563) from Klebsiella oxytoca (78%
identical and 87% similar), ␤-glucoside-specific PTS system compo-
nents IIABC subunits (GenBank accession no. YP 001336226) from
Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (75% iden-
tical and 86% similar), a ␤-glucoside transport protein (GenBank
accession no. ABG73228) from Klebsiella aerogenes (75% identi-
cal and 86% similar), a putative cellobiose-specific PTS permease
(GenBank accession no. YP 002920438) from K. pneumoniae NTUH-
K2044 (75% identical and 86% similar), and a ␤-glucoside-specific
phosphotransferase enzyme IIA component (GenBank accession
no. ZP 04635638) from Yersinia intermedia ATCC 29909 (63% iden-
tical and 77% similar).
2.8. Enzyme kinetic assays
Enzyme kinetic parameters of Bgl1T were obtained by mea-
suring the rate of hydrolysis of pNP-␤-d-glucoside at various
concentrations (0.1–10 mM) at 37 ◦C for 15 min in 50 mM Na-
phosphate buffer (pH 7.0). The enzymatic kinetic parameters, Km
and Vmax, were determined from Lineweaver–Burk plots using the
Enzyme Kinetics computer program [20].
3. Results and discussion
Multiple alignments of the deduced amino acids of Bgl1T with
the most homologous ␤-glucosidase proteins (above 60% similar-
ity) (NCBI database) are presented in Fig. 1. Amino acid sequence
comparison revealed that the deduced Bgl1T peptide shared the
conserved amino acid residues with other known ␤-glucosidases.
Fig. 2 presents the phylogenetic tree, showing the relationship
with other ␤-glucosidases, based on amino acids. The phyloge-
netic tree also revealed that Bgl1T was most closely related to PTS
system beta-glucoside-specific EIIBCA component from Cronobac-
ter turicensis and it clustered together with other ␤-glucosidases
from several bacterial species, including Pectobacterium atrosep-
ticum and Yersinia frederiksenii.
3.1. Construction of the metagenomic library and screening of the
library for ˇ-glucosidase gene
The approach of generating a metagenomic library from soils
or sediments that are known to harbor a high level of micro-
bial diversity has been used successfully to find a wide variety
of novel catalytic proteins and secondary metabolites [21]. The
metagenomic DNA in the current study was directly extracted
from samples of sludge from a bioreactor. The constructed library
was expected to contain a genome pool of all the microorgan-
isms in the reactor sludge, including those of uncultivable bacteria.
A cosmid library of ca. 30,000 clones was constructed using the
metagenomic DNA isolated from the bioreactor contents. Restric-
tion analysis of randomly chosen recombinant plasmids revealed
a high level of diversity of the foreign DNA fragments found
in pWEB::TNC. BamHI digestion of plasmids from 14 randomly
selected recombinants resulted in insert sizes ranging from 20
to 60 kb on agarose gels (data not shown), with an average size
of ca. 35 kb. The library possessed a capacity of approximately
1.05 × 109 bp of insertion DNA. One metagenomic DNA library
clone, named pGXN10, that expressed strong ␤-glucosidase activ-
ity was isolated using a function-based strategy. The positive clone
pGXN10 is surrounded by a black spot against a plain background
after 24 h at 37 ◦C, indicating active ␤-glucosidase. The positive
rate of ␤-glucosidase activity in the library was approximately
1/30,000, because sludge metagenomic DNA originated from thou-
sands of different types of microorganisms [22,23]. A recent paper
reported that the positive rate of cellulase in a cosmid library con-
structed from soil samples was approximately 1/70,000 [24], which
is somewhat lower than the final results obtained in the current
study.
3.3. Overexpression and purification of recombinant Bgl1T
protein
In order to characterize the function of Bgl1T, we subcloned the
gene in frame with a six-histidine tag sequence into expression vec-
tor pETBlue-2 and expressed it in E. coli Tuner (DE3) pLacI. Initial
analysis with crude cell lysates showed that the bacteria contain-
ing recombinant plasmid pETBlue-2-bgl1T produced a substantial
amount of the expected recombinant protein, while this protein
was not detectable in cultures of the bacteria containing the empty
vector pETBlue-2.
The recombinant Bgl1T protein was purified with Ni-NTA
Magnetic Agarose Chromatography, followed by the removal of
degraded and/or nonspecifically bound polypeptides with gel fil-
tration chromatography (GFC). As shown in Fig. 3, the recombinant
Bgl1T protein was purified to homogeneity. The recombinant pro-
tein eluted from the column had a molecular weight of ∼66 kDa,
which is consistent with the theoretical mass of 65 kDa.
3.2. Sequence analysis of the cloned ˇ-glucosidase
3.4. Functional characterization of recombinant Bgl1T protein
The plasmid from this positive clone of pGXN10 was extracted,
subcloned, and sequenced by primer-walking. The insert DNA
sequence had a length of 3041 base pairs (bp), and it had the
highest identical (79%) with a DNA fragment (GenBank accession
no. CP000653.1) from the genome of Enterobacter sp. 638 at DNA
level in the database. However, it shared moderate homology with
the other known putative ␤-glucoside-specific IIABC subunit com-
ponents at amino acids level, according to the BLASTx program.
Based on the sequence similarity results, we considered that the
cloned gene on pGXN10 could be a novel ␤-glucosidase gene and
named it bgl1T. The gene had an open reading frame of 1860 bp
and the deduced peptide was predicted to have a molecular mass
of ∼65 kDa and an isoelectric point of 5.69. The bgl1T nucleotide
sequence has been deposited in the GenBank under accession
number GQ507800. When the deduced amino acid sequence of
Bgl1T was searched against the NCBI and Expasy databases, it
was found that Bgl1T had the highest identical (85%) and simi-
lar (92%) with ␤-glucoside-specific PTS system components IIABC
We determined the product of enzymatic cellobiose hydrolysis
by HPLC. The retention time of enzymatic product was 4.75 min
and that of the substrate peak (d-(+)-cellobiose) was 8.48 min. The
enzymatic product peak eluted earlier than the substrate peak and
matched the retention time (4.57 min) of a glucose standard. These
results suggested that Bgl1T protein catalyzed the formation of
glucose from d-(+)-cellobiose. The functional characterization of
Bgl1T may provide new insights into the relationship between the
sequence, structure, and activity of the known ␤-glucoside-specific
PTS system component IIABC subunits [25].
3.5. Physico-chemical characterization of recombinant Bgl1T
protein
To determine the optimal pH for this putative ␤-glucosidase,
we measured the enzymatic activity of the purified Bgl1T protein at
various pH values from pH 4.0 to 9.5 using pNP-␤-d-glucoside as the
substrate. The data, presented in Fig. 4A, demonstrated that Bgl1T

14
C. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 11–16
Fig. 1. Sequence alignment of Bgl1T protein with other ␤-glucoside-specific PTS system components. The proteins are identified by their GenBank accession number.
Sequence similarity searches were performed with the BLAST 2.0 program. Amino acid sequence alignment of the target putative protein with homologous proteins was
performed with the Align X program, a component of the Vector NTI suite (Informax, North Bethesda, MD, USA) using the blosum62mt2 scoring matrix.
protein was active at a pH range of 6.0–9.0. The optimal pH of the
enzyme is around 7.0 because it achieves maximal enzymatic activ-
ity at this condition. Enzymatic activity remained at a high level
when the pH was between 6.0 and 9.0. However, the activity of the
recombinant enzyme was dramatically lost below pH 6.0 or above
pH 9.0. The pH range of the recombinant enzyme was consistent
with the reported properties of alkaline endoglucanase from Bacil-
lus circulans [26] and metagenome-derived ␤-glucosidase from the
contents of rabbit cecum [11].
To determine the optimal temperature for the enzymatic reac-
tion of Bgl1T, its activity was measured at pH 7.0 in the temperature
range from 20 to 60 ◦C. As shown in Fig. 4B, at a temperature lower
than 30 ◦C, the enzyme showed 65% of its maximum activity. Bgl1T
exhibited >70% of its maximum activity from 32 to 50 ◦C, and the
enzyme reached its highest activity at approximately 37 ◦C. The
temperature range of the recombinant enzyme was similar to that
reported for a ␤-glucosidase from corn stover [27].
The effects of metal ions, EDTA, and SDS on the hydrolytic activ-
ity of Bgl1T protein were determined with pNP-␤-d-glucoside as
the substrate by measuring activity in the presence of these com-
pounds. The enzyme activity of Bgl1T without added metal ions was
taken as 100%. The presence of CaCl2 and MgCl2 significantly stim-
ulated the enzyme activity to 142% and 136%, respectively, whereas
CuCl2, MnCl2, FeCl2 and FeCl3 dramatically reduced the enzymatic
activity to 73%, 63%, 41%, and 56%, respectively. In contrast, ZnCl2,
NaCl, KCl, LiCl, and KNO3 had no significant effect on the enzy-
matic activity. The chelating agent EDTA and the anionic surfactant
SDS reduced the activity to 63% and 50%, respectively. These results

C. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 11–16
15
Fig. 2. Phylogenetic relationship of Bgl1T with related proteins. The proteins are identified by their GenBank accession number. Sequence alignment was performed by using
ClustalW version 1.81 and the phylogenetic tree was constructed using the neighbor-joining method with Molecular Evolutionary Genetics Analysis 4.0 software (MEGA,
Version 4.0) [28]. Boot-strapping value was used to estimate the reliability of phylogenetic reconstructions (1000 replicates). The numbers associated with the branches refer
to bootstrap values (confidence limits) representing the substitution frequencies per amino acid residue.
Fig. 4. (A) Effects of pH on the enzymatic activity of Bgl1T protein; the buffers used
were Na-phosphate buffer (ꢀ) (pH 4.5–9.0), glycine-NaOH buffer (ꢁ) (pH 8.6–10.0).
The relative activities are the enzyme activities at each pH divided by the maximal
Fig. 3. SDS-PAGE analysis of Bgl1T. Proteins were separated by 12% SDS-PAGE and
activity. (B) Effects of temperature on the enzymatic activity (ꢀ) of Bgl1T protein.
then stained with Coomassie brilliant blue G-250. Lane 1, protein molecular weight
The relative activities are the enzyme activities at each temperature divided by the
markers; lane 2, the purified target protein; lane 3, total protein of E. coli Tuner (DE3)
maximal activity.
pLacI harboring empty pETBlue-2 as control; lane 4, total protein of E. coli Tuner
(DE3) pLacI harboring the recombinant bgl1T in pETBlue-2 induced by addition of
0.5 mM IPTG. The Bgl1T protein is indicated by the black arrow.
Table 1
Purification of Bgl1T protein from E. coli Tuner (DE3) pLacI.
Purification step
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification (fold)
Crude extract
Ni-NTA
GFC
1030
32.0
26.0
4800
474
432
4.66
14.8
16.6
100
9.88
9.0
1.0
3.18
3.56

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C. Jiang et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 11–16
suggested that a CaCl2 concentration of 10 mM was required for the
optimal activity of Bgl1T protein. As shown in Table 1, the enzyme
activity reached 16.6 U/mg protein using pNP-␤-d-glucoside as the
substrate under the optimal reaction conditions of pH 7.0, 37 ◦C,
and 10 mM CaCl2 for 15 min.
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from double reciprocal Lineweaver–Burk plots. The putative ␤-
glucosidase had an apparent Km value of 1.45 mM, a Vmax value
of 20.5 U/mg, a kcat value of 1370/min and a kcat/Km value of
943/mM/min. This Vmax value for Bgl1T protein was in agreement
with that recorded for ␤-glucosidase from uncultured microorgan-
isms [9]. Knowledge of these properties of Bgl1T protein should
allow better industrial production of glucose or ethanol by biolog-
ical processes under moderate conditions.
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Acknowledgements
This research was supported by the Open Research Fund Pro-
gram of the Key Laboratory of Ministry of Education for Microbial
and Plant Genetic Engineering (Grant No. J0801).
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