Cloning, expression and biochemical characterization of a GH1 β-glucosidase from Cellulosimicrobium cellulans

Article abstract of DOI:10.1080/10242422.2017.1395415

β-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel β-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence contained an open reading frame of 1494 bp, encoded a polypeptide of 497 amino acid residues. The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography and had a molecular weight of 57 kDa, as judged by SDS-PAGE. The optimum β-glucosidase activity was observed at 55 °C and pH 6.0. Recombinant CcBgl1B was found to be most active against aryl-glycosides p-nitrophenyl-β-D-glucopyranoside (pNPβGlc), followed by p-nitrophenyl-β-D-galactopyranoside (pNPβGal). Using disaccharides as substrates, the enzyme efficiently cleaved β-linked glucosyl-disaccharides, including sophorose (β-1,2-), laminaribiose (β-1,3-) and cellobiose (β-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellotetraose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode between the enzyme and the substrates driving the reaction was modelled using a molecular docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particularly its activity against cello-oligosaccharides, would be potentially useful for biotechnological applications of cellulose degradation.

Full text of DOI:10.1080/10242422.2017.1395415

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Cloning, expression and biochemical
characterization of a GH1 β-glucosidase from
Cellulosimicrobium cellulans
Ye Yuan, Fenghua Xu, Jianzhuang Yao, Yanho Hu, Jiao Wang, Tianjiao Zhao,
Yifa Zhou & Juan Gao
To cite this article: Ye Yuan, Fenghua Xu, Jianzhuang Yao, Yanho Hu, Jiao Wang, Tianjiao
Zhao, Yifa Zhou & Juan Gao (2017): Cloning, expression and biochemical characterization of a
GH1 β-glucosidase from Cellulosimicrobium cellulans, Biocatalysis and Biotransformation, DOI:
10.1080/10242422.2017.1395415
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Date: 27 October 2017, At: 11:18

BIOCATALYSIS AND BIOTRANSFORMATION, 2017
https://doi.org/10.1080/10242422.2017.1395415
RESEARCH ARTICLE
Cloning, expression and biochemical characterization of a GH1 b-glucosidase
from Cellulosimicrobium cellulans
Ye Yuana,b, Fenghua Xuc, Jianzhuang Yaoa, Yanho Hub, Jiao Wangb, Tianjiao Zhaob, Yifa Zhoub and
Juan Gaoa
b
aSchool of Biological Science and Technology, University of Jinan, Jinan, PR China; Jilin Province Key Laboratory on Chemistry and
Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, Changchun, PR China;
cDepartment of Pharmaceutics, PLA General Hospital, Beijing, PR China
ABSTRACT
ARTICLE HISTORY
Received 9 May 2017
Revised 4 October 2017
Accepted 16 October 2017
b-Glucosidase plays an important role in the degradation of cellulose. In this study, a novel
b-glucosidase ccbgl1b gene for a glycosyl hydrolase (GH) family 1 enzyme was cloned from the
genome of Cellulosimicrobium cellulans and expressed in Escherichia coli BL21 cells. The sequence
contained an open reading frame of 1494 bp, encoded a polypeptide of 497amino acid residues.
The recombinant protein CcBgl1B was purified by Ni sepharose fastflow affinity chromatography
and had a molecular weight of 57 kDa, as judged by SDS-PAGE. The optimum b-glucosidase
activity was observed at 55 ꢀC and pH 6.0. Recombinant CcBgl1B was found to be most active
against aryl-glycosides p-nitrophenyl-b-D-glucopyranoside (pNPbGlc), followed by p-nitrophenyl-
b-D-galactopyranoside (pNPbGal). Using disaccharides as substrates, the enzyme efficiently
cleaved b-linked glucosyl-disaccharides, including sophorose (b-1,2-), laminaribiose (b-1,3-) and
cellobiose (b-1,4-). In addition, a range of cello-oligosaccharides including cellotriose, cellote-
traose and cellopentaose were hydrolysed by CcBgl1B to produce glucose. The interaction mode
between the enzyme and the substrates driving the reaction was modelled using a molecular
docking approach. Understanding how the GH1 enzyme CcBgl1B from C. cellulans works, particu-
larly its activity against cello-oligosaccharides, would be potentially useful for biotechnological
applications of cellulose degradation.
KEYWORDS
Cellulosimicrobium cellu-
lans; cello-oligosaccharide
degradation; cloning and
expression; GH1
b-glucosidase;
molecular modelling
1. Introduction
metabolites, which have improved pharmacological
activity (Gao et al. 2014; Yuan et al. 2015), as well as
being used in juice clarification and improving the fla-
vour of wine (Zheng and Shetty 2000; Swangkeaw
et al. 2011).
b-Glucosidases are widely distributed in different
organisms, microorganisms, such as bacteria and fungi,
are particularly important sources of these enzymes.
According to the amino acid sequence homology,
b-glucosidases are mainly divided into two glycoside
hydrolase (GH) families, GH1 and GH3 (Cantarel et al.
2009). To date, some GH family 1 b-glucosidases have
been cloned or purified from different microorganisms
Cellulose is the most abundant renewable resource on
earth, which complete enzymatic hydrolysis was
accomplished by three different types of enzymes:
endo-b-1,4-glucanase (EC 3.2.1.4), cellobiohydrolase
(EC 3.2.1.91) and b-glucosidase (EC 3.2.1.21) (Greene
et al. 2015; Bischof et al. 2016). Glucose, a product of
cellulose hydrolysis, can be further converted into
ethanol, which could be used as biofuel that is envir-
onmentally friendly (Su et al. 2016). b-Glucosidase
plays an important role in the process of hydrolysing
cellobiose into glucose and is the key rate-limiting fac-
tor in cellulose complex degradation (Pei et al. 2012;
Singh et al. 2016). Therefore, there is a need to screen
potential b-glucosidase enzymes for efficient biocon-
version of cellulose. b-Glucosidases are also used in
other industrial fields such as the production of food,
animal feed, detergents, as well as in pharmaceutical
and cosmetic production. For example, b-glucosidase
can convert ginseng saponins into deglycosylated
ꢀ
(Wierzbicka-Wos et al. 2013; Yang et al. 2013; Chamoli
et al. 2016). Previously, we isolated a bacterium
Cellulosimicrobium cellulans from soil, whose genome
has been released in the NCBI database (accession
number: GCA_002162335.1). A putative b-glucosidase
enzyme (accession number KR185312) belonging to
the GH1 family was identified (Yuan et al. 2015). In
CONTACT Juan Gao
bio_gaoj@ujn.edu.cn
School of Biological Science and Technology, University of Jinan, Jinan, PR China
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

2
Y. YUAN ET AL.
this work, we characterize the activity of another GH1
b-glucosidase gene ccbgl1b from C. cellulans. The gene
was cloned, the recombinant enzyme was expressed,
purified and characterized, and its potential use for
the conversion of cello-oligosaccharides was evaluated.
and 37 ꢀC, until the OD600 nm reached 0.5, after which
CcBgl1B expression was induced by the addition of
IPTG (final concentration 1 mM), expression continued
at 25 ꢀC for 20 h.
Expression cell cultures were centrifuged (6000 ꢁ g,
4 ꢀC, 10 min), the cell pellet was resuspended in 30 ml
binding buffer (50 mM phosphate buffer, 100 mM NaCl,
10 mM imidazole, pH 7.4) and disrupted by sonication
(3 s each with 4-s interval, 300 W). After clarification of
the lysate by centrifugation (13,000 ꢁ g, 4 ꢀC, 45 min),
the supernatant was applied to a Ni sepharose
fastflow column (GE Healthcare, Piscataway, NJ;
diameter ¼ 1 cm, column volume ¼ 10 ml). The column
was equilibrated with five column volumes of binding
buffer (50 mM phosphate buffer, 100 mM NaCl, 20 mM
imidazole, pH 7.4). After loading the sample, the col-
umn was washed with five column volumes of binding
buffer, then eluted with five column volumes of elution
buffer (50 mM phosphate buffer, 100 mM NaCl, 100 mM
imidazole, pH 7.4). The flow rate was 1 ml/min. Aliquots
containing b-glucosidase activity were pooled and dia-
lyzed against 50 mM phosphate buffer, 10% (v/v) glycerol
and 100 mM NaCl (pH 7.4). Protein concentration was
determined using a BCA protein assay kit (Beyotime
Institute of Biotechnology, Nantong, China).
2. Material and methods
2.1 Strains and cultivation conditions
Bacterium C. cellulans strain 21 (CGMCC 7587) was cul-
tivated in Luria-Bertani (LB) agar at 30 ꢀC for 24 h.
Escherichia coli BL21 (DE3) (Novagen, Madison, WI,
USA) harbouring the recombinant plasmid were grown
at 37 ꢀC with shaking in LB broth supplemented with
kanamycin (kan) at a concentration of 50 lg/ml. All of
the chemicals and reagents used were analytical grade
unless otherwise stated.
2.2 Gene cloning and recombinant enzyme
expression
Genomic DNA from C. cellulans strain 21 was prepared
using a TIANamp Bacteria DNA kit (TIANGEN, Beijing,
China) according to its manual. The b-glucosidase
gene (ccbgl1b) was amplified with Phusion High-
Fidelity PCR Master Mix using primers 50-
GGAATTCCATATGGTGACCACCGACACCACCGCC-30 and
50- CGGGATCCTTATCAGGCGGTCTCGTCGACCGTG-30 for
gene amplification in an ABI 2700 thermal cycler using
the following program: initial denaturation at 98 ꢀC for
30 s, followed by 30 cycles of 10 s at 98 ꢀC, 45 s at
62 ꢀC and 60 s at 72 ꢀC, and final extension at 72 ꢀC for
10 min. After amplification, the PCR fragment was
digested with NdeI and BamHI, and ligated with a pET-
28a vector digested with NdeI/BamHI (Novagen, WI) to
give the recombinant plasmid pET-ccbgl1b. The liga-
tion mixture was transformed into E. coli BL21 (DE3)
cells (Invitrogen, Carlsbad, CA) by heat shock and
spread on LB (þkan) agar. After incubated at 37 ꢀC for
16 h, positive clones were screened by colony PCR
with T7 promoter (50-TAATACGACTCACTATAGGG-30)
and T7 terminator (50-GCTAGTTATTGCTCAGCGG-30)
sequences as primers and confirmed by DNA sequence
analysis. All the molecular biology enzymes used in
this process were purchased from New England
Biolabs.
2.3 Enzyme activity assay
During purification, the b-glucosidase activity against
p-nitrophenyl-b-D-glucopyranoside
(pNPbGlc)
was
assayed as follows. A 100 ll reaction mixture contain-
ing 1 mM substrate (Sigma, St. Louis, MO), and 20 ll of
b-glucosidase preparation was incubated in 50 mM
phosphate buffer (pH 6.0). After 10-min reaction at
37 ꢀC, the reaction was stopped by the addition of
NaOH (100 ll, 1 M). The concentration of p-nitrophenol
(pNP) released from pNPbGlc was determined from the
absorbance at 405 nm (Uchiyama et al. 2015; Yuan
et al. 2015). Activities against other p-nitrophenyl
glycoside substrates were assayed under the same
conditions. One unit of b-glucosidase activity is
defined as the amount of enzyme needed to release
1 lmol of pNP/min under above conditions. Activity
against disaccharides were measured in 100 ll reaction
mixture containing 1 mM substrate, 20 ll of b-glucosi-
dase preparation and 50 mM phosphate buffer (pH
6.0). The amount of reducing sugar released from the
substrate was determined using the dinitrosalicylic
acid (DNS) reagent (Miller 1959). The enzymatic activity
of CcBgl1B against carboxymethyl cellulose (CMC),
Avicel, corn stover and rice straw were assayed with
5 mg/ml of substrate by measuring the amount of
reducing sugar with DNS method.
The E. coli BL21 (DE3) cells harbouring the recom-
binant plasmid pET-ccbgl1b were first incubated in a
50-ml flask containing 10 ml of LB broth (þkan) at
37 ꢀC overnight. After that, 5 ml of the overnight cul-
tures were transferred into 200 ml LB broth (þkan).
The culture was incubated with shaking at 200 rpm

BIOCATALYSIS AND BIOTRANSFORMATION
3
The kinetic parameters Km, Vmax and kcat were deter-
mined by measuring the initial rates at various
pNPbGlc concentrations (0.05–5.0 mM) at 55 ꢀC for
15 min in phosphate buffer (50 mM, pH 6.0) and calcu-
lated from Lineweaver–Burk curve using GraphPad
Prism5 software (GraphPad Software, Inc. CA).
with a flow rate of 0.5 ml/min (Khodaei and Karboune
2016; Maier et al. 2016).
2.6 Molecular modelling
The homology model of CcBgl1B was built by SWISS-
MODEL website with default setting (www.swissmodel.
expasy.org). The template for homology model was
based on the crystal structure (PDB ID 21JIE) of b-glu-
cosidase B from Bacillus polymyxa complexed with 2-F-
Glucose (Isorna et al. 2007). The sequence identity is
40%. To dock the substrate cellobiose (G2) into the
active site of CcBgl1B, firstly 2-F-Glucose was put into
CcBgl1B active site by superposition. Then, the 2-F-
Glucose was manually changed to G2. Finally, the
binding model of CcBgl1B complexed with G2 was
optimized using an energy minimization method
under stochastic boundary condition (Zheng et al.
2016).
2.4 Characterization of the recombinant CcBgl1B
The optimum temperature and pH for CcBgl1B activity
was determined using pNPbGlc as a substrate over the
temperature and pH ranges of 20–80 ꢀC and pH
2.0–11.0 (sodium acetate buffer, pH 2.0-6.0; phosphate
buffer, pH 6.0-8.0; glycine-NaOH buffer, pH 8.0–11.0),
respectively. To estimate the thermostability over this
temperature range, the purified CcBgl1B was incu-
bated at 30–55 ꢀC for up to 60 min, samples were
taken at 5, 10, 15, 30, 45 and 60 min, and the b-gluco-
sidase activity was assayed with pNPbGlc as described
before. The pH stability of CcBgl1B was determined by
pre-incubation of purified CcBgl1B in buffers with dif-
ferent pH values (4.0–10.0) at 4 ꢀC for 24 h. Then, the
residual activities were assayed under optimum tem-
perature and pH with pNPbGlc as a substrate and nor-
malized against the activity without preincubation,
which was defined as 100%.
2.7 Nucleotide sequence accession number
The nucleotide sequence of ccbgl1b has been submit-
ted to the GenBank database with the accession num-
ber KY471392.
The effect of metal ions and chemical additives on
the activity of CcBgl1B was evaluated by incubating
purified CcBgl1B in the presence of various ions or
chemicals (5 mM) for 1 h at 25 ꢀC, then the residual
activity against pNPbGlc was determined under the
optimum conditions as described above.
The effect of glucose on the b-glucosidase activity
against pNPbGlc was defined in the presence of differ-
ent concentrations of glucose (5–1000 mM), under
optimum pH and temperature conditions.
3. Results and discussion
3.1 Protein sequence analysis
b-Glucosidase is the key enzyme for converting cello-
biose and cello-oligosaccharides into glucose, plays an
important role in the efficient degradation of cellulose
(Wilson 2009). To date, several GH1 b-glucosidases
have been purified or overexpressed in recombinant
systems (Wakarchuk et al. 1986; Love et al. 1988;
€
Grabnitz et al. 1991). In this study, an ORF of 1494 bp
was identified from C. cellulans strain 21, which enco-
des a GH1 enzyme of 497 amino acids (aa). Multiple
alignments of the deduced amino acids for CcBgl1B
with several GH1 b-glucosidase family members
(Figure 1) showed sequence identities to putative
b-glucosidases from Oerskovia enterophila (OCI29953.1,
87%), Xylanimonas cellulosilytica (WP_012879197.1,
73%), Sanguibacter suarezii (WP_066464915.1, 71%)
and Micromonospora zamorensis (SCG57771.1, 71%).It
was observed that the catalytic motifs NEPW (amino
acid 184–187) and TENG (391–394) were conserved
compared to those of other GH1 b-glucosidases. It is
likely that the two conserved glutamic acids Glu185
and Glu392 in CcBgl1B act as the catalytic acid-base
and nucleophile, respectively, as described for other
GH1 b-glucosidases (Ly and Withers 1999; Gao and
2.5 Degradation of cello-oligosaccharides by
CcBgl1B
Enzymatic hydrolysis of cellobiose (G2), cellotriose
(G3), cellotetraose (G4) and cellopentaose (G5) by
CcBgl1B was determined in 500 ll reaction systems
composed of 50 mM phosphate buffer (pH 6.0), 1 mg/
ml substrate and 0.02 mg/ml purified CcBgl1B at 30 ꢀC.
During the 2-h incubation, aliquots were withdrawn at
different time points and analysed by ion chromatog-
raphy (IC) with a DIONEX ICS-5000 þ Gradient pump, a
CarboPac PA200 (250 ꢁ 3 mm) column and a pulsed
amperometric detector. Elution was conducted in buf-
fer with a high pH in 100 mM NaOH, using a
30–100 mM sodium acetate gradient from 0 to 7 min,

4
Y. YUAN ET AL.
Figure 1. Multiple amino acid sequence alignment of GH1 enzymes: b-glucosidases CcBgl1B (WP_051027856) and CcBgl1A
(WP_034653879) from C. cellulans, O. enterophila (Oen, OCI29953.1), S. keddieii (Ske, WP_012866035) and Streptomyces sp. (Str,
1GNX). Dark shading indicates highly conserved residues in all sequences. The sequences were aligned using Clustal Omega
(http://www.ebi.ac.uk/Tools/msa/clustalo/) and the image was prepared with Genedoc. Putative catalytic residues are labelled as
ꢂ
ꢂ
for nucleophile and A as acid/base catalyst.
N

BIOCATALYSIS AND BIOTRANSFORMATION
5
Table 1. Summary of purification of recombinant CcBgl1B.
Volume
(ml)
Total
Total
Specific activity
(U/mg)
Recovery
(%)
Purification
(fold)
Purification step
protein (mg)a
activity (U)b
Crude enzyme extract
Ni sepharose fastflow column
15.0
10.0
58
21.2
34.8
23.3
0.6
1.1
100
67
1.0
1.8
aProtein was quantified by BCA protein assay kit.
bThe activity was reported as activity on pNPbGlc (1 mM).
Wakarchuk 2014; Chamoli et al. 2016). The detail pro-
tein tree is shown in Figure S1. Previously, C. cellulans
was reported to be able to effectively degrade curdlan
(Fu et al. 2015) and yeast-glucan (Tanabe and Oda
2011), endo-1,3-b-glucanase was considered to be the
key enzyme. The full enzyme was cloned and charac-
terized, which comprised a catalytic domain of the GH
family 16 and a C-terminal carbohydrate-binding mod-
ule family 13 (Tanabe and Oda 2011). Our preliminary
work found that C. cellulans strain 21 could degrade
b-1,4-linked xylo-oligosaccharides by
a multifunc-
tional b-xylosidase/a-L-arabinofuranosidase/b-glucosidase
CcXyl3A belonging to GH family 3 (Yuan et al. 2016).
Also, C. cellulans could degrade ginsenosides, only the
outer glucose linked to the b-1,2 linkage of C-3 pos-
ition in ginsenosides Rb1, Rb2, Rc and Rd was
removed. The gene ccbgl1a encoding a GH1 ginseno-
side-hydrolyzing b-glucosidase CcBgl1A was cloned
from C. cellulans strain 21 (Yuan et al. 2015). Sequence
alignment revealed that the two genes ccbgl1a and
ccbgl1b have only 37% identity; however, CcBgl1A and
CcBgl1B shared the same conserved catalytic residues
as other GH1 enzymes.
Figure 2. SDS-PAGE analysis of recombinant protein. M,
PageRuler prestained protein ladder (Thermo Scientific); 1, lys-
ate of recombinant E. coli BL21 cells before induction; 2, super-
natant of the culture lysate after IPTG induction; 3, purified
CcBgl1B after affinity chromatography.
purified CcBgl1B was determined by measuring the
activity in the pH range 2.0–11.0, the maximum activ-
ity was observed at pH 6.0 (Figure 3(a)). However,
CcBgl1B exhibited poor pH stability, losing more than
80% of its activity below pH 5.5 (Figure 3(b)). CcBgl1B
showed high levels of activity at temperatures 40-
60 ꢀC, with the highest activity observed at 55 ꢀC
(Figure 3(c)). The thermostability results showed that
the activity of CcBgl1B slightly decreased with increas-
ing temperature (<40 ꢀC), while CcBgl1B dramatically
losing its activity after a 10-min incubation at 50 ꢀC
(Figure 3(d)). In our previous study, a GH1 b-glucosi-
dase CcBgl1A was cloned from C. cellulans strain 21
(Yuan et al. 2015). CcBgl1A exhibited optimal activity
at pH 5.5, which was similar with that of CcBgl1B (pH
6.0). Similarly, both CcBgl1A and CcBgl1B exhibited
poor thermostability, which activities significantly
decreased above 40 ꢀC. However, the optimal tempera-
ture of CcBgl1A against pNPbGlc was observed at
35 ꢀC, which was much lower than that of CcBgl1B
(55 ꢀC).
3.2 Expression and purification of the
recombinant CcBgl1B
To characterize the enzymatic activity, the target gene
ccbgl1b was cloned into pET-28a, then expressed with
a His6-tag in E. coli BL21 (DE3) cells. Recombinant
CcBgl1B was expressed as soluble protein, the enzym-
atic activity reached 0.6 U/mg after induction (Table 1).
As analysed by SDS-PAGE (Figure 2), recombinant
CcBgl1B showed high purity after affinity chromatog-
raphy, with the molecular weight of approximate
57 kDa, consistent with that calculated from the amino
acid sequence. The recombinant CcBgl1B was purified
to homogeneity at 1.8-fold, with a recovery yield of
67%, the specific activity of CcBgl1B increased from
0.6 U/mg to 1.1 U/mg.
The influence of metal ions and chemical additives
(5 mM) on CcBgl1B activity was investigated. CcBgl1B
was sensitive to Fe3þ, Cu2þ, Hg2þ and SDS (Table 2).
Mn2þ and Ca2þ were reported to be activators for
some b-glucosidases (Pei et al. 2012; Zhao et al. 2013),
3.3 Biochemical properties of CcBgl1B
The biochemical properties of purified recombinant
CcBgl1B were determined by using pNPbGlc as a sub-
strate. The optimal pH for the b-glucosidase activity of

6
Y. YUAN ET AL.
Figure 3. Effects of pH and temperature on the activity and stability of CcBgl1B were determined using pNPbGlc as the substrate.
(a) Optimal pH was determined by ranging pH from 2.0 to 11.0, the maximum activity obtained was defined as 100%. (b) pH sta-
bility was determined by pre-incubation of CcBgl1B in buffers with varying pH value for 24 h and then determining the percentage
of residual activity under standard assay conditions. The activity of CcBgl1B without pre-incubating was defined as 100%. (c)
Optimal temperature was determined from 20 to 80 ꢀC in 50 mM phosphate buffer (pH 6.0), the maximum activity obtained was
defined as 100%. (d) Thermostability of b-glucosidase CcBgl1B was determined by pre-incubating the enzyme for 1 h at different
temperatures and then the residual activities were determined under the standard assay conditions. The activity of the enzyme
before incubation was defined as 100%. The relative activity represents the average of triplicate measurements.
Table 2. Effects of metal ions and chemical
effect on its activity, which suggested that CcBgl1B is
agents on the activity of CcBgl1B.
not a metalloprotein.
Metal ions or reagents (5 mM)
Relative activity (%)a
Kþ
104.5 6.3
111.4 2.9
118.1 0.7
106.5 6.4
11.1 1.4
6.8 1.5
Mg2þ
Ca2þ
Naþ
3.4 Substrate specificity assay
Cu2þ
Fe3þ
Hg2þ
Ba2þ
Mn2þ
DTT
The substrate specificity of CcBgl1B was determined
using various glycosides and oligosaccharides as sub-
strates. When using p-nitrophenyl glycosides as sub-
strates, CcBgl1B was found to have the highest activity
against pNPbGlc followed by pNPbGal, while other p-
nitrophenyl b- or a- linked glycosides were not suit-
able substrates for CcBgl1B (Table 3). Using pNPbGlc
as a substrate, the kinetic parameters of CcBgl1B were
determined, with the Km value of 0.36 mM, a Vmax
value of 4.09 U/mg, and a kcat value of 3.86 sꢃ1 using
the Michaelis–Menten equation. CcBgl1B exhibited
similar Km but lower kcat and kcat/Km value compared
to that of GH1 enzyme CcBgl1A (Km 0.53 mM, kcat
30.08 sꢃ1 and kcat/Km 57.16 sꢃ1mMꢃ1 for CcBgl1A).
b
–
122.7 8.3
130.7 4.2
110.3 1.5
107.2 7.1
1.5 0.4
EDTA
SDS
aThe activity assayed in the absence of cations or reagents
was taken as 100%. Results are presented as means SD
(n ¼ 3).
bNot detected.
and similar observations were noted for CcBgl1B, while
the activity of CcBgl1B increased to 130.7% and
118.1% in the presence of 5 mM Mn2þ and Ca2þ
,
respectively. Addition of 5 mM EDTA had no significant

BIOCATALYSIS AND BIOTRANSFORMATION
7
Table 3. Relative activity of CcBgl1B on different substratesa.
Substrateb
Relative activity (%)c
Substrate
Relative activity (%)
pNPbGlc
pNPbXyl
100 0.0
2.7 0.5
1.0 0.5
41.4 0.6
1.4 0.7
0.2 0.1
0.2 0.1
0.3 0.1
86.4 2.5
97.7 2.3
gentiobiose
0.2 0.1
100.0 0.0
20.4 2.4
34.8 2.6
24.2 1.6
cellobiose (G2)
cellotriose (G3)
cellotetraose (G4)
cellopentaose (G5)
CMC
Avicel
corn stover
rice straw
pNPbMan
pNPbGal
pNPaAraf
pNPaGlc
pNPaGal
pNPaMan
sophorose
laminaribose
d
–
–
–
–
aReactions were performed with 1 mM (p-nitrophenyl glycosides and disaccharides) or 5 mg/ml (polysaccharides)
substrate, pH 6.0, at 37 ꢀC for 10 min.
bThe relative activity on pNPbGlc and cellobiose were defined as 100%.
cThe data are reported as means SD from the mean for three independent experiments.
dNot detected.
Figure 4. IC analysis of hydrolytic products released from G2(a), G3(b), G4(c) and G5(d) by purified CcBgl1B. Reactions (500 ll) con-
taining 1 mg/ml substrate were incubated at 30 ꢀC in 50 mM phosphate buffer (pH 6.0) with 0.02 mg/ml CcBgl1B for the indicated
times.
Using disaccharides as substrates, CcBgl1B exhibited
high activity against b-1,2-, b-1,3- and b-1,4-linked glu-
cosyl disaccharides, that is, sophorose, laminaribiose
and cellobiose, with the preferred linkage in order of
b-1,4-> b-1,3->b-1,2-. However, no activity against a
b-1,6-linked glucosyl disaccharide gentiobiose was
detected. b-Glucosidases are divided into three groups
in the CAZy database: broad range b-glucosidase, cel-
lobiase and aryl-b-glucosidase (Chan et al. 2016).
According to its hydrolytic behaviour, CcBgl1B was
classified as a broad range b-glucosidase.
for biomass conversion (Gowen and Fong 2010; Gao
and Wakarchuk 2014). In this work, the hydrolytic
activities of CcBgl1B towards cello-oligosaccharide
substrates were determined using a panel of G2-G5
substrates. As expected, CcBgl1B showed high activ-
ity against tested cello-oligosaccharides G2-G5. The
IC analysis results showed that glucose was the
predominant product from cello-oligosaccharide
degradation leaving a coproduct that is one gluco-
syl residue shorter than the starting substrate
(Figure 4(a–d)). However,
a
few shorter cello-
b-1,4-linked
cello-oligosaccharides,
commonly
oligosaccharides were observed, which suggested
found in plant cell walls, are important substrates
that CcBgl1B may also act as an exo-b-glucosidase,

8
Y. YUAN ET AL.
cleaving the non-reducing terminal residue of cello-
dextrin products.
not significantly affected by glucose concentrations up
to 50 mM when using pNPbGlc as substrate.
a
However, the enzymatic activity sharply decreased
when the concentration of glucose was higher than
50 mM. In the presence of 100 mM glucose, CcBgl1B
retained 61.93% of its original activity (Figure 5),
higher than the Novozyme 188 b-glucosidase and
Neurospora crassa b-glucosidase, which retained only
26% and 27.74% of their original activities under these
conditions, respectively (Pei et al. 2016).
3.5 Effect of glucose on CcBgl1B activity
Previous studies reported that the accumulation of
glucose inhibited the enzymatic activity of b-glucosi-
dases during biorefinery hydrolysis of cellulose bio-
mass (Singhania et al. 2013). As shown in Figure 5, it
was found that the enzymatic activity of CcBgl1B was
3.6 Homology modelling and substrate docking
Based on the optimized complex as shown in Figure 6(a),
the substrate G2 fits the active site of CcBgl1B very well.
Interestingly, the favourable binding interactions (e.g.
the hydrogen bonding interaction) are majorly formed
between the first glucose of G2. No hydrogen bonding
or other favourable interaction was found between the
second glucose of G2 that is consistent with our kinetic
result that CcBgl1B shows the same catalytic efficiency
towards its promiscuous substrates G2 and pNPbGlc. In
addition, the active site of CcBgl1B seems to expose to
the solution directly, as shown in Figure 6(a). Therefore,
CcBgl1B shows comparable hydrolysis activities
between G2 and G3 (owning one more glucose
than G2).
Figure 5. Effect of glucose on the b-glucosidase activity of
CcBgl1B. b-glucosidase activity against pNPbGlc was assayed
in the presence of different glucose concentrations
(5–1000mM), under the optimum pH and temperature. The
activity in the absence of glucose was defined as 100%.
The relative activity represents the average of triplicate
measurements.
The catalytic mechanism and the catalytic residues
of b-glucosidases were studied by both biochemical
and X-ray crystal structure studies (Hassan et al. 2015).
The Ca RMSD of the catalytic residues between the
Figure 6. Model structure of the CcBgl1B with docked cellobiose (G2) substrate. (a) Three dimensional model structure of CcBgl1B
in complex with G2 created with SWISS-MODEL using the X-ray structure of B. polymyxa b-glucosidase B complexed with 2-F-
Glucose (PDB ID: IJ1E) as starting model. The protein backbone shown as a Cartoon. (b) Key interactions predicted to occur
between the catalytic residues and G2 substrate atoms. Amino acid residues are shown in stick representation; G2 are in ball and
stick model.

BIOCATALYSIS AND BIOTRANSFORMATION
9
model (e.g. E185, E392 and E450 in Figure 6(b)) and
template (e.g. E167, E356 and E409; PDB ID. 1JIE) is
only 0.3 Å. The small RMSD value of the catalytic resi-
dues suggests that the active site of the model is
accurate and indicates a similar catalytic mechanism
with the general-believed catalytic mechanism of
b-glucosidases (Hassan et al. 2015; Isorna et al. 2007).
Overall, the computational model agrees well with the
previous experimental result (Hassan et al. 2015; Isorna
et al. 2007), which suggests that the computational
insights are reasonable.
strain 21, which effectively hydrolyzed cello-oligosac-
charides to release glucose. The discovery and charac-
terization of this new b-glucosidase CcBgl1B is useful
information for the application of C. cellulans for the
enzymatic degradation of cellulose to biofuel and
other industries.
Disclosure statement
The authors report no declarations of interest.
Depicted in Figure 6(b) is the important interactions
for the catalytic activity of CcBgl1B between catalytic
residues of CcBgl1B and G2. The oxygen atom of E392
side chain aligns very well with the carbon atom of G2
with a distance of 2.94 Å and is ready for the nucleo-
philic attack. The protonated side chain of E185 is in a
good position towards the breaking bond of G2 and is
expected to function as a general acid during the nucleo-
philic attack. The E450 seems to participate the transition
state stabilization by two strong hydrogen bonds with
the first glucose of G2. Taken together, these interactions
indicate a high catalytic efficiency of CcBgl1B-catalzyed
hydrolysis of G2, which agrees well with our experimen-
tal data (as seen in Table 3 and Figure 4).
b-Glucosidases are the most common known
enzymatic activities for GH1 enzymes. However, differ-
ent members in this family exhibited different linkage
preferences. For CcBgl1A, the linkage preference for
glucosyl disaccharides was b-1, 2 > b-1, 3 ꢄ b-1, 4.
Different from CcBgl1A, the preferred linkage for
CcBgl1B was b-1,4->b-1,3-> b-1,2. The reason why dif-
ferent GH1 members exhibit different linkage preferen-
ces may be that highly conserved amino acid residues
(E392, E185 and E450) of GH1 enzymes are located at
the -1 subsite of the catalytic center, which recognize
the non-reducing glucose of the disaccharide in the
catalytic reaction. The reducing glucose of the disac-
charide is recognized by the amino acids at þ1 sub-
site. For different GH1 members, amino acids at þ1
subsites exhibit great diversification, which leads to a
variety of substrate specificities (Matsuzawa et al.
2016). A similar situation is observed in GH3 b-glucosi-
dases. The substrate recognition residues for non-reduc-
ing glucose of the substrates are highly conserved
among GH3 b-glucosidases, while the substrate recogni-
tion sites in other moieties of substrates are not con-
served (Nakajima et al. 2012; Nakajima et al. 2016).
Funding
We thank Natural Science Foundation of China (31400299)
for supporting this study.
References
Bischof RH, Ramoni J, Seiboth B. 2016. Cellulases and
beyond: the first 70 years of the enzyme producer
Trichoderma reesei. Microb Cell Fact 15:106.
Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V,
Henrissat B. 2009. The Carbohydrate-Active EnZymes data-
base (CAZy): an expert resource for glycogenomics.
Nucleic Acids Res 37:D233–D238.
Chamoli S, Kumar P, Navani NK, Verma AK. 2016. Secretory
expression, characterization and docking study of glucose-
tolerant b-glucosidase from B. subtilis. Int J Biol Macromol
85:425–433.
Chan CS, Sin LL, Chan KG, Shamsir MS, Manan FA, Sani RK,
Goh KM. 2016. Characterization of a glucose-tolerant
b-glucosidase from Anoxybacillus sp. DT3-1. Biotechnol
Biofuels 22:174.
Fu Y, Cheng L, Meng Y, Li S, Zhao X, Du Y, Yin H. 2015.
Cellulosimicrobium cellulans strain E4-5 enzymatic hydroly-
sis of curdlan for production of (1 ! 3)-linked b-D-glucan
oligosaccharides. Carbohydr Polym 134:740–744.
Gao J, Wang J, Cui J, Wang N, Bai Y, Yuan Y, Zhou Y. 2014.
Purification and characterization of two novel b-glucosi-
dases from Penicillium oxalicum and their application in
bioactive ginsenosides production. Biocatal Biotransfor
32:199–207.
Gao J, Wakarchuk W. 2014. Characterization of five
b-glycoside hydrolases from Cellulomonas fimi ATCC 484.
J Bacteriol 196:4103–4110.
€
€
Grabnitz F, Seiss M, Rucknagel KP, Staudenbauer WL. 1991.
Structure of the beta-glucosidase gene bglA of Clostridium
thermocellum. Sequence analysis reveals a superfamily of
cellulases and beta-glycosidases including human lactase/
phlorizin hydrolase. Eur J Biochem 200:301–309.
Greene ER, Himmel ME, Beckham GT, Tan Z. 2015.
Glycosylation of cellulases: engineering better enzymes for
biofuels. Adv Carbohydr Chem Biochem 72:63–112.
Gowen CM, Fong SS. 2010. Exploring biodiversity for cellu-
losic biofuel production. Chem Biodivers 7:1086–1097.
Hassan N, Nguyen TH, Intanon M, Kori LD, Patel BK, Haltrich
D, Divne C, Tan TC. 2015. Biochemical and structural char-
4. Conclusions
In conclusion, we have successfully expressed soluble
and functional b-glucosidase CcBgl1B from C. cellulans
acterization of
a
thermostable b-glucosidase from

10
Y. YUAN ET AL.
Halothermothrix orenii for galacto-oligosaccharide synthe-
sis. Appl Microbiol Biotechnol 99(4):1731–1744.
hydrolysis of cellulose for bioethanol production.
Bioresour Technol 127:500–507.
ꢀ
~
ꢀ
Isorna P, Polaina J, Latorre-Garcıa L, Canada FJ, Gonzalez B,
Sanz-Aparicio J. 2007. Crystal Structures of Paenibacillus
polymyxa beta-glucosidase
Su H, Lin J, Wang G. 2016. Metabolic engineering of
Corynebacterium crenatium for enhancing production of
higher alcohols. Sci Rep 6:39543.
B
complexes reveal the
molecular basis of substrate specificity and give new
insights into the catalytic machinery of family I glycosi-
dases . J Mol Biol 371:1204–1218.
Swangkeaw J, Vichitphan S, Butzke C, Vichitphan K. 2011.
Characterization of b-glucosidases from Hanseniaspora sp.
and Pichia anomala with potentially aroma-enhancing
Khodaei N, Karboune S. 2016. Enzymatic generation of gal-
actose-rich oligosaccharides/oligomers from potato rham-
capabilities in juice and wine. World
Biotechnol 27:423–430.
J
Microbiol
nogalacturonan
197:406–414.
I
pectic polysaccharides. Food Chem
Tanabe Y, Oda M. 2011. Molecular characterization of endo-
1,3-b-glucanase from Cellulosimicrobium cellulans: effects
of carbohydrate-binding module on enzymatic function
and stability. Biochim Biophys Acta 1814(12):1713–1719.
Uchiyama T, Yaoi K, Miyazaki K. 2015. Glucose-tolerant b-glu-
cosidase retrieved from a Kusaya gravy metagenome.
Front Microbiol 6:548.
Love DR, Fisher R, Bergquist PL. 1988. Sequence structure
and expression of a cloned beta-glucosidase gene from an
extreme thermophile. Mol Gen Genet 213:84–92.
Ly HD, Withers SG. 1999. Mutagenesis of glycosidases. Annu
Rev Biochem 68:487–522.
Maier M, Reusch D, Bruggink C, Bulau P, Wuhrer M, Mølhøj
M. 2016. Applying mini-bore HPAEC-MS/MS for the charac-
terization and quantification of Fc N-glycans from hetero-
Wakarchuk W, Kilburn D, Miller R, Warren RA. 1986. The
molecular cloning and expression of a cellobiase gene
from an Agrobacterium in Escherichia coli. Molec Gen
Genet 205:146–152.
geneously glycosylated IgGs.
J Chromatogr B Analyt
ꢀ
ꢀ ꢀ
Technol Biomed Life Sci 15:342–352.
Wierzbicka-Wos A, Bartasun P, Cieslinski H, Kur J. 2013.
Cloning and characterization of a novel cold-active glyco-
Matsuzawa T, Jo T, Uchiyama T, Manninen JA, Arakawa T,
Miyazaki K, Fushinobu S, Yaoi K. 2016. Crystal structure
and identification of a key amino acid for glucose toler-
ance, substrate specificity, and transglycosylation activity
of metagenomic b-glucosidase Td2F2. FEBS J 283(12):
2340.
side hydrolase family
b-fucosidase and b-galactosidase
Biotechnol 15:22.
Wilson DB. 2009. Cellulases and biofuels. Curr Opin
Biotechnol 20:295–299.
1
enzyme with b-glucosidase,
activities. BMC
Miller GL. 1959. Use of dinitrosalicylic acid reagent for the
determination of reducing sugar. Anal Chem 31:426–428.
Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T.
2012. Identification, cloning, and characterization of b-glu-
cosidase from Ustilago esculenta. Appl Microbiol
Biotechnol 93(5):1989–1998.
Nakajima M, Yoshida R, Miyanaga A, Abe K, Takahashi Y,
Sugimoto N, Toyoizumi H, Nakai H, Kitaoka M, Taguchi H.
2016. Functional and structural analysis of a b-glucosidase
involved in b-1,2-glucan metabolism in Listeria innocua.
PLoS One 11(2):e0148870.
Pei J, Pang Q, Zhao L, Fan S, Shi H. 2012. Thermoanaerobac-
terium thermosaccharolyticum b-glucosidase: a glucose-
tolerant enzyme with high specific activity for cellobiose.
Biotechnol Biofuels 5:31.
Pei X, Zhao J, Cai P, Sun W, Ren J, Wu Q, Zhang S, Tian C.
2016. Heterologous expression of a GH3 b-glucosidase
from Neurospora crassa in Pichia pastoris with high purity
and its application in the hydrolysis of soybean isoflavone
glycosides. Protein Expr Purif 119:75–84.
Yang S, Hua C, Yan Q, Li Y, Jiang Z. 2013. Biochemical prop-
erties of a novel glycoside hydrolase family 1 b-glucosi-
dase (PtBglu1) from Paecilomyces thermophila expressed in
Pichia pastoris. Carbohydr Polym 30:784–791.
Yuan Y, Hu Y, Hu C, Leng J, Chen H, Zhao X, Gao J, Zhou Y.
2015. Overexpression and characterization of a glycoside
hydrolase family 1 enzyme from Cellulosimicrobium cellu-
lans sp.21 and its application for minor ginsenosides pro-
duction. J Mol Catal B: Enzym 120:60–67.
Yuan Y, Hu Y, Zhang H, Leng J, Li F, Zhao X, Gao J, Zhou Y.
2016. Characterization of a recombinant multifunctional
glycosidehydrolase family 3 b-xylosidase/a-L-arabinofura-
nosidase/b-glucosidase fromCellulosimicrobium cellulans
sp. 21. J Mol Catal B: Enzym 131:67–72.
Zhao L, Xie J, Zhang X, Cao F, Pei J. 2013. Overexpression
and characterization of a glucose-tolerant b-glucosidase
from Thermotoga thermarum DSM 5069T with high cata-
lytic efficiency of ginsenoside Rb1 to Rd. J Mol Catal B:
Enzym 95:62–69.
Zheng X, Deng J, Zhang T, Yao J, Zheng F, Zhan CG. 2016.
Potential anti-obesity effects of a long-acting cocaine
hydrolase. Chem Biol Interact 259:99–103.
Zheng Z, Shetty K. 2000. Solid-state bioconversion of phe-
nolics from cranberry pomace and role of Lentinus edodes
beta-glucosidase. J Agric Food Chem 48:895–900.
Singh G, Verma AK, Kumar V. 2016. Catalytic properties, func-
tional attributes and industrial applications of b-glucosi-
dases. J Biotech 6:3–16.
Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A.
2013. Role and significance of beta-glucosidases in the