A novel trifunctional, family GH10

enzyme from Acidothermus cellulolyticus

11B, exhibiting endo-xylanase,
arabinofuranosidase and acetyl xylan
esterase activities
Saher Shahid, Razia Tajwar &
Muhammad Waheed Akhtar

Extremophiles
Microbial Life Under Extreme
Conditions

ISSN 1431-0651
Volume 22
Number 1

Extremophiles (2018) 22:109-119

DOI 10.1007/s00792-017-0981-8

1 23
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 Author's personal copy
Extremophiles (2018) 22:109–119
https://doi.org/10.1007/s00792-017-0981-8

ORIGINAL PAPER

A novel trifunctional, family GH10 enzyme from Acidothermus

cellulolyticus 11B, exhibiting endo‑xylanase, arabinofuranosidase
and acetyl xylan esterase activities
Saher Shahid1 · Razia Tajwar1 · Muhammad Waheed Akhtar1

Received: 2 May 2017 / Accepted: 14 November 2017 / Published online: 23 November 2017
© Springer Japan KK, part of Springer Nature 2017

Abstract
A novel, family GH10 enzyme, Xyn10B from Acidothermus cellulolyticus 11B was cloned and expressed in Escherichia
coli. This enzyme was purified to homogeneity by binding to regenerated amorphous cellulose. It had higher binding on
Avicel as compared to insoluble xylan due to the presence of cellulose-binding domains, CBM3 and CBM2. This enzyme
was optimally active at 70 °C and pH 6.0. It was stable up to 70 °C while the CD spectroscopy analysis showed thermal
unfolding at 80 °C. Xyn10B was found to be a trifunctional enzyme having endo-xylanase, arabinofuranosidase and acetyl
xylan esterase activities. Its activities against beechwood xylan, p-Nitrophenyl arabinofuranoside and p-Nitrophenyl acetate
were found to be 126,480, 10,350 and 17,250 U μmol−1, respectively. Xyn10B was highly active producing xylobiose and
xylose as the major end products, as well as debranching the substrates by removing arabinose and acetyl side chains. Due to
its specific characteristics, this enzyme seems to be of importance for industrial applications such as pretreatment of poultry
cereals, bio-bleaching of wood pulp and degradation of plant biomass.

Keywords Endo-xylanase · Acidothermus cellulolyticus 11B · Arabinofuranosidase · Acetyl xylan esterase · Trifunctional

enzyme

Introduction methyl-glucopyranosyl residues. These side groups hinder

Lignocellulosic materials including grasses, rice straw,
wheat straw, hardwood, softwood, bagasse, etc., are abun-
dantly available as renewable energy resources (Anwar et al.
2014). Hemicellulose contains a linear backbone of xylose
residues linked via β-(1 → 4) linkages. These linkages can
be efficiently hydrolyzed by endo-xylanases and/or exo-
xylanases (Subramaniyan and Prema 2002). However, the
xylan backbone is substituted with various side chain groups
including acetyl, α-l-arabinofuranosyl, d-galactosyl and
Communicated by S. Albers.
* Muhammad Waheed Akhtar
mwa.sbs@pu.edu.pk
Saher Shahid
saher.sbs@pu.edu.pk
Razia Tajwar
razia.sbs@pu.edu.pk
1
School of Biological Science, University of the Punjab,
Lahore 54590, Pakistan
the activity of endo-xylanases by decreasing the accessibil-
ity of xylan backbone to the xylanases (Thomson 1993).
The angiosperm xylan is usually substituted with 50–70% of
acetyl groups that cause hindrance in plant biomass degrada-
tion by the enzymes (Timell 1967). These acetyl groups can
be removed by the acetyl xylan esterases thereby improv-
ing the accessibility of xylan to the endo-xylanases. The
improvement of xylan hydrolysis by the addition of acetyl
xylan esterases has also been reported to increase cellu-
lose hydrolysis by improving the cellulose accessibility for
cellulose-degrading enzymes (Zhang et al. 2011b). In addi-
tion to the acetyl groups, the xylan backbone also contains
α-l-arabinofuranosyl units at the ­O2 and/or ­O3 positions
that restrict the hydrolysis of xylan. Arabinoxylan is one of
the major components of the cereal grains used in poultry
and feedstocks (Kambourova et al. 2007). Arabinofuranosi-
dases have gained great importance in recent years because
of their ability to remove the arabinose side chains from
xylan, thereby improving xylan hydrolysis (Margolles and
Clara 2003).

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110
The synergistic effect of xylanases, esterases and arabino-
furanosidases has been reported to improve hemicellulosic
degradation for industrial purposes (Lei et al. 2016). Using
a mixture of enzymes each with a single type of activity
would be uneconomical as compared to the use of multifunc-
tional enzymes having the required activities in one protein
(Himmel et al. 2010). This study describes the first family
10 enzyme from Acidothermus cellulolyticus 11B having
endo-xylanase, esterase and arabinofuranosidase activities
in one protein.
Acidothermus cellulolyticus 11B is a thermophilic bacte-
rium that produces various industrially significant enzymes.
It was isolated from acidic hot springs in Yellowstone
National Park and its genome has been reported to contain
43 genes encoding glycoside hydrolases, two of which are
xylanases (Barabote et al. 2009). Previously, a xylanase
Xyn10A from A. cellulolyticus 11B has been purified and
characterized (Barabote et al. 2010). This study reports the
purification and characterization of the second xylanase
(Xyn10B) from A. cellulolyticus 11B, having endo-xylanase,
esterase and arabinofuranosidase activities.
Materials and methods
Bacterial strains and vectors
The genomic DNA of A. cellulolyticus 11B provided by Prof.
D. B. Wilson (Cornell University, Ithaca, NY, USA) was
used to amplify the xylanase gene. E. coli DH5α strain was
used as a host for cloning while E. coli BL21 Codon Plus
(DE3) RIPL strain was used as a host for expression. The
gene of interest was ligated in the pET22b(+) vector (Nova-
gen, Madison, USA) to construct the recombinant plasmids.
Cloning and expression
Sequence of xylanase 10B (Acel_0180) was obtained from
GenBank accession no. ABK51955.1. Protein sequence
of the enzyme was analyzed using BLAST software at the
National Center for Biotechnology Information (NCBI). The
selected homologous sequences were further analyzed by
pairwise alignment on COBALT server on NCBI to iden-
tify the domains of Xyn10B that find homology with the
sequences of other enzymes. The nucleotide sequence was
analyzed for the presence of signal peptide via Signal P. 4.1
server (Petersen et al. 2011). The forward (GCAT​ATG​ACC​
TTGA ​ AAC​ AGG​ GGG ​ CG)​ and reverse primers (CAAGC ​ TT​
TCA​TCA​GGA​GGT​GGT​GCA​G) were designed by using
the NEBcutter (Vincze et al. 2003), Primer 3.0 (Rozen and
Skaletsky 2000) and Oligocalc (Kibbe 2007) softwares
as described previously (Shahid et al. 2015). The forward
primer contained NdeI, while the reverse primer contained
13
Extremophiles (2018) 22:109–119
HindIII restriction site. The gene sequence was amplified
using Taq polymerase and the resulting PCR products were
purified using gel DNA recovery kit (Vivantis Technolo-
gies, Malaysia) according to the recommended protocol. The
purified PCR products were ligated into pET22b(+) vector
followed by the transformation of E. coli DH5α cells with
the ligated products. The positive construct was confirmed
with restrcition analysis and DNA sequencing of the plas-
mid extracted from positive colony. The competent cells
of E. coli BL21 CodonPlus (RIPL) were transformed with
the confirmed purified recombinant plasmid. The trans-
formed cells were grown at 37 °C in 400 ml TB medium
until an ­OD600 of 0.7–0.8 was obtained. These cells were
then induced with 0.5 mM IPTG and incubated at 16 °C
overnight; cells were collected by centrifugation for further
processing.
Purification of Xyn10B
The harvested cells were resuspended in 0.05 M phosphate
buffer (pH 6.0) and sonicated using UP400S ultraschall pro-
cessor (Dr. Hielscher GmbH, Teltow, Germany). The cells
lysed by sonication were then centrifuged for 15 min at
8000 rpm. Soluble fractions of Xyn10B obtained after soni-
cation were mixed with regenerated amorphous cellulose
(RAC) slurry (Hong et al. 2008). Adsorption was allowed
for 15 min at room temperature followed by centrifugation
at 8000 rpm for 10 min. The RAC pellet was washed several
times using 0.05 M potassium phosphate buffer (pH 6.0) to
remove all unadsorbed proteins. Finally, the RAC pellet was
mixed with 4 volumes of 100% glycerol followed by centrif-
ugation. Glycerol was then removed from purified protein by
dialysis and the diluted sample was concentrated by Corning
Spin-X UF concentrator. Protein samples were analyzed on
12% SDS-PAGE by the method of Laemmli (1970). Expres-
sion levels of recombinant proteins were analyzed by densi-
tometric analysis via Syngene Gene Tool Software.
Enzyme assays
Enzyme assays were performed by mixing 0.5 ml appropri-
ately diluted enzyme samples with an equal volume of 1%
solution of beechwood xylan (Sigma-Aldrich, Germany) in
0.05 M phosphate buffer (pH 6.0) at 70 °C for 10 min in
a shaking water bath. The resulting reducing sugars were
determined by the DNS method (Ghose 1987). Activities
were also determined against insoluble oat spelt xylan,
solubilized birchwood xylan, Avicel, carboxy methylcellu-
lose (CMC), p-Nitrophenyl acetate (pNPA), p-Nitrophenyl
glucopyranoside (pNPGlu), p-Nitrophenyl galactopyra-
noside (pNPGal) (all from Sigma-Aldrich, Germany) and
p-Nitrophenyl-α- l-arabinofuranoside (pNPAra) (Mega-
zyme International, Ireland). The final concentration of the
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Extremophiles (2018) 22:109–119 111
insoluble oat spelt xylan, solubilized birchwood, CMC and
Avicel were 1%. pNPAra was used at the final concentration
of 50 mM, while the final concentrations of pNPA, pNPGlu
and pNPGal were 1 mM. Solubilized xylan was prepared by
alkali treatment (Kamble and Jadhav 2012), while insoluble
oat spelt was prepared as described previously (Zhang et al.
2011a). For the enzyme assays using p-Nitrophenol (pNP) as
substrates, the reactions were stopped by the addition of 1 M
­Na2CO3 and the absorbance was measured at 410 nm. One
unit of enzyme activity against xylan substrates, CMC and
Avicel was defined as the amount of enzyme that released
1 µmol of reducing sugars per minute under assay condi-
tions, whereas enzyme activity against pNPA, pNPGlu,
pNPGal and pNPAra was defined as the amount of enzyme
that released 1 µmol of nitrophenol per minute under the
assay conditions (Wongratpanya et al. 2015).
Enzyme characterization
Enzyme assays were performed in 0.05 M sodium acetate
buffer (pH 5.0), potassium phosphate buffers (pH 6.0–7.0)
and Tris-Cl buffer (pH 8.0). The effect of temperature on
activity was determined by performing the enzyme assays at
temperatures between 50 and 100 °C using beechwood xylan
as a substrate. Optimum pH for activity of the enzyme was
determined by suitably diluting the enzyme samples in the
above described buffers. The substrate was also dissolved in
the corresponding buffer.
Activities on beechwood xylan, oat spelt xylan, birch-
wood xylan, CMC and Avicel were performed at 70 °C in
0.05 M potassium phosphate buffer (pH 6.0). Km and Vmax
values were calculated by determining the rates of reac-
tion using the amounts of beechwood xylan between 1 and
18 mg ml−1 in phosphate buffer (pH 6.0) at 70 °C.
Temperature stability was determined by incubating the
enzyme at various temperatures (between 20 and 80 °C)
for 30 min, followed by assaying the residual activities as
described above. pH stability was monitored by incubat-
ing the purified enzyme in the buffers of various pH val-
ues (5.0–8.0) for 30 min, followed by enzyme assays as
described above.
Circular dichroism analysis
Circular dichroism (CD) spectrum of the purified Xyn10B
was obtained at different temperatures using Chirascan Plus
CD spectrophotometer (Applied Biophysics) equipped with
a Peltier thermal-controlled cuvette holder. Prior to obtain-
ing the spectra, the instrument was calibrated with an aque-
ous solution of IS-(+)-10-camphosulfonic acid. The solution
containing 0.20 mg enzyme/ml dissolved in 10 mM potas-
sium phosphate buffer (pH 6.0) was scanned after incubation
for 30 min at 20, 30, 40, 50, 60, 70 and 80 °C at a bandwidth
of 1 nm. Two consecutive scans were averaged to obtain the
spectrum for each wavelength. CD spectrum deconvolution
software CDNN (Bohm et al. 1992) was used to calculate
the secondary structure content of the protein.
Effect of metal ions
The effect of metal ions, SDS and EDTA was determined
by applying 1 mM of metal ions ­(Mn2+, ­Ni2+, ­Ca2+, ­Mg2+,
­Fe3+ and ­Cu2+), 0.1% SDS and 1, 10 and 50 mM EDTA to
the purified Xyn10B in phosphate buffer (pH 6.0). Enzymes
were incubated with each reagent for 2 h at room tempera-
ture, followed by assaying the residual activity. The activi-
ties of the enzyme without metal ions, SDS or EDTA were
defined as 100%.
Binding assays
For substrate-binding studies, the enzyme sample contain-
ing 0.02 µmol of purified Xyn10B was mixed with 30 mg
of each insoluble oat spelt xylan or Avicel to a final volume
of 1 ml of phosphate buffer (pH 6.0) taken in 15 ml conical
falcon tubes (Thermo Fischer Scientific, USA). After gen-
tle shaking for 2 h at 4 °C, the mixture was centrifuged at
5000×g for 10 min and the residual activity was assayed in
the supernatant.
Analysis of xylan hydrolysis products
The hydrolysis products of xylo-oligosaccharides (XOs)
by Xyn10B were analyzed using thin-layer chromatogra-
phy. XOs (X2–X4) (Megazyme International, Ireland) were
incubated with 120 U of Xyn10B at 50 °C for 15 min. Thin-
layer chromatography (TLC) was performed on silica gel 60
F245 plates (Merck 1.05554; 20 × 20 cm) using a solvent
mixture of n-butanol, acetic acid and water (2:1:1). Plates
were sprayed with 5% H ­ 2SO4 followed by heating at 100 °C
for 3 min (Sornyotha et al. 2007).
To analyze the hydrolysis products from various xylan
substrates, 60 mg of pre-treated wheat straw [prepared as
described previously (Sajjad et al. 2012) except using 2.5%
­H2SO4 instead of 0.5% NaOH], beechwood xylan, oat spelt
xylan and birchwood xylan was incubated with 120 U of
the purified Xyn10B, respectively, for 2 and 4 h at 50 °C.
Controls for all the substrates were incubated at the same
conditions without the enzyme. The hydrolysis products
obtained were centrifuged and the resulting supernatant was
passed through 0.2 µm filters. The total reducing sugars in
the hydrolysates of each substrate were calculated by the
DNS method as described previously (Ghose 1987).
These filtered samples obtained after 4  h were also
analyzed by high-performance liquid chromatography
(HPLC) using HPX-42A column (300 by 78 mm; Bio-Rad
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112 Extremophiles (2018) 22:109–119

Laboratories, Inc., Hercules, CA). Signals were observed
with a differential refractive index detector (S3580; Sykam
GmbH, Germany). Double-distilled deionized water was
used as the mobile phase/solvent. The temperature of the
column was maintained at 85 °C, while the temperature of
the detector was adjusted at 45 °C. Xylose from Sigma and
xylobiose, xylotriose and xylotetarose, all from Megazyme
International, Ireland, were used as standards.
with the corresponding domains of the bifunctional xyla-
nase-arabinofuranosidase from Actinoplanes sp. N902-109.
Similarly, Xyn10B consisting of the catalytic domain along
with both CBM3 and CBM2, showed 32% homology with
the bifunctional acetyl xylan esterase/xylanase of Sporocy-
tophaga myxococcoides (Table 1). These homologies indi-
cated the possibility of Xyn10B showing arabinofuranosi-
dase and esterase activities as well.

Quantification of liberated arabinose and acetic acid Expression and purification

Arabinose and acetic acid assays were performed on the
hydrolysates of all the substrates, obtained after incubation
of 60 mg of substrates with 120 U of the purified Xyn10B
for 2 and 4 h at 50 °C, using K-ARGA and K-ACETRM kits
(Megazyme International, Ireland), respectively, according
to the protocol recommended by the manufacturer.
Results and discussion
Gene cloning and sequence analysis
SDS-PAGE analysis of the total proteins of E. coli cells
transformed with the recombinant vector showed successful
expression of 69 kD protein (Fig. 1). The expression level of
Xyn10B was found to be 23% of the total cell proteins. After
ultrasonic disintegration of the transformed cells, Xyn10B
was obtained in the soluble fraction of the cell lysate. The
soluble fraction of the cell lysate was mixed with regener-
ated amorphous cellulose (RAC) which resulted in binding
of ~ 80% of the total enzyme activity. The enzyme bound
to RAC was successfully eluted with glycerol. The enzyme

The complete sequence of Xyn10B gene encoding the cata-

lytic domain with CBM3 and CBM2 attached to the C-ter-
minus, but without the signal sequence, was cloned. The
presence of the cloned sequence was confirmed by colony
PCR and restriction analysis of the recombinant plasmid.
The correctness of the gene sequence was confirmed by
DNA sequencing.
The catalytic domain of Xyn10B on the basis of homol-
ogy study was predicted to belong to the GH10 family. The
catalytic domain of only Xyn10B showed 45% sequence
homology with chain A of xylanase Xyn10 of Thermoascus
aurantiacus that specifically binds to the arabinose-deco- Fig. 1  SDS-PAGE showing the expression of Xyn10B. Lane 1, unin-
duced; lane 2, total cell protein; lane 3, pellet after cell lysis; lane 4,
rated xylan (Vardakou et al. 2005). Xyn10B consisting of the supernatant after cell lysis; lane 5, RAC supernatant (unbound pro-
catalytic domain along with CBM2 showed 45% homology teins); lane 6, purified protein; lane M, protein markers

Table 1  BLAST search results for proteins having sequences similar to that of  Xyn10B
Microorganism Accession % identity Identical region Predicted functions

A. cellulolyticus 11B ABK51954.1 98 CBM3 & CBM2 Esterase

Streptomyces sp. 2131.1 SEE20922.1 52 GH10 & CBM3 Endo-1,4-beta-xylanase
Streptomyces sp. ATexAB-D23 WP_026250303.1 51 GH10 & CBM3 Glycoside hydrolase
Actinoplanes sp. N902-109 WP_015621245.1 45 GH10 and CBM2 Xylanase-arabinofuranosidase bifunctional
Thermoascus aurantiacus (chain 2BNJ_A 45 GH10 Xylanase specific for arabinose decorated xylan
A of xylanase Ta)
Sporocytophaga myxococcoides GAL84846.1 32 GH10 Bifunctional acetylxylan esterase/xylanase
Alteromonadales bacterium BS08 WP_075185067.1 30 GH10 Acetyl xylan esterase
Geobacillus stearothermophilus AAD45520.2 33 GH10, CBM2 & CBM3 α-l-Arabinofuranosidase
Aspergillus awamori Q92194.2 75 GH10 & CBM3 Acetyl xylan esterase

The accession numbers shown in bold have been aligned with the sequence of Xyn10B in Fig. 1

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Extremophiles (2018) 22:109–119 113

thus obtained was ~ 95% pure. The purified enzyme had
specific activity, yield and purification fold of 1833 U mg−1,
27% and 10, respectively, as shown in Table 2.
Enzyme characteristics
The activities of Xyn10B on beechwood, birchwood and
oat spelt xylan were 126,480, 30,636 and 46,023 U µmol−1,
respectively (Table  3). Km and Vmax values for Xyn10B
against 0.5% beechwood xylan were 2.5  mg  ml −1 and
2857 U mg−1, respectively. Beechwood xylan was found to
be the best substrate for Xyn10B as reported for the various
other xylanases previously, as for example, xylanase from
thermophilic bacterium Anoxybacillus flavithermus BC
(Hollaender 2012). Xyn10B showed low activity on alkali
treated birchwood xylan, which may be due to the loss of
acetyl groups from xylan during alkali treatment (Bastawde
1992). However, the activity of Xyn10B on birchwood xylan
(30,636 U μmol−1) was higher than the activity of XynZ-
C of Clostridium thermocellum (14,820 U µmol−1), one of
the highly active xylanases reported previously (Sajjad et al.
2010). The activity of Xyn10B toward p-Nitrophenyl acetate
and p-Nitrophenyl-α-l-arabinofuranosidase was found to be
17,250 and 10,350 U µmol−1, respectively. No activity was
observed on CMC, Avicel, p-Nitrophenyl glucopyranoside
and p-Nitrophenyl galactopyranoside (Table 3).
Sequence alignment showed that the two active site
glutamate residues of Xyn10 from T. aurantiacus that are
highly conserved among the GH10 xylanases aligned with
E161 and E266 in the Xyn10B (Fig. 2). Xyn10B consisting
of the catalytic domain, CBM3 and CBM2, showed 33%
sequence homology with α-l-arabinofuranosidase AbfCelf
(family GH51) of Geobacillus stearothermophilus. The
sequence of α-l-arabinofuranosidase AbfCelf has two con-
served glutamate residues, typical of arabinofuranosidase
activity (Shallom et al. 2002). However, Xyn10B did not
show the presence of these residues at the corresponding
positions (Fig. 2). Likewise, the catalytic domain along
with CBM3 of Xyn10B showed 75% sequence identity
with acetyl xylan esterase belonging to family CE1 from
Aspergillus awamori (Table 1). Acetyl xylan esterase of
A. awamori showed the presence of serine in consensus
Table 3  Substrate specificity of Xyn10B
Substrate Activity (U μmol−1)
Beechwood xylan 126,480 ± 400
Birchwood xylan 30,636 ± 200
Oat spelt xylan 46,023 ± 100
p-Nitrophenyl arabinofuranoside 10,350 ± 150
p-Nitrophenyl acetate 17,250 ± 200
p-Nitrophenyl galactopyranoside NDa
p-Nitrophenyl glucopyranoside NDa
CMC NDa
Avicel NDa
a
 No activity was detected
sequence motif GXSXG, as well as aspartate and histidine
residues which formed a catalytic triad in the active site
of the enzyme (Koseki et al. 2005). Again, Xyn10B did
not seem to possess these residues in the corresponding
positions (Fig. 2). It is, therefore, likely that all the three
activities in Xyn10B are being performed by a single cata-
lytic domain. Many of the previously reported bifunctional
GH10 xylanase/β-glucanase (Shi et al. 2010) and xylanase/
cellulase (Sermsathanaswadi et al. 2017) also showed two
catalytic activities within a single active site having two
conserved glutamates as the active site residues.
Xyn10B showed activity at a broad temperature
(50–100 °C) and pH range (5.0–8.0) under the assay con-
ditions used. However, the highest activity was observed
at 70 °C (Fig. 3a) and pH 6.0 (Fig. 3b) under the assay
conditions used. The enzyme was found to be stable at pH
5.0–8.0 with a slight decrease in the residual activity at pH
5.0 as shown in Fig. 3c. This might be due to precipitation
of some of the enzymes at pH 5.0 which is close to its pI
(pH 4.8), calculated from ExPASy server (Gasteiger et al.
2005). The thermostability profile of Xyn10B showed that
the enzyme remained completely stable at 40 and 50 °C,
while ~ 90 and 70% of the activity was retained at 60 and
70 °C after 30 min as shown in Fig. 3d. The results of
thermostability were further validated by CD spectrometry
as discussed below.

Table 2  Purification of Fraction Protein (mg) Activity Yield (%) Purifi-

recombinant Xyn10B expressed cation
−1
in  E. coli  Units Units ­mg fold

Soluble cell lysate 360 66,000 183 100 1

Unbound protein 240 14,000 58 – –
Adsorbed ­proteina 120 52,000 433 79 2.4
Eluted protein 10 18,330 1833 27 10
a
 The values shown are those of the enzyme activity left unbound after incubation with RAC

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114 Extremophiles (2018) 22:109–119

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Extremophiles (2018) 22:109–119 115
◂Fig. 2  Sequence alignment of Xyn10B from Acidothermus cel-
lulolyticus 11B (Ac.Xyn10B) with previously characterized α-l-
arabinofuranosidase from Geobacillus stearothermophilus (Gs.
AbfCelf; AAD45520.2), endo-1,4-β-xylanase from Thermoascus
aurantiacus (Ta.Xyn10; 2BNJ_A) and esterase from Aspergillus
awamori (Aw.AxeA; Q92194.2) using Clustal Omega (http://www.
ebi.ac.uk/Tools/msa/clustalo/) and ESPript3.0 (Robert and Gouet
2014). The GH10 catalytic domain, CBM3 and CBM2 of Xyn10B
are boxed purple, blue and red, respectively. The active site residues
of each sequence are shown in yellow background while the strictly
conserved residues close to the active sites are shown in green back-
ground. The residues that are identical in all sequences are shown in
blue background
Circular dichroism analysis
The circular dichroism spectrum of the protein at 20 °C
in the far UV range showed maximum positive ellipticity
at 194  nm and maximum negative ellipticity at 216  nm
as shown in Fig. 4. There was no pronounced change in
positive or negative ellipticities until 70  °C. The mild
changes in protein confirmation with increasing tempera-
ture (20–70 °C) may be required for optimal activity of the
protein as described previously (Santos et al. 2010). How-
ever, at 80 °C, the negative ellipticity decreased from − 10.9
to − 7.9 at 217 nm, while the positive ellipticity decreased
from 6.5 to 2.0 at 194 nm, respectively. Previous studies
have shown that decrease in maximum negative and posi-
tive ellipticities after incubation at higher temperatures may
be due to thermal unfolding of the protein (Hurlbert and
Preston 2001). Similar results were obtained in the case of
heating Xyn10B at 80 °C. This finding corresponds to the
decreased activity of enzyme at this temperature. CD spec-
trum deconvolution software CDNN showed the presence
of 23% α-helices, 20% antiparallel β-sheets, 9% parallel
β-sheets, 14% β-turn and 34% random coils in the protein at
20 °C. After incubation at 80 °C, the α-helices, antiparallel
β-sheets, parallel β-sheets and random coil were 18, 23, 10
and 36%, respectively. Heating decreased the α-helix con-
tent, while there was a slight increase in random coils and
β-sheets as already reported for some other proteins whose
β-sheet content increased on heating leading to aggregation
of proteins at higher temperatures (Tajwar et al. 2017).
Effect of metal ions
Metal ions had almost no effect on the activity of Xyn10B
in Fig. 5, except ­Mn2+ which inhibited its activity. SDS also
decreased the activity of enzyme, while EDTA inhibited
activity only at high concentrations (50 mM EDTA).
Binding assays
Binding assay for Xyn10B showed that 57 and 82% of activ-
ity was bound to oat spelt xylan and Avicel, respectively.
The presence of the CBMs would explain strong binding
of the enzyme to Avicel. However, binding of the enzyme
to the insoluble oat spelt may be due to several surface-
exposed aromatic rings of the catalytic domain, CBM3 and
CBM2, which form hydrogen bonds and stacking interac-
tions with polysaccharide chains, thereby facilitating binding
of the enzyme to the substrate (Tenkanen et al. 1995). Even
a single domain xylanase from Bacillus D3 strain could bind
Avicel as well as insoluble xylan owing to the presence of
the aromatic residues (Connerton et al. 1999).
Hydrolysis products
Thin-layer chromatogram (Fig. 6) shows that Xyn10B was
able to hydrolyze xylotetarose and xylotriose, but could
not hydrolyze xylobiose, as reported for many GH10 endo-
xylanases (Tan et al. 2008). Xyn10B showed endo-xyla-
nase activity on various hemicellulosic substrates, includ-
ing beechwood xylan, birchwood xylan, oat spelt xylan
and wheat straw, resulting in the release of xylobiose and
xylose. HPLC analysis showed that Xyn10B almost com-
pletely hydrolyzed beechwood xylan (~ 95%) in 4 h with
xylobiose and xylose as the major hydrolysis products
(Fig. 7b). Xylose formation can be attributed to the action
of the enzymes on xylotriose releasing xylobiose and xylose,
respectively (Biely et al. 1997). Birchwood xylan was the
least hydrolyzed, as ~ 50% of the substrate remained unhy-
drolyzed while the remaining was hydrolyzed mainly to
xylobiose (Fig. 7c). Oat spelt was also almost completely
(~ 90%) hydrolyzed releasing xylobiose as the major hydrol-
ysis product (Fig. 7d). Hydrolysis of hemicellulose in wheat
straw released significant amounts of xylobiose with lower
amounts of xylose as shown in Fig. 7e. Xylobiose is used in
anti-obesity diets, as it possesses low calorie content and it is
also reported to prevent dental caries (Vázquez et al. 2000).
Xylobiose is also used as a food supplement, as it promotes
the growth of some intestinal bacteria that are beneficial to
human health (Moure et al. 2006). Degradation of oat spelt
and wheat straw produced more of xylobiose than xylose.
Therefore, application of Xyn10B in producing xylobiose
deserves further attention for commercial applications.
Quantification of liberated arabinose and acetic acid
Various xylan substrates are reported to have different
amounts of arabinose as side chain substituent on the xylan
backbone that hinder the access of endo-xylanases to the
xylan backbone (Satyanarayana and Johri 2005). Beechwood
xylan and birchwood xylan are known to contain > 90%
xylose residues with a small fraction of arabinose and acetyl
groups substituted on the xylose backbone (Bastawde 1992;
Wagschal et al. 2008). Oat spelt xylan contains around 70%
xylose residues that are mainly substituted by arabinose
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116 Extremophiles (2018) 22:109–119

Fig. 3  Effect of pH and tem-

perature on activity and stability
of Xyn10B. a Activity of the
enzyme at various pH values,
b activity of the enzyme at
various temperatures, c stability
of the enzyme at various pH
values. The enzyme was incu-
bated with buffers of various pH
values for 30 min, followed by
assaying the residual activity, d
stability of the enzyme at vari-
ous temperatures. The enzyme
was incubated at various tem-
peratures for 30 min followed
by assaying the residual activity.
All assays were performed in
triplicates and variation from
the mean was less than 5%

Fig. 4  Circular dichroism of Xyn10B dissolved in 10 mM potassium

phosphate buffer (pH 6.0). The samples were incubated at 20–80 °C
for 10 min, followed by a scan at 185–260 nm

Fig. 6  Thin-layer chromatography analysis of hydrolysis products of

xylo-oligosaccharides (X2–X4) by Xyn10B. Lane 1, standards X1
xylose, X2 xylobiose, X3 xylotriose, X4 xylotetarose; lane 2, X2
hydrolysates; lane 3, X3 hydrolysates; lane 4, X4 hydrolysates

(maximum 10% of total mass) and only a few acetyl groups

Fig. 5  Effect of metal ions on the activity of Xyn10B. The enzyme
was incubated with metal ions (1 mM), SDS (0.5%) and EDTA (1, 10
and 50 mM) for 30 min, followed by assaying the enzyme activities.
The activity of the control without any cation or reagent was defined
as 100%. Experiments were performed in triplicate and variation
about the mean was less than 5%
(Wagschal et al. 2008; Zhang et al. 2011a). Wheat straw is
known to be heavily substituted with arabinose and acetyl
side chains. The hot water-soluble fraction of wheat straw
has been reported to have 3.17% of acetyl groups. The
xylose to arabinose ratio in the pentosan fraction of wheat
straw hemicellulose was found to be 5.2:1. These substitu-
tions inhibit the hydrolysis of wheat straw and decrease its
nutritional content (Adams and Castagne 1952).

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Extremophiles (2018) 22:109–119 117

Fig. 7  HPLC profiles of the

sugars produced on the hydroly-
sis of the substrates (60 mg
each) with 120 U of Xyn10B.
a Standards X4 xylotetarose,
X3 xylotriose, X2 xylobiose,
X1 xylose and A arabinose,
b hydrolysis products of
beechwood xylan, c hydrolysis
products of birchwood xylan, d
hydrolysis products of oat spelt
xylan and e hydrolysis products
of wheat straw. Control of each
substrate is shown as dotted
line and the enzyme-hydrolyzed
product is shown in red line

Xyn10B showed arabinofuranosidase activity by releas-
ing arabinose from the xylan backbone that was validated
by HPLC analysis and arabinose assay kit (K-ARGA, Meg-
azyme International, Ireland). Arabinose released from
beechwood, birchwood and oat spelt xylan was found to be
7.5, 4.5 and 5 mg ml−1, respectively, after 4 h incubation
(Table 4). The highest amount of arabinose (27.5 g 100 g−1)
was released from the wheat straw as evident from a promi-
nent peak in HPLC (Fig. 7e). Arabinoxylan is the major anti-
nutritional component of wheat, especially for monogastric

Table 4  Amount of the liberated Substrate Total reducing sugars (g Arabinose (g 100 g−1) Acetic acid (g 100 g−1)
total reducing sugars, 100 g−1)
arabinose and acetic acid in
the hydrolysates obtained after 2 h 4 h 2 h 4 h 2 h 4 h
incubation of the substrates with
Xyn10B Beechwood xylan 64 ± 3 86 ± 5 4.8 ± 0.5 7.5 ± 1 0.5 ± 0.05 0.73 ± 0.01
Birchwood xylan 22 ± 2 40 ± 3 2.1 ± 0.02 4.5 ± 1 NDa NDa
Oat spelt xylan 53 ± 2.5 85 ± 4 3 ± 0.01 5 ± 0.5 NDa 0.05
Wheat straw 33 ± 2 60 ± 2 15 ± 1 27.5 ± 3 0.9 ± 0.02 1.5 ± 0.05
a
 No acetic acid was detected

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118
animals like broilers (Lei et al. 2016). Pretreatment of broiler
feed with Xyn10B can release the arabinose side chains in
wheat, in addition to xylan hydrolysis, thereby improving
the nutritional content of the cereals without using multiple
enzymes.
In addition to arabinose, the xylan backbone contains
acetyl side chains that hinder the activity of endo-xylanases.
Xyn10B showed acetyl xylan esterase activity, in addition
to endo-xylanase and arabinofuranosidase activities. The
release of acetic acid in the hydrolytic products of the vari-
ous xylan substrates was detected after 2 and 4 h incubation
and quantified by the acetic acid kit (K-ACETRM, Mega-
zyme International, Ireland), as shown in Table 4. Very low
amount of acetic acid was detected in the hydrolysate of oat
spelt, because the insoluble oat spelt mostly contains arab-
inose substitutions (Beg et al. 2001). The amount of acetic
acid from beechwood xylan was found to be 0.73 g 100 g−1,
while the highest amount of acetic acid was released from
the wheat straw (1.5 g 100 g−1) (Table 4). No acetic acid
was detected in the birchwood xylan’s hydrolysate as
expected, because the acetyl groups would be removed dur-
ing the alkali treatment of the substrate. However, low endo-
xylanase activity of Xyn10B on this deacetylated substrate
indicates that the catalytic site preferably recognizes and
hydrolyzes the acetylated xylan substrates as compared to
the deacetylated xylan substrates.
In conclusion, this study reported a highly active and
thermostable GH10 endo-xylanase with the ability to cleave
arabinose and acetyl side chains that usually hinder the activ-
ity of endo-xylanases on the xylan backbone. This enzyme
can be a valuable candidate for the industrial applications.
Acknowledgements  This research was funded by the Higher Education
Commission of Pakistan.
Compliance with ethical standards  Appl Microbiol 102(6):1586–1593
Conflict of interest  The authors declare that they have no conflict of
interest.
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