Characterization of a thermostable, specific GH10 xylanase from Caldicellulosiruptor bescii with high catalytic activity

Article abstract of DOI:10.1016/j.molcatb.2015.04.003

Xylanase (EC 3.2.1.8) is one of the most important enzymes for the biodegradation of xylan. Since many industrial processes utilizing xylanase are operated at elevated temperatures, thermostable xylanases are highly desirable. In the present study, xyn10B gene from thermophilic bacterium Caldicellulosiruptor bescii that encodes a glycoside hydrolase (GH) family 10 xylanase was overexpressed in Escherichia coli and systematically characterized. CbXyn10B exhibited optimal activity at pH 7.2 and 70 °C. It had a half-life of about 7.7 h at 60 °C, and retained over 85% of maximal activity after incubation at pH 4.0-12.0. The activity of this xylanase was not affected by most divalent cations, but inhibited by Fe³⁺ and Zn²⁺. CbXyn10B exhibited high activity on beech wood xylan, oat spelt xylan, and birch wood xylan, with specific activities of about 450 U mg^-1. Compared with other GH10 xylanases, CbXyn10B was highly specific for xylan and showed low catalytic efficiency toward sodium carboxymethyl cellulose and p-nitrophenyl-β-d-xylopyranoside. HPLC analysis of the products released from xylo-oligosaccharides and xylan revealed that xylobiose was the predominant hydrolytic product. The action mode of the enzyme was studied by product analysis, homology modeling and molecular docking to gain an insight into the structural basis for its substrate recognition mechanism.

Full text of DOI:10.1016/j.molcatb.2015.04.003

Accepted Manuscript
Title: Characterization of a thermostable, specific GH10
xylanase from Caldicellulosiruptor bescii with high catalytic
activity
Author: Jiao An Yuan Xie Yong Zhang Dongsheng Tian
Shuhao Wang Guangyu Yang Yan Feng
PII:
S1381-1177(15)00101-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.04.003
MOLCAB 3143
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
14-10-2014
17-3-2015
4-4-2015
Please cite this article as: J. An, Y. Xie, Y. Zhang, D. Tian, S. Wang, G. Yang, Y. Feng,
Characterization of a thermostable, specific GH10 xylanase from Caldicellulosiruptor
bescii with high catalytic activity, Journal of Molecular Catalysis B: Enzymatic (2015),
http://dx.doi.org/10.1016/j.molcatb.2015.04.003
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Characterization of a thermostable, specific GH10 xylanase from
Caldicellulosiruptor bescii with high catalytic activity
Jiao An 1,2, Yuan Xie1, Yong Zhang1, Dongsheng Tian1, Shuhao Wang1, Guangyu
Yang1*, Yan Feng1, 2*
1
State Key Laboratory of Microbial Metabolism, School of Life Sciences and
Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, P.R. China
2
Key Laboratory for Molecular Enzymology and Engineering of Ministry of
Education, Jilin University, Changchun 130021, P.R. China
*Corresponding author
To whom correspondence should be addressed:
Guangyu Yang, State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong
University, 800 Dongchuan Rd. Shanghai 200240, China, Tel: +86-21-34207248; Fax:
+86-21-34207189, E-mail: yanggy@sjtu.edu.cn
Yan Feng, State Key Laboratory of Microbial Metabolism, College of Life Sciences
and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai
200240, P. R. China. Tel & Fax: 86-21-34207189, E-mail: yfeng2009@sjtu.edu.cn
Abstract
Xylanase (EC 3.2.1.8) is one of the most important enzymes for the biodegradation of
xylan. Since many industrial processes utilizing xylanase are operated at elevated
temperatures, thermostable xylanases are highly desirable. In the present study,
xyn10B gene from thermophilic bacterium Caldicellulosiruptor bescii that encodes a
glycoside hydrolase (GH) family 10 xylanase was overexpressed in Escherichia coli
and systematically characterized. CbXyn10B exhibited optimal activity at pH 7.2 and
70 °C. It had a half-life of about 7.7 h at 60 °C, and retained over 85% of maximal
activity after incubation at pH 4.0-12.0. The activity of this xylanase was not affected
by most divalent cations, but inhibited by Fe3+ and Zn2+. CbXyn10B exhibited high
activity on beech wood xylan, oat spelt xylan, and birch wood xylan, with specific
activities of about 450 U mg-1. Compared with other GH10 xylanases, CbXyn10B
1
Page 1 of 29

was highly specific for xylan and showed low catalytic efficiency towards sodium
carboxymethyl cellulose and p-nitrophenyl-β-D-xylopyranoside. HPLC analysis of
the products released from xylo-oligosaccharides and xylan revealed that xylobiose
was the predominant hydrolytic product. The action mode of the enzyme was studied
by product analysis, homology modeling and molecular docking to gain an insight
into the structural basis for its substrate recognition mechanism.
Keywords
GH10 xylanase; Caldicellulosiruptor bescii; Substrate specificity; Thermostability;
Xylans; Xylooligosaccharides.
1. Introduction
Xylan, as the major constituent of plant cell walls, is the second most abundant
polysaccharide in nature and accounts for approximately one-third of all renewable
organic carbon on earth [1]. Biodegradation of xylan requires the coordinate action of
several enzymes, such as endo-β-1,4-xylanase (xylanase for short), β-D-xylosidase,
α-L-arabinofuranosidase, α-D-glucuronidase, acetylxylan esterase and feruloyl
esterases [2]. Among these, xylanases (EC 3.2.1.8) that cleave internal linkages on the
β-1,4-xylose backbone play a leading role. Xylanases are produced by many
microorganisms, including bacteria, actinomycetes, protozoa and fungi [3, 4]. These
enzymes vary in primary sequences, structure folds, substrate specificities and
catalytic mechanism [5, 6]. They have been mainly classified into glycoside hydrolase
(GH) families 5, 7, 8, 10, 11 and 43 [7]. GH10 xylanases, as one of the most well
studied family, typically also have considerable activity towards glucose-derived
substrates such as aryl-cello-oligosaccharides [8-10].
Xylanases have immense potential in various biotechnological industries,
including the food, feed, textile, waste treatment, bioethanol production and pulp
bleaching [11]. However, most known xylanases show maximal activity at
temperatures between 40 °C and 60 °C [12], whereas industrial processes favor higher
temperatures. In addition, thermostable xylanases that function at elevated
temperatures lower the likelihood of microbial contamination, increase reaction rates
and substrate solubility and simplify the downstream protein purification [13]. Thus,
2
Page 2 of 29

the requirement for thermostable xylanases is progressively increased.
Caldicellulosiruptor bescii DSM 6725 is an anaerobic thermophilic bacterium
that can efficiently utilize various types of untreated plant biomass. This strain can
grow at temperatures as high as 90 °C, which makes it the most thermophilic
cellulose-degrading organism known to date [14]. Based on its genome information,
C. bescii DSM 6725 produces three types of xylanases, including two GH10
xylanases (CbXyn10A and CbXyn10B) and a GH11 xylanase [15]. CbXyn10A is
composed of an N-terminal signal-peptide, two carbohydrate-binding domains and a
C-terminal catalytic domain, while CbXyn10B possesses only a catalytic domain. It
has recently been reported that these two GH10 xylanases are highly thermostable and
could work synergistically with other xylan-degrading enzymes from C. bescii to
hydrolyzed xylan at 65 to 80 °C [16]. To enable CbXyn10B to function under
well-defined conditions, a fine characterization of CbXyn10B appears to be desirable.
Here, the gene encoding CbXyn10B was overexpressed in Escherichia coli, and
recombinant enzyme was purified and further characterized. The mode of action of
the enzyme on xylo-oligosaccharides (XOS) was analyzed in detail. In addition,
homology modeling and molecular docking were performed to further dissect the
structural basis for CbXyn10B action manner.
2. Materials and methods
2.1. Chemicals, strains and plasmids
Xylose
(X1),
p-nitrophenyl-β-xylopyranoside
(pNPX),
p-nitrophenyl-β-glucopyranoside (pNPG), beech wood xylan, birch wood xylan and
oat spelt xylan were purchased from Sigma-Aldrich (St. Louis, USA).
Beta-1,4-D-xylooligosaccharides from xylobiose to xylohexaose (X2-X6) were
obtained from Megazyme International Ireland Ltd. The pET-28a vector was
purchased from Novagen (Darmstadt, Germany). E. coli TOP10 and E. coli
BL21-CodonPlus (DE3)-RIL strains were purchased from Invitrogen (Carlsbad, CA,
USA) and they were used for DNA manipulation and recombinant protein production,
respectively. Caldicellulosiruptor bescii DSM 6725 was obtained from Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).
3
Page 3 of 29

2.2. Multiple sequence alignment and phylogenetic analysis
Homology search was performed with BLAST program at the NCBI web server
(http://www.ncbi.nlm.nih.gov/BLAST) [17]. The phylogenetic tree was constructed
by Mega 5 software using the neighbor joining method [18]. Sequence alignments
were performed with ClustalX [19].
2.3. Cloning and plasmid construction
The CbXyn10B gene (GenBank No. ACM59337) was amplified from genomic
DNA of C. bescii DSM 6725 using the primers CbXyn10B_F
(5′-CCAGTCCCATGGAGAGCGAAGATTATTATGAAAA-3′) and CbXyn10B_R
(5′-CGACGACTCGAGAAAGTCAATTATTCTGAAAAATGCC-3′).
The
PCR
products were digested with FD NcoI and FD XhoI (Fermentas, Shenzhen, China) and
inserted into pET-28a expression vector.
2.4. Overexpression and purification of CbXyn10B
The pET28-CbXyn10B was expressed in E. coli BL21-CodonPlus (DE3)-RIL
cells. Cells were grown at 37 °C in 2YT medium supplemented with 100 μg/ml
kanamycin until the optical density at 600 nm (OD600) reached 0.6 to 0.8. Gene
expression was induced for 16 h at 24 °C by the addition of 1.0 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG). The cells were harvested and suspended in 30
mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and 30 mM imidazole, and
then disrupted by sonication. The cell lysates were heated at 60 °C for 30 min, and the
resulting precipitate was removed by centrifugation. The recombinant protein was
purified by Ni-NTA affinity chromatography (Qiagen, Hilden, Germany). Protein
concentration was measured by the Bradford method with a Bovine Serum Albumin
standard set (Fermentas, Shenzhen, China).
2.5. Determination of enzyme activities and properties
The standard assay for xylanase activity was performed at 65 °C in 40 mM pH
7.2 sodium phosphate buffer in the presence of 1.0% (w/v) xylans for 5 min. For
CMCase activity, the assay was performed in the same buffer containing 1.0% (w/v)
4
Page 4 of 29

CMC-Na for 30 min. The amount of reducing sugars released was determined with
the 3,5-dinitrosalicylic acid (DNS) reagent, using xylose or glucose as standard. After
incubation, DNS reagent was added and the samples were heated in a boiling water
bath for 5 min followed by cooling on ice. The absorbance was then measured at 540
nm. Each assay was performed in triplicate. One unit of xylanase activity is defined as
the amount of enzyme required to release one μmole of xylose/glucose reducing-sugar
equivalents per minute at 65 °C and pH 7.2. For pNPX and pNPG, the assay was
performed with a final concentration of 0.2 mM at 65 °C for 30 min and the
absorbance was measured at 405 nm. One unit of xylanase activity is defined as the
amount of enzyme required to release one μmole of p-nitrophenol per minute at 65 °C
and pH 7.2.
The effect of temperature on enzyme activities was determined by assaying the
enzymes at temperatures from 40 to 85 °C using 1% (w/v) beech wood xylan as
substrate. Thermal stability of xylanase was assessed by incubated enzyme solutions
(0.2 mg/ml) for 4 h at 60 °C, 65 °C, 70 °C and 75 °C, respectively. The relative
activities of heat-treated CbXyn10B were measured at definite time intervals.
The effects of pH on enzyme activity were determined at 60 °C under pH
ranging from 4 to 11 using 1% (w/v) beech wood xylan as substrate. The reaction
buffer contained 30 mM each of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES),
3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid
(TAPS), 3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS),
2-(N-morpholino)ethanesulfonic acid (MES) and acetic acid [20], and was adjusted to
the appropriate pH with 1 M NaOH. To exclude the possibility of thermo-inactivation
at high temperature, the pH stability of the purified enzyme was diluted 100-fold
using respective buffers having pH values ranging from 3.0 to 13.0 as described
above and were incubated for 4 h at 37 °C [21, 22]. The relative activity of the
incubated enzyme was estimated following the procedure described above.
The kinetic parameters were determined by incubating CbXyn10B in 40 mM
sodium phosphate buffer (pH 7.2) with birch wood xylan, beech wood xylan and oat
spelt xylan at concentrations ranging from 0.2 to 10 mg/ml. All constants were
determined at 65 °C to avoid the activity loss during the reaction process. The extent
of hydrolysis for each substrate was kept consistent, so that the change of the
5
Page 5 of 29

inhomogeneous substrate was minimized. Kinetic parameters Vmax(app) and Km(app)
were acquired by fitting enzymatic activities as a function of substrate concentrations
to the Michaelis-Menten equation using non-linear regression of the software Origin
8.0. The kcat(app) was obtained by using the equation kcat(app)=Vmax(app)/[E], where
[E] was the molar concentration of the enzymes.
The effects of metal ions on purified xylanase enzyme activity were determined
by incubating the enzyme with a 1, 5 or 10 mM solution of Ca2+, Mg2+, Ni2+, Zn2+,
Fe3+, Co2+, Cu2+ or EDTA for 1 h at 25 °C. Xylanase activity was measured using 1%
(w/v) beech wood xylan as substrate under the standard condition.
2.6. Reaction product analysis
To analyze the hydrolysis mode of xylo-oligosaccharides (XOS), 0.15 mg/ml
XOS were incubated with 0.06 μM recombinant xylanase and aliquot samples were
collected at different time intervals. For the end products analysis, the reactions were
carried out by incubation the XOS and xylans with 12.5 μM CbXyn10B in 40 mM
sodium phosphate buffer pH 7.2 at 65 °C for 2 h. All the reactions were terminated by
boiling for 5 min. After centrifugation, the soluble products released from the
substrates were analyzed using an Agilent 1260 HPLC apparatus (Santa Clara, CA,
USA) equipped with a Rezex RPM-monosaccharide column (Phenomenex, CA, USA)
and a refractive index detector at a flow rate of 0.3 ml/min water at 75 °C.
2.7. Homology modeling and molecular docking
The 3D structure model of CbXyn10B was generated using the Build Homology
Models (MODELER) in Discovery Studio 3.0 (DS 3.0, Accelrys Software Inc., CA,
USA). A xylanase from Paenibacillus barcinonensis (PbXyn10B, PDB ID 3EMC,
54% identity with CbXyn10B) was selected as the template. The geometry of the loop
regions was corrected using Refine Loop/MODELER. The quality of the final model
was evaluated by PROCHECK [23] and Profile-3D of DS 3.0.
The molecular docking was performed using AutoDock 4.2 software package
[24]. A grid of 60 Å ×50 Å ×50 Å with 0.375 Å spacing was calculated using
AutoGrid. Xylopentaose was chosen as the ligand for docking. The results were
ranked on the basis of predicted free binding energy. Structural figures were prepared
using PyMOL (http://www.pymol.org/).
6
Page 6 of 29

3. Results
3.1. Sequence analysis of CbXyn10B
CbXyn10B shared the highest sequence identity (96%) with a putative xylanase
from Caldicellulosiruptor kronotskyensis 2002 (GenBank No. ADQ47206), which is
uncharacterized to date. Among characterized xylanases, CbXyn10B shared the
highest sequence identity (78%) with XynA from Caldicellulosiruptor
saccharolyticus (GenBank No. AAA23059), followed by PbXyn10B from
Paenibacillus barcinonensis (GenBank No. CAA07174, 54% identity). To further
analyze the CbXyn10B sequence, phylogenetic trees generated from 21 xylanase
sequences were constructed using the neighbor joining method (Fig. 1). These
enzymes are grouped into two subfamilies, intracellular xylanases and extracellular
xylanases [25]. CbXyn10B has no signal peptide sequence and possesses two typical
conserved motifs of the intracellular xylanases: (AIE_YASL) and (RTDL__PT_EM).
Alignment of CbXyn10B and its homologues revealed that Glu139 and Glu247 were
putative catalytic residues, and residues along the binding cleft were also highly
conserved (Fig. 2).
3.2. Overexpression and purification of CbXyn10B
Recombinant CbXyn10B was abundantly expressed in E. coli. As much as 500
mg protein can be obtained from 1 L shaking flask cell culture. The recombinant
protein was approximately 40 kDa on a 15% SDS-PAGE gel, in agreement with
calculated molecular mass (40.2 kDa) of CbXyn10B (Fig. 3). After heat treatment of
the cell lysate at 60 °C for 30 min, most of the native E.coli protein was denatured and
the target protein was purified to approximately 80% purity. Such a simple
purification procedure would reduce the overall costs for industrial applications.
3.3. Biochemical characterization of CbXyn10B xylanase
The optimal temperature of CbXyn10B activity was 70 °C (Fig. 4a), with >60%
of maximum activity retained at 60-75 °C. CbXyn10B had a half-life of about 7.7 h
and 3h at 60 °C and 65 °C, respectively (Fig. 4b). It showed a half-life of about 30
7
Page 7 of 29

min at 70 °C, which was obviously lower than the previous reported data (longer than
24 h) [16]. As it is known that thermostability of a protein may be affected by its
concentration [26-29], we might use lower protein concentration (0.2 mg/ml) than that
of the previous research, which was not clearly reported [16]. At 75 °C, CbXyn10B
lost more than 90% of its activity after 5 min.
The optimal pH of CbXyn10B activity was 7.2 (Fig. 4c), with more than 50% of
maximum activity retained at pH 6.0-8.0. It also showed high pH stability: more than
85% of the maximum activity retained after incubation at pH values from 4.0 to 12.0
for 4 h (Fig. 4d).
CbXyn10B showed high activity towards various xylan substrates (Table 1). The
maximum activity (497 U/mg, 100%) was observed for beech wood xylan, followed
by oat spelt xylan (482 U/mg, 96.9%) and birch wood xylan (448 U/mg, 90.1%).
CbXyn10B showed similar apparent kinetic parameters for beech wood xylan, oat
spelt xylan and birch wood xylan, with K (app) values of 1.90 mg/ml, 1.94 mg/ml and
m
2.16 mg/ml, respectively. In contrast, CbXyn10B exhibited very low activity towards
pNPX (0.002 U/mg, 0.0004% relative to xylan) and CMC-Na (0.05 U/mg, 0.01%
relative to xylan), revealing its high specificity for xylan.
Addition of 5 mM Mg2+, Ca2+, Co2+, Ni2+, Cu2+ or Mn2+ had no obvious effect on
the activity of CbXyn10B (Table 2). However, 1 mM of Zn2+ and Fe3+ inhibited
20-40% enzymatic activity of CbXyn10B, and the effects were dramatic enhanced
(69-100% inhibition) when the concentration of the cations increased to 10 mM.
CbXyn10B displayed almost full enzyme activity in the presence of 10 mM EDTA,
indicating that cation is not necessary for the catalysis.
3.4. CbXyn10B mode of action
To gain an insight into the mode of enzyme action, xylo-oligosaccharides (XOS)
with increasing degrees of polymerization (DP) were hydrolyzed by CbXyn10B, and
the released products were analyzed by HPLC at different time intervals (Fig. 5).
CbXyn10B showed low activity towards xylotriose (X3) (Fig. 5a). Nevertheless, X3
could be slowly converted into X2 and X1 with excess of enzyme in 2h (Fig. 6a). The
hydrolysis rate was obviously enhanced as the substrate DP increased. The hydrolysis
of xylotetraose (X4) was much faster with respect to X3, as 45% of X4 was
hydrolyzed in 4 min (Fig. 7a). For xylopentaose (X5), the hydrolysis rate was even
8
Page 8 of 29

faster than X4, with about 80% of X5 hydrolyzed in 4 min. CbXyn10B hydrolyzed
xylohexaose (X6) at a similar rate to that of X5.
Analysis of the reaction products profile provides detailed information for the
hydrolytic patterns of different XOS substrates. CbXyn10B showed no activity
towards xylobiose (X2), even with excess of enzyme (12.5 μM, 0.5 mg/ml) (Fig. 6a).
X3 could be completely hydrolyzed into X2 and X1 under the same reaction
conditions. Thus, X3 was the smallest substrate for CbXyn10B. For X4, the released
product was primary in the form of X2 with a slight amount of X3 (Fig. 5b).
CbXyn10B showed preferential action mode on X5 that the released product was
predominantly X2 and X3 (Fig. 5c). The accumulated products released from X6
hydrolysis was X3, and to a lesser extent of X2 and X4 (Fig. 5d).
The ultimate hydrolytic products of all the XOS were similar to that for natural
xylans (Fig. 6). CbXyn10B could efficiently hydrolyze beech wood xylan, oat spelt
xylan and birch wood xylan (Fig. 6b) into X2 and a small amount of X1. This feature
makes CbXyn10B to be useful in the enzymatic production of X2.
3.5. Homology modeling and molecular docking
To investigate the structural basis for the action mode of CbXyn10B, we
performed a homology modeling and molecular docking study. The overall structure
model of CbXyn10B displayed a typical (α/β)8-barrel fold (Fig. 8a) [30]. The
substrate-binding cleft of CbXyn10B was located on the surface of the protein and
consisted of five subsites. The X5 ligand was well docked into the binding cleft (Fig.
8b). Conventionally, the substrate binding subsites are labeled from –3 to +2, with -n
at the non-reducing end and +n at the reducing end, while the cleavage occurs
between -1 and +1 subsites [8]. At the non-reducing end, the xylose residue at –3
subsite had no obvious interaction with the protein. The xylose residue at the –2
subsite formed hydrogen bonds with side chains of Glu50, Asn51 and Trp297 (Fig.
8c). And the xylose moiety at the –1 subsite formed hydrogen bonds with side chains
of Lys54, His87, Asn138, Glu139, Gln215 and Glu247. On the other hand, the xylose
residues at the non-reducing end mainly stacked against aromatic residues: the xylose
moieties at +1 and +2 subsites interacted with Tyr184 and Phe255, respectively.
9
Page 9 of 29

4. Discussion
In this study, CbXyn10B was found to be a highly stable xylanase. Compared
with its close homologues, it exhibited very high catalytic activity for xylans and
showed high substrate specificity (Table 3). The catalytic activities of CbXyn10B
towards beech wood xylan and birch wood xylan were approximately 8- to 11-fold
higher than iXylC from C. laeviribosi HY-21 [31] and 2-fold higher than PbXyn10B
from P. barcinonensis BP-23 [25]. The catalytic activity of CbXyn10B towards oat
spelt xylan was approximately 74-fold higher than XynA from C. saccharolyticus
[32], and was 5.5- to 8-fold higher than iXylC and XynBE18 from Paenibacillus sp.,
respectively [33]. Interestingly, CbXyn10B showed extremely low activity (about
0.002 U/mg) toward pNP-xylopyranoside (pNPX), which was approximately 143-fold
and 28,900-fold lower than that of PbXyn10B and iXylC, respectively. In addition,
the specific activity of CMC-Na was only 0.05 U/mg, which was 240-fold lower than
that of TmxB from Thermotoga maritima MSB8 [34].
Unlike some xylanases [35, 36], CbXyn10B was insensitive to a variety of
divalent ions (Table 2). EDTA also did not affect CbXyn10B activity, suggesting that
no metal is essential for this enzyme. This is also different from some xylanases that
require Mg2+ and Ca2+. For example, addition of MgCl2 to a GH10 xylanase from
Bacillus sp. NG-27 (BSX; PDB code 2F8Q) greatly enhanced its activity [37]. A Ca2+
ion was found in the Pseudomonas fluorescens sp. xylanase (PFX; PDB code 1CLX)
to protect it from proteinase inactivation and thermal unfolding [38].
As commonly observed in GH10 xylanases, the catalytic efficiency of
CbXyn10B was improved with increasing the polymerization degree of XOS. The
activities towards various XOS had the following order: X6=X5>X4>X3>>X2,
implying there might be five xylose-binding subsites in CbXyn10B active site (Fig.
8b).
The hydrolytic pattern analysis allows a more detailed understanding of the
substrate recognition mechanism. CbXyn10B was completely ineffective on X2
substrate, which implied that X2 nonproductively bound at the -2 and -1 subsite
instead of the productive -1 and +1 subsites. This is consistent with our docking result
that xylose moieties formed much stronger interactions with the residues at the -2 and
10
Page 10 of 29

-1 subsites compared with other subsites (Fig. 8c). CbXyn10B exhibited low activity
towards X3, suggesting that only a small number of X3 molecules could form
productive complex. It is possible most X3 molecules may be trapped in the region
from –3 and –1 subsites and form a nonproductive complex (Fig. 7b).
X4 was cleaved primarily into X2, indicating X4 preferred to bind from -2 to +2
subsites of CbXyn10B. Meanwhile, a small amount of X1 and X3 also formed,
suggesting a minor amount of X4 also bound at -3 to +1 subsites. Molecular docking
result revealed that the xylose residue at +2 subsite stacked against the phenyl group
of Phe255 and formed hydrogen bonds with side chain of Asn185. In contrast, no
obvious interaction was observed at –3 subsite. The tightly binding of subsite +2 over
-3 may explain why the binding at -2 to +2 subsites was favored.
X5 had preferential mode of cleavage that released X2 and X3, suggesting a
predominant binding position from -3 to +2 subsites (Fig. 7b). This was consistent
with the molecular docking result that the carbohydrate chain of X5 lined well in the
binding cleft of CbXyn10B. The docked complex didn’t show any direct
xylose-enzyme interaction at -3 subsite. However, the faster hydrolysis rate of X5
compared to X4 suggested a catalytically important role of this subsite. We speculated
that residues at the -3 subsite possibly indirectly interact with xylose moiety via water
molecules. This kind of water-mediated interaction was found in a xylanase from G.
stearothermophilus T-6 [39].
The majority of accumulated product for X6 hydrolysis was X3. This reveals that
the major binding mode of the substrate is from -3 to +2 subsites with a free xylose
moiety at the reducing end (Fig. 7b). Meanwhile, a smaller amount of X2 and X4 also
formed, suggesting a minor binding mode that with a free xylose moiety at the
non-reducing end.
CbXyn10B can efficiently hydrolyze various xylans into X2, which has been
proven to have potential to improve health by maintaining a balanced intestinal
microflora [40]. The enzymatic production of X2 is highly advantageous compared
with chemical methods because of energy conservation, environment-friendly and less
byproducts [41, 42]. Moreover, CbXyn10B functions efficiently at elevated
temperatures, which in turn reduces the risk of microbial contamination, improves
reaction rates, and increases substrate solubility, which highlights its potential in the
11
Page 11 of 29

large-scale production of X2.
5. Conclusion
In this study, xyn10B gene from C. bescii sp. nov. DSM 6725 was overexpressed
in E. coli and systematically characterized. The recombinant xylanase has high
activity for xylans and high substrate specificity. It shows high thermostability and a
wide range of pH stability. No metal ion is required for its catalysis and
thermostability. The enzyme hydrolyzes various xylans into xylobiose, which
highlights its potential in the large-scale production of xylobiose. The action mode of
the enzyme was studied by product analysis, homology modeling and molecular
docking, which provide an insight into the structural basis for its substrate
recognition.
Abbreviations
pNPX: p-nitrophenyl-β-D-xylopyranoside; GH: glycoside hydrolase; CMC-Na:
Sodium carboxymethyl cellulose; XOS: xylo-oligosaccharides; X1-X6: xylose,
xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose.
Acknowledgements
This work was supported by the National High Technology Research and
Development Program of China (863 Program), the National Basic Research Program
of China (973 Program), Natural Science Foundation of China (31470788), and
Research Fund for the Doctoral Program of Higher Education of China (No.
20110073120062).
References
[1] R.A. Prade, Biotech. Genet. Eng. Rev. 13 (1995) 100-131.
[2] R. Chávez, P. Bull, J. Eyzaguirre, J. Biotechnol. 123 (2006) 413-433.
[3] S. Ahmed, S. Riaz, A. Jamil, Appl. Microbiol. Biotechnol. 84 (2009) 19-35.
12
Page 12 of 29

[4] D. Shallom, Y. Shoham, Curr. Opin. Microbiol. 6 (2003) 219-228.
[5] K.K. Wong, L.U. Tan, J.N. Saddler, Microbiol. Rev. 52 (1988) 305-317.
[6] C.S. Rye, S.G. Withers, Curr. Opin. Chem. Biol. 4 (2000) 573-580.
[7] T. Collins, C. Gerday, G. Feller, FEMS Microbiol. Rev. 29 (2005) 3-23.
[8] A. Pollet, J.A. Delcour, C.M. Courtin, Crit. Rev. Biotechnol. 30 (2010) 176-191.
[9] S.R. Andrews, S.J. Charnock, J.H. Lakey, G.J. Davies, M. Claeyssens, W.
Nerinckx, M. Underwood, M.L. Sinnott, R.A. Warren, H.J. Gilbert, J. Biol. Chem.
275 (2000) 23027-23033.
[10]V. Ducros, S.J. Charnock, U. Derewenda, Z.S. Derewenda, Z. Dauter, C. Dupont,
F. Shareck, R. Morosoli, D. Kluepfel, G.J. Davies, J. Biol. Chem. 275 (2000)
23020-23026.
[11]Q.K. Beg, M. Kapoor, L. Mahajan, G.S. Hoondal, Appl. Microbiol. Biotechnol.
56 (2001) 326-338.
[12]S. Subramaniyan, P. Prema, Crit. Rev. Biotechnol. 22 (2002) 33-64.
[13]D. Verma, T. Satyanarayana, Bioresour. Technol. 117 (2012) 360-367.
[14]S.J. Yang, I. Kataeva, S.D. Hamilton-Brehm, N.L. Engle, T.J. Tschaplinski, C.
Doeppke, M. Davis, J. Westpheling, M.W. Adams, Appl. Environ. Microbiol. 75
(2009) 4762-4769.
[15]I.A. Kataeva, S.J. Yang, P. Dam, F.L. Poole II, Y. Yin, F. Zhou, W.C. Chou, Y.
Xu, L. Goodwin, D.R. Sims, J.C. Detter, L.J. Hauser, J. Westpheling, M.W.
Adams, J. Bacteriol. 191 (2009) 3760-3761.
[16]X. Su, Y. Han, D. Dodd, Y.H. Moon, S. Yoshida, R.I. Mackie, I.K. Cann, Appl.
Environ. Microbiol. 79 (2013) 1481-1490.
[17]S.F. Altschul, T.L. Madden, A.A. Schäffer, J. Zhang, Z. Zhang, W. Miller, D.J.
Lipman, Nucleic Acids Res. 25 (1997) 3389-3402.
[18]K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S. Kumar, Mol. Biol.
Evol. 28 (2011) 2731-2739.
[19]M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H.
McWilliam, F. Valentin, I.M. Wallace, A. Wilm, R Lopez, J.D. Thompson, T.J.
Gibson, D.G. Higgins, Bioinformatics 23 (2007) 2947-2948.
[20]T. Juhász, Z. Szeltner, L. Polgár, FEBS Lett. 580 (2006) 3493-3497.
[21]K. Ratanakhanokchai, K.L. Kyu, M. Tanticharoen, Appl. Environ. Microbiol. 65
(1999) 694-697.
13
Page 13 of 29

[22]S. Wu, B. Liu, X. Zhang, Appl. Microbiol. Biotechnol. 72 (2006) 1210-1216.
[23]R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, J. Appl.
Crystallogr. 26 (1993) 283-291.
[24]G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J.
Olson, J. Comput. Chem. 19 (1998) 1639-1662.
[25]O. Gallardo, P. Diaz, F.I. Pastor, Appl. Microbiol. Biotechnol. 61 (2003)
226-233.
[26]S. Iametti, S. Cairoli, B. De Gregori, F. Bonomi, J. Agric. Food Chem. 43 (1995)
53-58.
[27]V. Consalvi, R. Chiaraluce, L. Politi, A. Pasquo, M. De Rosa, R. Scandurra,
Biochim. Biophys. Acta. 1202 (1993) 207-215.
[28]B.I. Kurganov, B.A. Kornilaev, N.A. Chebotareva, V.P. Malikov, V.N. Orlov,
A.E. Lyubarev, N.B. Livanova, Biochemistry 39 (2000) 13144-13152.
[29]M.L. Galisteo, P.L. Mateo, J.M. Sanchez-Ruiz, Biochemistry 30 (1991)
2061-2066.
[30]G. Davies, B. Henrissat, Structure 3 (1995) 853-859.
[31]Y. Kim do, M.K. Han, H.W. Oh, K.S. Bae, T.S. Jeong, S.U. Kim, D.H. Shin, I.H.
Kim, Y.H. Rhee, K.H. Son, H.Y. Park, Bioresour. Technol. 101 (2010)
8814-8821.
[32]E. Lüthi, N.B. Jasmat, P.L. Bergquist, Appl. Environ. Microbiol. 56 (1990)
2677-2683.
[33]P. Shi, J. Tian, T. Yuan, X. Liu, H. Huang, Y. Bai, P. Yang, X. Chen, N. Wu, B.
Yao, Appl. Environ. Microbiol. 76 (2010) 3620-3624.
[34]Z.Q. Jiang, W. Denga, Y.P. Zhua, L.T. Li, Y.J. Sheng, K. Hayashi, Journal of
Molecular Catalysis B: Enzymatic 27 (2003) 207-213.
[35]S. Wu, B. Liu, X. Zhang, Appl. Microbiol. Biotechnol. 72 (2006) 1210-1216.
[36]A. Khasin, I. Alchanati, Y. Shoham, Appl. Environ. Microbiol. 59 (1993)
1725-1730.
[37]K. Manikandan, A. Bhardwaj, N. Gupta, N.K. Lokanath, A. Ghosh, V.S. Reddy,
S. Ramakumar, Protein Sci. 15 (2006) 1951-1960.
[38]T.D. Spurway, C. Morland, A. Cooper, I. Sumner, G.P. Hazlewood, A.G.
O'Donnell, R.W. Pickersgill, H.J. Gilbert, J. Biol. Chem. 272 (1997)
17523-17530.
14
Page 14 of 29

[39]G. Zolotnitsky, U. Cogan, N. Adir, V. Solomon, G. Shoham, Y. Shoham, Proc.
Natl. Acad. Sci. U S A 101 (2004) 11275-11280.
[40]A.A. Aachary, S.G. Prapulla, Comprehensive Reviews in Food Science and Food
Safety 10 (2011) 2-16.
[41]D. Chapla, P. Pandit, A. Shah, Bioresour. Technol. 115 (2012) 215-221.
[42]H. Yang, K. Wang, X. Song, F. Xu, Bioresour. Technol. 102 (2011) 7171-7176.
Figure captions
Fig. 1. Neighbor-Joining tree of 21 xylanases sequences. Numbers on nodes
correspond to percentage bootstrap values for 1000 replicates.
Fig. 2. Multiple sequence alignment of CbXyn10B with its homologues. The two
catalytic glutamate residues of CbXyn10B are marked with black asterisks. The
residues along the binding cleft are marked by arrows. The motifs a (AIE_YASL) and
b (RTDL__PT_EM) of signal peptide-less xylanases are highlighted. C. bescii DSM
6725: GenBank No. ACM59337; C. Saccharolyticus: GenBank No. AAA23059; P.
barcinonensis BP-23: GenBank No. CAA07174; Paenibacillus sp. E18: GenBank No.
ACY69972; C. laeviribosi HY-21: GenBank No. ADE08352; Alicyclobacillus sp. A4:
GenBank No. ACY69980; B. halodurans S7: GenBank No. Q17TM8; Bacillus sp.
NG-27: GenBank No. AAB70918.
Fig. 3. SDS-PAGE analysis of purified recombinant CbXyn10B. Lane M protein
marker; Lane 1 supernatant of crude enzyme incubated at 60 °C for 30 min; Lane 2
CbXyn10B purified by Ni-NTA affinity chromatography.
Fig. 4. Enzymatic properties of recombinant CbXyn10B. (a) Temperature effect on
CbXyn10B activity; (b) pH effect on CbXyn10B activity at defined pH ranging from
4 to 11; (c) Thermal stability of CbXyn10B at 60 °C, 65 °C and 70 °C; (d) pH
stability of CbXyn10B.
Fig. 5. HPLC analysis of hydrolytic products released from X3 (a), X4 (b), X5(c), and
X6 (d). Reactions (300 μl) containing 0.15 mg/ml substrate were incubated at 65 °C in
40 mM sodium phosphate buffer pH 7.2 with 0.06 μM CbXyn10B for the indicated
15
Page 15 of 29

times.
Fig. 6. HPLC analysis of the ultimate products released from XOS (a) and xylan (b)
hydrolysis by 12.5 μM CbXyn10B at 65 °C in 40 mM sodium phosphate buffer (pH
7.2) for 2 h.
Fig. 7. Activity of CbXyn10B against XOS and the mode action of enzyme. (a) XOS
(X3-X6) consumption during the reaction process. The initial amount of XOS was
taken as 100%. (b) Suggested binding form of XOS in the enzyme-substrate
complexes. XOS shown are: X3 (■), X4 (●), X5 (▲), and X6 (◆).
Fig. 8. Molecular modeling and substrate docking of CbXyn10B with X5. (a) The
overall structure of the modeled CbXyn10B. The putative catalytic residues are shown
as sticks. (b) Solvent-accessible surface representing the substrate-binding cleft of
CbXyn10B. The binding subsites (–3 to +2) are labelled and the substrate X5 is
shown by stick and ball. (c) The CbXyn10B-X5 complex structure. The hydrogen
bonding network was shown in dashed line.
16
Page 16 of 29

Table 1 Substrate specificity of recombinant CbXyn10B
Specific activity
(IU mg−1)a
497b
Km(app)f
(mg ml-1)
1.90
kcat(app)f
(s-1)
378.4
355.8
321.6
ND
kcat/Km(app)f
(ml mg-1 s-1)
199.2
Substrate
Beech wood xylan
Oat spelt xylan
Birch wood xylan
CMC-Na (low viscosity)
pNPX
482b
1.94
183.4
148.8
ND
ND
448b
2.16
0.05c
NDg
0.002d
ND
ND
ND
ND
e
pNPG
-
ND
a
One international unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol reducing sugar (for xylans and
CMC-Na) or p-nitrophenol (for pNPX and pNPG) min-1 under standard assay conditions.
b The data were obtained using 0.03 μM CbXyn10B with 1% xylan for 5 min at 65 °C and pH 7.2.
c The data were obtained using 12.5 μM CbXyn10B with 1% CMC for 30 min at 65 °C and pH 7.2.
d The data were obtained using 12.5 μM CbXyn10B with 0.2 mM pNPX for 30 min at 65 °C and pH 7.2.
e Not detectable.
f The (app) designations are used because the inhomogeneous substrates changes during the reaction, so the actual value observed depends upon
the source and batch of xylan used, as well as the extent of reaction followed. These parameters were kept consistent in all studies described
here.
g Not detected.
17
Page 17 of 29

Table 2 Effect of metal ions on CbXyn10B activity
Relative activity (%)
1 mM
5 mM
10mM
Control
MgCl2
CaCl2
CoCl2
NiCl2
CuCl2
MnCl2
ZnCl2
FeCl3
100.0
100.0
100.0
100±1.9
102±0.9
99±0.3
96±0.7
100±0.9
94±3.0
88±0.5
65±0.2
98±1.3
101±0.7 97±0.6
100±0.3 101±0.9
98±0.4 76±1.8
88±1.0 82±0.3
93±1.8 88±1.5
81±1.5 68±0.6
52±1.0 31±0.9
41±0.7
98±2.1 97±1.2
0
EDTA
18
Page 18 of 29

Table 3 Comparison of enzymatic properties and specific activities of CbXyn10B and its homologues
Specific activity (IU mg−1)
Optimum
Optimum
Enzyme
Source
temperature pH
(°C)
Beech wood Birch wood Oat spelt
References
pNPX
pNPG2
xylan
xylan
xylan
a
2×10-3
NA
CbXyn10B C. bescii DSM 6725
XynA C. saccharolyticus
PbXyn10B P. barcinonensis BP-23
497
448
NA
482
6.5
-
70-72
70
40
7.2
5.5-6
5.5
this study
[32]
[25]
[33]
[31]
NAb
NA
249×10-3
NA
43.6
233×10-3
61.6
313×10-3 286×10-3 42×10-3
XynBE18 Paenibacillus sp. E18
59.6
88.6
NA
NA
57.8
NA
NA
140.5
NA
50
50
7.5-9
7.5
iXylC
BSX
C. laeviribosi HY-21
Bacillus sp. NG-27
52.4
NA
NA
70
8.4
[37]
a Not detected.
b Not available.
19
Page 19 of 29

Highlights
 A xylanase gene (xyn10B) from the thermophilic bacterium Caldicellulosiruptor bescii was cloned, overexpressed and the recombinant
enzyme CbXyn10B was systematically charaterized.
 CbXyn10B exhibited high xylanase activity and thermo-/pH stability.
 CbXyn10B displayed high substrate specificity and released high levels of physiologically important xylooligosaccharides from xylans.
20
Page 20 of 29

*Graphical Abstract (for review)
Page 21 of 29

Figure 1
Page 22 of 29

Figure 2
Page 23 of 29

Figure 3
Page 24 of 29

Figure 4
Page 25 of 29

Figure 5
Page 26 of 29

Figure 6
Page 27 of 29

Figure 7
Page 28 of 29

Figure 8
Page 29 of 29