High level expression of a novel family 3 neutral β-xylosidase from Humicola insolens Y1 with high tolerance to D-xylose

Article abstract of DOI:10.1371/journal.pone.0117578

A novel β-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, e

Full text of DOI:10.1371/journal.pone.0117578

RESEARCH ARTICLE
High Level Expression of a Novel Family 3
Neutral β-Xylosidase from Humicola insolens
Y1 with High Tolerance to D-Xylose
Wei Xia1,2, Pengjun Shi2, Xinxin Xu3, Lichun Qian1*, Ying Cui2, Mengjuan Xia2, Bin Yao2*
1 College of Animal Science, Zhejiang University, Hangzhou 310058, P. R. China, 2 Key Laboratory for
Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural
Sciences, Beijing 100081, P. R. China, 3 Biotechnology Research Institute, Chinese Academy of Agricultural
Sciences, Beijing 100081, P. R. China
* binyao@caas.cn (BY); lcqian@zju.edu.cn (LQ)
Abstract
A novel β-xylosidase gene of glycosyl hydrolase (GH) family 3, xyl3A, was identified from
the thermophilic fungus Humicola insolens Y1, which is an innocuous and non-toxic fungus
that produces a wide variety of GHs. The cDNA of xyl3A, 2334 bp in length, encodes a 777-
residue polypeptide containing a putative signal peptide of 19 residues. The gene fragment
without the signal peptide-coding sequence was cloned and overexpressed in Pichia pas-
toris GS115 at a high level of 100 mg/L in 1-L Erlenmeyer flasks without fermentation optimi-
zation. Recombinant Xyl3A showed both β-xylosidase and α-arabinfuranosidase activities,
but had no hydrolysis capacity towards polysaccharides. It was optimally active at pH 6.0
and 60°C with a specific activity of 11.6 U/mg. It exhibited good stability over pH 4.0–9.0 (in-
cubated at 37°C for 1 h) and at temperatures of 60°C and below, retaining over 80% maxi-
mum activity. The enzyme had stronger tolerance to xylose than most fungal GH3 β-
xylosidases with a high Ki value of 29 mM, which makes Xyl3A more efficient to produce xy-
lose in fermentation process. Sequential combination of Xyl3A following endoxylanase
Xyn11A of the same microbial source showed significant synergistic effects on the degrada-
tion of various xylans and deconstructed xylo-oligosaccharides to xylose with high efficiency.
Moreover, using pNPX as both the donor and acceptor, Xyl3A exhibited a transxylosylation
activity to synthesize pNPX2. All these favorable properties suggest that Xyl3A has good po-
tential applications in the bioconversion of hemicelluloses to biofuels.
OPEN ACCESS
Citation: Xia W, Shi P, Xu X, Qian L, Cui Y, Xia M, et
al. (2015) High Level Expression of a Novel Family 3
Neutral β-Xylosidase from Humicola insolens Y1 with
High Tolerance to D-Xylose. PLoS ONE 10(2):
e0117578. doi:10.1371/journal.pone.0117578
Academic Editor: Linsheng Song, Institute of
Oceanology, Chinese Academy of Sciences, CHINA
Received: September 28, 2014
Accepted: December 28, 2014
Published: February 6, 2015
Copyright: © 2015 Xia et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by the National
High-Tech Research and Development Program of
China (863 Program, 2012AA022105) and the
National Science Foundation for Distinguished Young
Scholars of China (31225026) and the 948 program
of the Ministry of Agriculture (2014-S1) and the China
Modern Agriculture Research System (CARS-42).
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Introduction
Hemicelluloses, mainly composed of hetero-1,4-β-D-xylans and hetero-1,4-β-D-mannans, are
the second most abundant renewable polysaccharides in nature [1]. As the major component
of hemicellulose, xylan is composed of a backbone of β-1,4-linked D-xylopyranosyl residues
and side chains of different substituents. Thu2s, the degradation of xylan requires action of a
battery of debranching and depolymerizing activities, which is achieved by the synergic
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
1 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
Competing Interests: The authors have declared
that no competing interest exists.
hydrolysis of multiple enzymes. Specifically, xylanase attacks internal xylosidic linkages on the
backbone, β-xylosidase releases xylosyl residues by endwise attack of xylooligosaccharides, and
other debranching enzymes including α-L-arabinofuranosidase, α-glucuronidase and esterases
remove branched chains [2]. Single xylosyl residues released from xylooligosaccharides and re-
ducing end product inhibit xylanases, which makes β-xylosidase a key enzyme in the xylanoly-
tic system with a great potential in many biotechnological applications.
β-Xylosidases (EC 3.2.1.37) have been classified into the glycoside hydrolase (GH) families
3, 39, 43, 52 and 54 based on the amino acid sequence similarities (http://www.cazy.org/
Glycoside-Hydrolases.html) [3]. Their catalytic mechanisms are distinct; for instance, those of
GH3 employ retaining catalysis and GH43 ones have inverting catalysis. Up to the present
days, β-xylosidases have been described from a variety of microorganisms including bacteria
and fungi, and fungal β-xylosidases are mostly grouped into GH3 [4–8]. Although β-xylosidase
has been a research hotspot in the biofuel field in recent years due to its fermentation ability on
pentose sugars [9], few fungal β-xylosidases of GH3 have yet been identified or heterologously
expressed. Therefore, a better understanding of the deconstruction of xylan to constituent sug-
ars, mainly xylose, for subsequent fermentation by ethanol producing microbes is critical to the
efficient use of plant biomass for biofuel production [10].
Thermophilic and thermostable enzymes from thermophilic fungi are usually used in the
conversion of hemicellulose to reduce the risk of contamination and increase the solubility of
substrate [11]. Of the fungal sources, thermophilic Humicola is widely considered to be an in-
nocuous and non-toxic fungus that produces a wide variety of GHs. Cellulases and hemicellu-
lases from this genus have favorable characteristics and are utilized in various commercial
applications, such as enzyme detergents (Novo Industri A/S., Enzymatic detergent additive, U.
S. Patent 4810414) and enzyme preparations (Ultraflo L, Novozymes A/S, mainly composed of
β-glucanase and xylanase). Humicola insolens Y1 has been reported to be an excellent producer
of xylanolytic enzymes [12, 13], in which three thermophilic GH10 xylanases, one GH11 xyla-
nase, and two bifunctional GH43 xylosidase/arabinosidases have been characterized. In this
study, a novel β-xylosidase gene of GH3 was identified in H. insolens Y1 and overexpressed in
Pichia pastoris. Biochemical characterization and synergy test with xylanase on xylan degrada-
tion were also carried out. This is a prior article for now through exhaustively describing the se-
quence identity, enzymatic properties, xylose tolerance, transxylosylation activity and
synergistic action with xylanase of a fungal GH3 β-xylosidase.
Materials and Methods
Strains, media, vectors and chemicals
H. insolens Y1 GCMCC 4573 was routinely cultured in wheat bran medium as described previ-
ously [12]. Escherichia coli Trans1-T1 and vector pEASY-T3 (TransGen, Beijing, China) were
used for gene cloning. The heterologous protein expression system of pPIC9 and P. pastoris
GS115 (Invitrogen, Carlsbad, CA) was employed as the gene expression vector and expression
host, respectively. The DNA purification kit, restriction endonucleases and LA Taq DNA poly-
merase were purchased from TaKaRa (Otsu, Japan). T4 DNA ligase and the total RNA isola-
tion system kit were purchased from Promega (Madison, WI). The cDNA synthesis kit was
purchased from TransGen.
Beechwood xylan, 4-nitrophenyl β-D-xylopyranoside (pNPX), 4-nitrophenyl α-L-arabino-
furanoside (pNPAf), 4-nitrophenyl α-D-galactopyranoside (pNPGal), 4-nitrophenyl β-D-gluco-
pyranoside (pNPG), 4-nitrophenyl α-L-arabinopyranoside (pNPAb) and 4-nitrophenyl β-D-
cellobioside (pNPC) were purchased from Sigma-Aldrich (St. Louis, MO). Soluble wheat
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
2 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
arabinoxylan was obtained from Megazyme (Wicklow, Ireland). All other chemicals used were
of analytical grade and commercially available.
Cloning of the cDNA of xyl3A
Total RNA of H. insolens Y1 was extracted from the mycelia after 3 days’ growth in the induc-
ing medium [12], and was reverse transcribed into cDNA by RT-PCR following the protocol of
One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen). Based on the partial ge-
nome sequence of strain Y1 (whole genome sequencing in progress), a β-xylosidase encoding
gene of GH3, xyl3A, was identified, and the gene fragment without the signal peptide-coding
sequence was amplified with the specific primer set (Xyl3A-F: 50-GGGGAATTCGGGGTCC
TCCCCGACTGCAGCAAGCCG-30, and Xyl3A-R: 50-GGGGCGGCCGCTCAATTCTTG
AGGTTGGGGTCAACAG-30, restriction sites underlined). H. insolens Y1 cDNA was used as
the template, and the annealing temperature was 60°C. The PCR products were purified and li-
gated into the pEASY-T3 vector for sequencing.
Sequence analysis
DNA and protein sequence were aligned using the BLASTn and BLASTp programs (http://
www.ncbi.nlm.nih.gov/BLAST/), respectively. Vector NTI Advance 10.0 software (Invitrogen)
was used to analyze DNA sequence and to predict the molecular weight of protein. Genes,
introns, exons and transcription initiation sites were predicted using the online software
FGENESH (http://linux1.softberry.com/berry.phtml). Multiple sequence alignments were per-
formed with the ClustalW software. The signal peptide and the potential N-glycosylation sites
were predicted by the SignalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/) and the
NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/), respectively.
High level expression and purification of recombinant Xyl3A
The cDNA of xyl3A without the signal peptide-coding sequence and the pPIC9 vector were
both digested by EcoRI and NotI and ligated into in-frame fusion of the α-factor signal peptide
to construct the recombinant plasmid pPIC9-xyl3A. The recombinant plasmid was linearized
using BglII and transformed into P. pastoris GS115 competent cells by electroporation using
Gene Pulser X cell Electroporation System (Bio-Rad, Hercules, CA). Minimal dextrose medium
(MD) plates were prepared for the screening of positive transformants. The positive transfor-
mants were transferred to buffered glycerol complex medium (BMGY) and grown at 30°C for
2 days. The cells were collected by centrifugation and resuspended in buffered methanol com-
plex medium (BMMY) containing 0.5% methanol for induction. The β-xylosidase activities of
the culture supernatants were assayed as described below, and the transformant exhibiting the
highest β-xylosidase activity was subjected to high level expression in 1-L Erlenmeyer flasks ac-
cording to the Pichia expression manual (Invitrogen).
The culture supernatants of aforementioned recombinant strain were collected by centrifu-
gation (12,000 × g, 4°C, 10 min) to remove cell debris and undissolved materials, followed by
concentration through a Vivaflow ultrafiltration membrane (Vivascience, Hannover, Ger-
many) with a molecular weight cut-off of 5 kDa. The crude enzyme was loaded onto a FPLC
HiTrap Q Sepharose XL 5 mL column (GE Healthcare, Uppsala, Sweden) that was equilibrated
with 20 mM Tris-HCl (pH 8.0). Proteins were eluted using a linear gradient of NaCl (0–1.0 M)
in the buffer mentioned above at a flow rate of 3.0 mL/min. Fractions exhibiting β-xylosidase
activities were pooled and subjected to sodium dodecyl sulfate-polyacrylamide gel electropho-
resis (SDS-PAGE). The protein concentration was determined by a Bradford assay with bovine
serine albumin as a standard. To remove N-glycosylation during heterologous expression in
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
3 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
P. pastoris, purified recombinant Xyl3A was incubated with 500 U of endo-β-N- acetylglucosa-
minidase H (Endo H) at 37°C for 2 h according to the manufacturer’s instructions (New En-
gland Biolabs, Ipswich, MA). The deglycosylated enzyme was also analyzed by SDS-PAGE.
Enzyme activity assay
The β-xylosidase activity was assayed using pNPX as the substrate. The standard reaction sys-
tem consisted of 250 μL of appropriately diluted enzyme and 250 μL of McIlvaine buffer (pH
6.0) containing 2 mM pNPX. After incubation at 60°C for 10 min, 1.5 mL of 1.0 M Na2CO3
was added into the system to terminate the reaction. The amount of p-nitrophenol released
was determined spectrophotometrically by reading the absorbance at 405 nm. One unit of β-
xylosidase activity was defined as the amount of enzyme that released 1 μmol of p-nitrophenol
per minute under the assay conditions. Each experiment was performed in triplicate.
Biochemical characterization
The optimal pH for Xyl3A activity was determined at 60°C for 10 min over a pH range of 2.0–
11.0 using the following buffers: 100 mM Na2HPO4-citric acid (pH 2.0–8.0), 100 mM Tris-HCl
(pH 8.0–9.0), and 100 mM glycine-NaOH (pH 9.0–11.0). To estimate pH stability, the enzyme
was pre-incubated in the buffers mentioned above without substrate at 37°C (physiological
temperature) or 60°C (optimal temperature) for 1 h, and the residual activities were measured
under the standard conditions (pH 6.0, 60°C, 10 min).
The optimal temperature was examined at the optimal pH by measuring the enzyme activity
over the temperature range of 30 and 90°C. The thermostability was investigated by determin-
ing residual enzyme activities after preincubation at 60°C or 70°C and optimal pH without sub-
strate for various periods. The samples were collected at 0, 2, 5, 10, 20, 30 and 60 min,
respectively. Enzyme activity assays were performed under the standard conditions.
The enzyme activity of Xyl3A was measured in the presence of 5 mM of various metal ions
and chemical reagents (Ag+, Ca2+, Li+, Co2+, Cr3+, Ni2+, Cu2+, Mg2+, Fe3+, Mn2+, Hg2+, Pb2+,
EDTA, SDS or β-mercaptoethanol) to estimate their effect on Xyl3A activity. The reaction
without any additive was used as a blank control.
Substrate specificity and kinetic parameters
To investigate the substrate specificity of Xyl3A, activity assays were performed under opti-
mum conditions with 2 mM of p-nitrophenyl derivatives (pNPX, pNPAf, pNPG, pNPGal,
pNPAb, pNPC) or 1% (w/v) polysaccharides (beechwood xylan, water-soluble wheat arabinox-
ylan, sugar beet arabinan and debranched AZCL-arabinan) as the substrate.
The Km, Vmax and Kcat values of Xyl3A were determined at pH 6.0 and 60°C for 5 min in
100 mM Na2HPO4-citric acid containing 1–10 mM pNPX as the substrate. The data were plot-
ted according to the Lineweaver-Burk method.
Xyl3A tolerance to xylose
The tolerance of Xyl3A to xylose was investigated as described by Yan et al. [14]. The β-xylosi-
dase activity of Xyl3A in each reaction system containing xylose of different concentrations
(5–50 mM) was determined in the presence of two final concentrations of pNPX (0.75 mM
and 1 mM). The residual β-xylosidase activities were measured according to the standard assay
method. The data were analyzed by a Dixon plot. The Ki value of xylose was defined as the
amount of xylose required to inhibit 50% of the Xyl3A activity.
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
4 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
Transxylosylation of Xyl3A
For transxylosylation assay, pNPX was added to serve as both the donor and acceptor. The re-
action system containing 75 mM of pNPX and 3 U/mL of Xyl3A was incubated at pH 6.0 and
37°C for 0–24 h. After the removal of Xyl3A by ultrafiltration, the transxylosylation products
were analyzed by thin layer chromatography (TLC) using the mixture of xylose, xylobiose and
xylotriose as standards. TLC was performed on aluminum-coated silica gel 60 sheets (Merck,
Darmstadt, Germany), developed in a system of n-butyl alcohol/acetic acid/water 2:1:1 (v/v/v),
and colored by saturating the plate in 5% sulphuric acid (v/v) and then heating at 110°C for
5 min.
Enzyme synergy
Xyl3A and Xyn11A of the same microbial source were selected to study their synergic actions
on the hydrolysis of beechwood xylan and water soluble wheat arabinoxylan [13]. Basic manip-
ulation consulted to the method reported by Raweesri et al. [15]. Each reaction containing
900 μL of 0.5% (w/v) substrate and 100 μL of enzyme(s) in 100 mM Na2HPO4-citric acid
(pH 6.0) was incubated at 37°C for 12 h. Reactions containing substrate only were treated as
blank controls. Enzymes, 0.25 U of Xyl3A and/or 0.25 U of Xyn11A, were added alone, simul-
taneously or sequentially. For sequential reactions, the first reactions were terminated by
10-min boiling water bath, and the second reactions were performed as described above. The
xylose equivalents (mM) released were determined using the DNS method at room tempera-
ture, with xylose as the standard.
The synergy degree was defined as the ratio of xylose equivalents released by enzyme combi-
nations to the sum of the xylose equivalents released by each enzyme alone. One-way ANOVA
with a Tukey’s test was used for statistical analysis in OriginPro 8.
Results and Discussion
Gene cloning and sequence analysis
The full-length cDNA of xyl3A from H. insolens Y1 contains 2334 bp and encodes a polypep-
tide of 777 amino acid residues and a termination codon. Deduced Xyl3A consists of a putative
signal peptide at the N-terminus (residues 1–19) and a catalytic domain of GH3 (residues 20–
777). The calculated molecular mass and pI value were estimated to be 83.2 kDa and 6.21, re-
spectively. The pI value of Xyl3A is higher than that of most fungal β-xylosidases (4.0–5.0)
[16]. Five potential N-glycosylation sites (Asn110, Asn252, Asn314, Asn555, and Asn570) were
identified in deduced Xyl3A.
Deduced Xyl3A exhibits highest sequence identities of 64% with a putative (trans)glycosi-
dase from Glarea lozoyensis ATCC 20868 and relative low sequence identities with functionally
characterized GH3 β-xylosidases (Fig. 1), i.e. 41% with Xyl from Aspergillus awamori K4 [4]
and XylA from Aspergillus japonicas [5], 40% with XlnD from Aspergillus nidulans [6], Bxl1
from Talaromyces emersonii [7] and XylA from Aspergillus oryzae [8]. Blast analysis against
the PDB database indicates that Xyl3A shares a fairly low sequence similarity to crystal-re-
solved counterparts (29% identity the highest), thus no available template was found to predict
the tertiary structure of Xyl3A.
The catalysis mechanism of GH3 β-xylosidases has been proved to proceed via a retaining
reaction mechanism under the operation of a nucleophile Glu and an acid/base residue Asp.
Based on the amino acid sequence alignments, the two putative catalytic residues of Xyl3A
were predicted to be Asp282 and Glu488, corresponding to Asp307 and Glu516 of XylA from
A. japonicas [5] and Asp242 and Glu441 of Bxl1 from T. emersonii [7].
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
5 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
6 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
Fig 1. Sequence alignment of deduced Xyl3A from H. insolens Y1 with other GH3 β-xylosidases from
A. awamori K4 (Q4AEG8), A. nidulans (CAA73902), A. oryzae (T00392), A. niger (CAB06417) and T.
emersonii (JC7966). Identical and similar residues are shaded in black and grey, respectively. The
putative catalytic residues are marked with asterisks.
doi:10.1371/journal.pone.0117578.g001
Overexpression and purification of recombinant Xyl3A
Bacterial enzymes are generally heterologously expressed in E. coli [17], while that of fungi are
usually purified from original cultures [18] or produced in high-level expression systems like P.
pastoris [19] or Aspergillus [8]. In this study, we successfully overexpressed mature Xyl3A with-
out the signal peptide in P. pastoris GS115 with methanol induction, which was secreted into
culture supernatant by the α-factor signal peptide. Without fermentation optimization, the
yield of Xyl3A reached approximately 100 mg/L. The result suggests its great potential for cost-
saving mass production.
Crude Xyl3A was concentrated by nearly ten fold in volume and purified by a one-step anion
exchange chromatography. The purified enzyme solution exhibited 8.1 U/mL of β-xylosidase ac-
tivity under its optimum condition with a concentration of 0.698 mg/mL. Purified Xyl3A migrat-
ed one single band of about 85.0 kDa on SDS-PAGE (Fig. 2). After deglycosylation with Endo H,
Xyl3A showed a slight decrease in molecular mass, which was in agreement with the calculated
molecular weight. Similar molecular masses have been reported for some GH3 β-xylosidases [4,
18]. Mozolowski and Connerton [20] purified a GH3 β-xylosidase from H. insolens culture,
which was composed of two protein subunits of 64 kDa and 17 kDa with a total molecular mass
of approximately 81 kDa. Deduced Xyl3A showed high sequence identities (91–100%) to the di-
gested peptide fragments of both subunits, but no supposed fracture occurred at the C-terminus
of recombinant Xyl3A when heterologously expressed in P. pastoris GS115.
Enzymatic properties of purified recombinant Xyl3A
Similar to the two GH43 β-xylosidases of H. insolens Y1 [13] but different from other fungal β-
xylosidases that function at pH 3.5–5.0 (Table 1), purified recombinant Xyl3A was active in
weak acid pH, showing optimal activity at pH 6.0, 65.5% activity at pH 6.5 and 27.4% at pH 7.0
(Fig. 3A). The pH stability of Xyl3A was assayed at both 37°C and 60°C (Fig. 3B). The enzyme
had good pH stability at 37°C, retaining more than 80% activity after incubation at pH 4.0–9.0
for 1 h. After pre-incubation at 60°C for 1 h, the enzyme showed a narrower pH stability range
(pH 5.07.0). When assayed Xyl3A activity at pH 6.0 (the optimal pH), it acted the best at a rela-
tively high temperature of 60°C and remained 60% activity even at 70°C (Fig. 3C). This temper-
ature optimum falls within the range of other fungal GH3 β-xylosidases (50–70°C; Table 1),
but higher than that of β-xylosidase from Neocallimastix frontalis [21]. Xyl3A was thermosta-
ble at 60°C, retaining about 80% activity after 1 h incubation at 60°C without substrate, and
lost activity rapidly when treated at 70°C (Fig. 3D). The enzymatic properties of Humicola
GHs are much similar [12, 14, 20, 22], which might be due to their grown surroundings of neu-
tral pH and high temperature.
The effects of metal ions and chemical reagents on the activity of Xyl3A were determined at
the concentrations of 5 mM (Table 2). Xyl3A was highly resistant to EDTA, β-mercaptoethanol
and almost all of tested cations except for Ag+ and SDS. Mn2+ enhanced the activity slightly.
Substrate specificity and kinetic parameters
Xyl3A exhibited the highest activity towards pNPX (100%), moderate on pNPAf (26.9%) and
weak on pNPGal (2.57%). It has been reported that GH3 β-xylosidases have broad substrate
specificity. For example, when the activity against pNPX was defined as 100%, β-xylosidase
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
7 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
Fig 2. SDS-PAGE analysis of the purified recombinant Xyl3A. Lanes: 1, the purified recombinant Xyl3A after deglycosylation with Endo H; 2, the
purified recombinant Xyl3A; M, the standard protein molecular weight markers.
doi:10.1371/journal.pone.0117578.g002
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
8 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
Table 1. Property comparison of Xyl3A from H. insolens Y1 with other fungal β-xylosidases.
Species
MW (kDa)
Optimum activity
Temperature (°C)
Specific
activity
(U/mg)
K
m (mM) Vmax
Ki (mM) References
(μmol/min/mg)
pH
Humicola insolens Y1a
H. insolens Y1b
83.2
37.0
62.0
68
6.0
6.5
7.0
5.5
5.0
3.5
4.0
4.0
60
50
50
70
50
70
70
60
11.6
20.5
1.7
2.51
12.2
1.29
1.74
1.1
37.33
29
This work
[14]
[14]
[20]
[6]
203.8
79
H. insolens Y1b
2.18
292
H. insolensc
Aspergillus nidulansc
Aureobasidium pullulans ATCC 20524c 88.5
Aspergillus awamori K4c
Aspergillus oryzaed
193
22.17
-
85
107.1
288
25.6
-
3.5
263
-
[18]
[4]
84.6
110
132
91.2
90.0
103.7
97
19.58
76
-
-
-
-
-
-
[8]
Aspergillus phoenicisc
4.0–4.5 75
820.6
53
2.36
0.77
122
0.12
2.37
0.04
920.75
-
[24]
[29]
[21]
[25]
[26]
[23]
Fusarium proliferatumc
Neocallimastix frontalisc
Penicillium herquei IFO 4674c
Talaromyces thermophilusc
Trichoderma koningii G-39c
4.5
6.5
4
60
35
50
50
75
5
-
4.3
-
353.6
147.5
3.02
-
-
7
0.049
-
-
104
3.5–4.0 55–60
5
a Expressed in P. pastoris GS115.
b Belonging to GH43 and expressed in E.coli.
c Purified from the original fungi.
d Expressed in A. oryzae.
doi:10.1371/journal.pone.0117578.t001
from Asperigus pullulans ATCC20524 showed 35.8% relative activity towards pNPAf [5], and
β-xylosidase from A. japonicus strain MU-2 showed 31.6% β-glucosidase activity and 17.7% α-
arabinfuranosidase activity [18]. However, the 64 kDa subunit of H. insolens β-xylosidase had
no activity towards pNPAf [20], which was probably due to the deletion of the C-terminus. No
activity was detected when using other polysaccharides as the substrate. In contrast, several bi-
functional GH43 β-xylosidase-arabinosidases have higher α-arabinfuranosidase and xylanase
activities [13]. The underlying mechanism might be ascribed to the spatial similarity of D-xylo-
pyranose and L-arabinofuranose [23].
Using pPNX as the substrate, the specific activity of purified recombinant Xyl3A was deter-
mined to be 11.6 U/mg, which was higher than that of GH3 β-xylosidases from Neocallimastix
frontalis (4.3 U/mg) [21] and Trichoderma koningii G-39 (3.02 U/mg) [23], but lower than that
from Aspergillus phoenicis (820.6 U/mg) [24] and Penicillium herquei IFO 4674 (353.6 U/mg)
[25]. The same conclusion applied to the comparison among four β-xylosidases produced by
H. insolens [13, 20].
The Km and Vmax values of purified recombinant Xyl3A were 2.51 mM and 37.33
μmol/min/mg, respectively, when pNPX was used as the substrate. The Km value of Xyl3A
is similar to that of β-xylosidases from Talaromyces thermophiles (2.37 mM) [26] and
A. phoenicis (2.36 mM) [24], suggesting its moderate affinity to substrate. In comparison
with the kinetic values of other H. insolens isoenzymes (Xyl43A, 12.2 mM and 203.8 μmol/
min/mg; Xyl43B, 1.29 mM and 2.18 μmol/min/mg; a 64 kDa β-xylosidase subunit, 1.74 mM
and 22.17 μmol/min/mg), Xyl3A had a middle affinity and Vmax towards pNPX.
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
9 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
Fig 3. Properties of purified recombinant Xyl3A. (A) Effect of pH on Xyl3A activities. (B) pH stability of Xyl3A. (C) Effect of temperature on Xyl3A
activities. (D) Thermostability of Xyl3A. Each value in the panel represents the means SD (n = 3).
doi:10.1371/journal.pone.0117578.g003
Xyl3A tolerance to xylose
D-Xylose is the principal end-product of xylooligosaccharide hydrolysis. It performs competi-
tive inhibition on the activity of β-xylosidase. Thus a xylose-tolerant β-xylosidase is essential to
maintain the efficiency of hemicellulose conversion by removing xylobioses, the natural sub-
strate of β-xylosidase [27]. Based on the Dixon plot, Xyl3A had a Ki value of 29 mM, which is
significantly higher than almost all reported fungal GH3 β-xylosidases (2 to 10 mM) [23, 28,
29]. Higher tolerance to xylose has been reported in fungal GH43 xylosidases, such as 79 mM
and 292 mM for Xyl43A and Xyl43B of H. insolens Y1, respectively [13] and 139 mM for
Table 2. Effect of metal ions and chemical reagents (5 mM) on the activity of purified Xyl3A.
Chemicals
Relative activity (%)a
Chemicals
Relative activity (%)a
Control
Ag+
Li+
Pb2+
Ca2+
Cu2+
Cr3+
Co3+
Co3+
100.0 0.4
5.3 0.2
Mn2+
Fe3+
Ni2+
Mg2+
114.9 0.2
83.9 1.9
99.5 0.2
94.7 0.1
93.4 0.4
87.5 0.2
46.4 0.2
92.0 1.1
103.5 0.4
95.3 0.3
101.8 0.3
93.4 0.3
94.4 1.1
94.9 0.4
94.9 0.4
Zn2+
EDTA
SDS
β-Mercaptoethanol
a Values represent the means of triplicates relative to the untreated control samples.
doi:10.1371/journal.pone.0117578.t002
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
10 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
PtXyl43 [17]. However, the GH3 TthXynB3 from archaebacterium Thermotoga thermarum
has an extremely high Ki value of 1 M [30]. Moreover, the enzymatic activity was even activated
by xylose at concentrations less than 500 mM.
Transxylosylation of Xyl3A
Preparation of useful oligosaccharides and glycosyl compounds has attracted broad attention
in recent years. Compared with chemical approach, transglycosylation activities of glycosidases
have advantages on the high specificity for substrate, the selectivity of the bonding, and the
mildness of reaction conditions. Therefore transglycosylation ability has been regarded as an
important characteristic of a glycosidase for potential applications [31]. Several studies have
addressed the transxylosylation capacities of several GH3 β-xylosidases with oligosaccharides,
alcohols and even pNPX as acceptors [32–34]. In the present study, the transxylosylation abili-
ty of Xyl3A was determined by TLC (Fig. 4). After co-incubation of Xyl3A and pNPX for 1 h,
two products appeared on the TLC plate, which are conjectured to be pNPX2 and xylobiose.
Along with time, the amount of xylobiose increased while that of pNPX2 decreased. The possi-
ble reason might be that pNPX acts as the primary xylosyl acceptor when the concentration of
xylose is relatively low in the early stage of the reaction, but is slowly hydrolyzed when pNPX
runs out. As the time went on, pNPX2 was hydrolyzed, and the released xylosyl residues formed
Fig 4. TLC analysis of the transxylosylation products of Xyl3A with pPNX as both the donor and acceptor. S, the xylooligosaccharide standards;
0–24, the time course of transxylosylation (h).
doi:10.1371/journal.pone.0117578.g004
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
11 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
xylobiose in turn. No larger oligosaccharide or pNP-oligosaccharide was observed, indicating
that transxylosylation by Xyl3A tended to produce short chain oligosaccharides with low poly-
merization degree. It was quite different from Aspergillus β-xylosidase that was able to catalyze
the synthesis of p-nitrophenyl (pNP)-(1,4)-D-xylooligosaccharides with a degree of polymeriza-
tion of 2–7 [33].
Xylan degradation by enzyme synergy
β-Xylosidase is usually used in combination with xylanase to fully deconstruct hemicelluloses
and β-1,4-xylooligosaccharides, which products can be fermented to biofuels like ethanol and
butanol [35]. In the current study, a synergy value was used to quantify the cooperation be-
tween Xyl3A and Xyn11A (Table 3). Significant differences (p < 0.05) were observed among
different enzyme combinations on xylan degradation. Birchwood xylan with less side chains
acted as a better substrate for synergism. No matter which substrate was used, the sequential
enzyme combination of Xyn11A followed by Xyl3A exhibited the highest synergy degrees (1.25
and 1.08, respectively), and Xyl3A alone released the least xylose equivalents. This result sug-
gests that Xyl3A works better on xylooligosacchardies rather than on xylan polysaccharides.
In comparison with Xyl43A and Xyl43B of H. insolens Y1 [13], Xyl3A displayed almost
equal synergistic effects with xylanase at a quarter of enzyme dosage. Considering the absence
of standard signal peptides in Xyl43A and Xyl43B, Xyl3A may act as the primary extracellular
β-xylosidase for xylan degradation in H. insolens Y1 under natural conditions.
Conclusions
A novel GH3 β-xylosidase gene was identified in H. insolens Y1, and its gene fragment without
the signal peptide-coding sequence was overexpressed in P. pastoris GS115 with a high level of
100 mg/L. Recombinant Xyl3A exhibited β-xylosidase, α-arabinosidase and transxylosylation
activities. Using pPNX as the substrate, the enzyme had maximum activity at pH 6.0 and 60°C
and retained stable over pH 4.0−9.0 (after incubation at 37°C for 1 h) and at temperatures of
60°C and below. Moreover, it was highly tolerant to xylose (up to 29 mM) and had significant
synergistic action with xylanase on the degradation of various xylans. These properties make
Xyl3A prospective for application in the bioconversion of hemicellulose and biofuel industry.
Table 3. Synergetic reactions by Xyn11A and Xyl3A against two xylan substrates. a
Beechwood xylanb
Xylose equivalents (mM)
Enzyme added
First reaction Second reaction
Wheat arabinoxylanc
Xylose equivalents (mM)
Synergy d
Synergy
Xyn11A
Xyl3A
None
6.63 0.13
0.12 0.06
7.63 0.09
8.42 0.03
7.95 0.13
-
6.36 0.06
0.06 0.04
6.08 0.02
6.94 0.23
6.80 0.14
-
None
-
-
Xyn11A+Xyl3A
Xyn11A
Xyl3A
None
1.13*
1.25*
1.18*
0.95*
1.08*
1.06*
Xyl3A
Xyn11A
a Simultaneous reactions refer to the reactions with two enzymes added simultaneously; sequential reactions refer to the reactions with enzymes
added sequentially.
b Beechwood xylan:the arabinose:xylose ratio of *1:90.
c Water-soluble wheat arabinoxylan: the arabinose:xylose:other sugars ratio of *37:61:2.
d Synergy degree is defined as the ratio of xylose equivalents from enzyme combinations to the sum of that released by the individual enzymes;
the data marked with* means significant difference at p < 0.05 (One-way ANOVA with a Tukey’s test by OriginPro 8).
doi:10.1371/journal.pone.0117578.t003
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
12 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
Acknowledgments
The authors are grateful to lab assistants Zhaohui Zhang and Rui Ma at the Key Laboratory for
Feed Biotechnology of the Ministry of Agriculture for their help in protein purification and En-
glish polishing, respectively.
Author Contributions
Conceived and designed the experiments: WX PS. Performed the experiments: WX YC MX.
Analyzed the data: WX PS XX. Contributed reagents/materials/analysis tools: WX PS LQ.
Wrote the paper: WX PS. Guided the experiment design: BY. Provided the research platform:
BY.
References
1. Puls J, Schuseil J (1993) Chemistry of hemicelluloses: relationship between hemicellulose structure
and enzymes required for hydrolysis. Portland Press London. pp. 1–27. PMID: 10133702
2. Rahman AK, Sugitani N, Hatsu M, Takamizawa K (2003) A role of xylanase, α-L-arabinofuranosidase,
and xylosidase in xylan degradation. Can J Microbiol 49: 58–64. PMID: 12674349
3. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases.
Curr Opin Struct Biol 7: 637–644. PMID: 9345621
4. Kurakake M, Osada S, Komaki T (1997) Transglycosylation of β-xylosidase from Aspergillus awamori
K4. Biosci Biotechnol Biochem 61: 2010–2014.
5. Wakiyama M, Yoshihara K, Hayashi S, Ohta K (2008) Purification and properties of an extracellular β-
xylosidase from Aspergillus japonicus and sequence analysis of the encoding gene. J Biosci Bioeng
106: 398–404. doi: 10.1263/jbb.106.398 PMID: 19000618
6. Kumar S, Ramón D (1996) Purification and regulation of the synthesis of a β-xylosidase from Aspergil-
lus nidulans. FEMS Microbiol Lett 135: 287–293.
7. Reen FJ, Murray PG, Tuohy MG (2003) Molecular characterization and expression analysis of the first
hemicellulase gene (bxl1) encoding β-xylosidase from the thermophilic fungus Talaromyces emersonii.
Biochem Bioph Res Co 305: 579–585.
8. Kitamoto N, Yoshino S, Ohmiya K, Tsukagoshi N (1999) Sequence analysis, overexpression, and anti-
sense inhibition of a β-xylosidase gene, xylA, from Aspergillus oryzae KBN616. Appl Environ Microbiol
65: 20–24. PMID: 9872754
9. Li Z, Qu H, Li C, Zhou X (2013) Direct and efficient xylitol production from xylan by Saccharomyces cer-
evisiae through transcriptional level and fermentation processing optimizations. Bioresour Technol
149: 413–419. doi: 10.1016/j.biortech.2013.09.101 PMID: 24128404
10. Dodd D, Cann IK (2009) Enzymatic deconstruction of xylan for biofuel production. GCB Bioenergy 18:
2–17.
11. Turner P, Mamo G, Karlsson EN (2007) Potential and utilization of thermophiles and thermostable en-
zymes in biorefining. Microb Cell Fact 6: 9. PMID: 17359551
12. Du Y, Shi P, Huang H, Zhang X, Luo H, et al. (2013) Characterisation of three novel thermophilic xyla-
nases from Humicola insolens Y1 with application potentials in the brewing industry. Bioresour Technol
130: 161–167. doi: 10.1016/j.biortech.2012.12.067 PMID: 23306124
13. Yang X, Shi P, Huang H, Luo H, Wang Y, et al. (2014) Two xylose-tolerant GH43 bifunctional β-xylosi-
dase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degra-
dation of xylan. Food Chem 148: 381–387. doi: 10.1016/j.foodchem.2013.10.062 PMID: 24262572
14. Yan QJ, Wang L, Jiang ZQ, Yang SQ, Zhu HF, et al. (2008) A xylose-tolerant β-xylosidase from Paeci-
lomyces thermophila: characterization and its co-action with the endogenous xylanase. Bioresour
Technol 99: 5402–5410. doi: 10.1016/j.biortech.2007.11.033 PMID: 18180153
15. Raweesri P, Riangrungrojana P, Pinphanichakarn P (2008) α-L-Arabinofuranosidase from Streptomy-
ces sp. PC22: purification, characterization and its synergistic action with xylanolytic enzymes in the
degradation of xylan and agricultural residues. Bioresour Technol 99: 8981–8986. doi: 10.1016/j.
biortech.2008.05.016 PMID: 18606539
16. Knob A, Terrasan CRF, Carmona EC (2010) β-Xylosidases from filamentous fungi: an overview. World
J Microbiol Biotechnol 26: 389–407.
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
13 / 14

A Xylose-Tolerant β-Xylosidase from Humicola insolens Y1
17. Teng C, Jia H, Yan Q, Zhou P, Jiang Z (2011) High-level expression of extracellular secretion of a β-
xylosidase gene from Paecilomyces thermophila in Escherichia coli. Bioresour Technol 102: 1822–
1830. doi: 10.1016/j.biortech.2010.09.055 PMID: 20970996
18. Ohta K, Fujimoto H, Fujii S, Wakiyama M (2010) Cell-associated β-xylosidase from Aureobasidium
pullulans ATCC 20524: Purification, properties, and characterization of the encoding gene. J Biosci
Bioeng 110: 152–157. doi: 10.1016/j.jbiosc.2010.02.008 PMID: 20547381
19. Chen X, Meng K, Shi P, Bai Y, Luo H, et al. (2012) High-level expression of a novel Penicillium endo-
1,3(4)-β-D-glucanase with high specific activity in Pichia pastoris. J Ind Microbiol Biotechnol 39: 869–
876. doi: 10.1007/s10295-012-1087-z PMID: 22354732
20. Mozolowski GA, Connerton IF (2009) Characterization of a highly efficient heterodimeric xylosidase
from Humicola insolens. Enzyme Microb Tech 5: 436–442.
21. Hebraud M, Fevre M (1990) Purification and characterization of an extracellular β-xylosidase from the
rumen anaerobic fungus Neocallimastix frontalis. FEMS Microbiol Lett 60: 11–16. PMID: 2126511
22. Almeida EM, Polizeli MDL, Terenzi HF, Atilio JJ (1995) Purification and biochemical characterisation of
β-xylosidase from Humicola grisea var. thermoidea. FEMS Microbiol Lett 130: 171–175.
23. Li Y, Yao H, Cho Y (2000) Effective induction, purification and characterization of Thrichoderma konin-
gii G-39 β-xylosidase with high transferase activity. Biotechnol Appl Biochem 31: 119–125. PMID:
10744957
24. Rizzatti A, Jorge J, Terenzi H, Rechia C, Polizeli M (2001) Purification and properties of a thermostable
extracellular β-xylosidase produced by a thermotolerant Aspergillus phoenicis. J Ind Microbiol Biotech-
nol 26: 1–5. PMID: 11548743
25. Ito T, Yokoyama E, Sato H, Ujita M, Funaguma T, et al. (2003) Xylosidases associated with the cell sur-
face of Penicillium herquei IFO 4674. J Biosci Bioeng 96: 354–359. PMID: 16233536
26. Guerfali M, Gargouri A, Belghith H (2008) Talaromyces thermophilus β -D-xylosidase: purification, char-
acterization and xylobiose synthesis. Appl Biochem Biotechnol 150: 267–279. doi: 10.1007/s12010-
008-8260-x PMID: 18592411
27. Sunna A, Antranikian G (1997) Xylanolytic enzymes from fungi and bacteria. Crit Rev Biotechnol 17:
39–67. PMID: 9118232
28. Saha BC (2001) Purification and characterization of an extracellular β-xylosidase from a newly isolated
Fusarium verticillioides. J Ind Microbiol Biotechnol 27: 241–245. PMID: 11687937
29. Saha BC (2003) Purification and properties of an extracellular β-xylosidase from a newly isolated Fu-
sarium proliferatum. Bioresour Technol 90: 33–38. PMID: 12835054
30. Shi H, Li X, Gu H, Zhang Y, Huang Y, et al. (2013) Biochemical properties of a novel thermostable and
highly xylose-tolerant β-xylosidase/α-arabinosidase from Thermotoga thermarum. Biotechnol Biofuels
6: 27. doi: 10.1186/1754-6834-6-27 PMID: 23422003
31. Wang L, Huang W (2009) Enzymatic transglycosylation for glycoconjugate synthesis. Curr Opin Chem
Biol 13: 592–600. doi: 10.1016/j.cbpa.2009.08.014 PMID: 19766528
32. Dilokpimol A, Nakai H, Gotfredsen CH, Appeldoorn M, Baumann MJ, et al. (2011) Enzymatic synthesis
of β-xylosyl-oligosaccharides by transxylosylation using two β-xylosidases of glycoside hydrolase fami-
ly 3 from Aspergillus nidulans FGSC A4. Carbohyd Res 346: 421–429. doi: 10.1016/j.carres.2010.12.
010 PMID: 21215963
33. Eneyskaya VE, Brumer H, Backinowsky LV, Ivanen DR., Kulminskaya AA, et al. (2003) Enzymatic syn-
thesis of β-xylanase substrates: transglycosylation reactions of the β-xylosidase from Aspergillus sp.
Carbohyd Res 338: 313–325. PMID: 12559729
34. Puchart V, Biely P (2007) A simple enzymatic synthesis of 4-nitrophenyl β-1,4-D-xylobioside, a chromo-
genic substrate for assay and differentiation of endoxylanases. J Biotechnol 128: 576–586. PMID:
17223215
35. Jordan DB, Wagschal K (2010) Properties and applications of microbial β-D-xylosidases featuring the
catalytically efficient enzyme from Selenomonas ruminantium. Appl Microbiol Biotechnol 86: 1647–
1658. doi: 10.1007/s00253-010-2538-y PMID: 20352422
PLOS ONE | DOI:10.1371/journal.pone.0117578 February 6, 2015
14 / 14