Enzyme and Microbial Technology 54 (2014) 1–7

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Enzyme and Microbial Technology

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A new bi-modular endo-␤-1,4-xylanase KRICT PX-3 from whole

genome sequence of Paenibacillus terrae HPL-003
Ha Young Song a , Hee Kyung Lim a , Dal Rye Kim a , Kee In Lee a,b , In Taek Hwang a,b,∗
a
Biorefinery Research Group, Green Chemistry Division, Korea Research Institute of Chemical Technology, Yusoeong P.O. Box 107, Daejon 305-600, Republic
of Korea
b
Department of Green Chemistry and Environmental Biotechnology, University of Science and Technology, 217 Gajungro, Yuseong-gu, Daejon 305-350,
Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: A new bi-modular, wide pH spectrum and highly active xylanase KRICT PX3 (JF320814) isolated from
Received 2 May 2013 Paenibacillus terrae HPL-003 (KCTC11987BP) has been cloned and expressed in Escherichia coli. Purified
Received in revised form 4 September 2013 recombinant xylanase KRICT PX-3 (1,620 bp, 540aa, NCBI accession number JF320814) showed highly
Accepted 5 September 2013
active at 55 ◦ C in pH 4.0–11.0, and stability for at least 24 h at 50 ◦ C, and exhibited Km and Vmax of
0.2 mg/mL and 153.8 U/mg on birchwood xylan. Most common ions did not affect the enzyme activity at
Keywords:
1 mM concentration. This enzyme could belong to glycoside hydrolase family 10 because hydrolyzed glu-
Multi-domain
curonoxylan and arabinoxylan substrate to xylobiose, xylotriose, and some traces of xylose as hydrolysis
Paenibacillus terrae
Xylanase
products. Model 3-D structure was composed of two domains, the catalytic domain of a (␤/␣)8 barrel
Xylan binding domain fold while the small domain probably functions as a xylan binding domain, and the two domains are
connected by a flexible linker peptide (PPLAIEKDIPSL). However, sequence alignment between xylan-
binding module in this xylanase KRICT PX3 and CBM22 showed 21% of identity and 35% of similarity.
This xylanase structure showed a distinctive group of enzyme cluster separately from the rest of GH10
xylanases, and seems to constitute a new type of xylanases.
© 2013 The Authors. Published by Elsevier Inc. Open access under CC BY-NC-ND license.

1. Introduction processes and equipment to produce fuels, power, and chemi-

Enzymes are extremely efficient and highly specific biocatalysts.
Since decades they have been used extensively in industrial pro-
cesses. Because environmental pollution due to toxic wastes has
been a major irritant to industrial development, enzymes are more
readily used in many industrial processes. With the advancement in
biotechnology, the increasing demand to amend or to replace tradi-
tional chemical processes with biotechnological processes includes
xylanases and ligninase in pulp and paper industry, cellulases for
fiber refining and deinking, pectinases in leather and food industry,
proteases for detergents, and lipases in food, dairy and pharmaceu-
tical industries [1–5].
A new and promising field for industrial application is the
biorefinery which is a facility that integrates biomass conversion
∗ Corresponding author at: Biorefinery Research Group, Green Chemistry Division,
Korea Research Institute of Chemical Technology, Yusoeong P.O. Box 107, Daejon
305-600, Republic of Korea. Tel.: +82 42 860 7447; fax: +82 42 860 7034.
E-mail address: ithwang@krict.re.kr (I.T. Hwang).
cals from biomass. It has gained considerable interest to develop
cost-effective, environmentally friendly procedures for the pro-
duction of chemicals from renewable feedstocks as a consequence
of increasing oil prices and diminishing reserves. Most agricul-
tural residues contain 30–50% cellulose, 20–40% hemicellulose, and
10–20% lignin based on their dry matter. Xylan is the major com-
ponent of hemicellulose, and its basic structure consists of a main
chain of ␤-1,4-linked d-xylopyranosyl residues; they can be con-
verted into soluble sugars either by acid or enzymatic hydrolysis,
so they can be used as a plentiful and cheap source of renewable
energy in the world [4–7].
Xylan is a complex polysaccharide comprising a backbone of
xylose residues linked by ␤-1,4-glycosidic bonds with the main
chain composed of ␤-xylopyranose residues. It is the second most
abundant biopolymer after cellulose and the most common hemi-
cellulosic polysaccharide in cell walls of land plants, representing
up to 15–30% and 7–12% of the total dry weight in hardwood from
angiosperms and softwood from gymnosperms, respectively, and
xylan is the most abundant hemicelluloses [3,6–9].
Bioconversion with xylan-degrading enzymes has received
much attention because of chemical hydrolysis of lignocellu-
loses results in hazardous byproducts, phenolic compounds from
lignin degradation, furan derivatives (furfural and HMF) from

0141-0229 © 2013 The Authors. Published by Elsevier Inc. Open access under CC BY-NC-ND license.
http://dx.doi.org/10.1016/j.enzmictec.2013.09.002
2 H.Y. Song et al. / Enzyme and Microbial Technology 54 (2014) 1–7
sugar degradation and aliphatic acids (acetic acid, formic acid, and
levulinic acid) are considered to be fermentation inhibitors gener-
ated from chemical conversion of lignocellulosic biomass [10–12].
Recently, the use of microbial enzymes which are specific in action
for xylan hydrolysis has been accepted as an environmentally
friendly option. Also, the systems of cellulose-free xylanases have
been receiving more attention, especially in systems which the
degradation of xylan without breaking cellulose fiber is required,
like in the paper and pulp industries [1–5].
Xylanases (EC. 3.2.1.8) hold great promise in saccharifying
xylans from various pretreated agricultural and forestry residues
to fermentable 5-carbon sugars, owing to numerous possibilities
of five-carbon (C5) chemistry for industrial applications. There are
different types of xylanases varying in substrate specificities, pri-
mary sequences, folds and physicochemical properties and these
are produced by a number of bacteria and fungi [5–7,11,13]. Xylan-
degrading enzymes of micro-organisms are potentially important
in various industrial processes at high temperature and broad range
of pH condition. Owing to the increasing biotechnological impor-
tance of xylanases, interest in industrially applicable xylanases
has markedly increased. Thus, many attempts are being made
to isolate new strains and to discover more relevant xylanases.
Various strains isolated from soil in a variety of locations have
been screened to detect bacteria with high xylanase activities
[7,11,13,14]. We also isolated bacteria with high xylanase activi-
ties from soil found in forest residue on Gara Mountain in Korea.
This strain was identified as Paenibacillus terrae by 16S rRNA
gene sequencing and physiological analysis, showing the high-
est 16S rRNA gene sequence identity, 99.4%, to strain AM141T
(NCBI accession number AF391124). P. terrae HPL-003 is a Gram-
positive, endospore-forming, xylanase-producing bacterium, and
whole-genome sequencing was carried out in order to facilitate
understanding of the unique properties of P. terrae HPL-003 at the
genomic level [15].
In the present study, we cloned the KRICT PX3 gene (Gen-
Bank accession code: JF320814) from P. terrae and expressed in
Escherichia coli. We also examined some biochemical properties
and predicted the biomolecular 3D structure of KRICT PX3 xylanase
with a deduced amino acid sequence as a homology model struc-
ture of the purified enzyme.
2. Materials and methods
2.1. Cloning and construction of xylanase expression system
Whole genome sequence of P. terrae HPL-003 used in this study as a source
of the xylanase gene was sequenced during our previous study [15]. By sequence
similarity search with referred to as xylanase, we detected seven putative xylanase-
encoding genes (xy1–xy7) within the P. terrae HPL-003 genome. For amplifying these
genes from chromosomal DNA by PCR, all seven sets of specific oligo-nucleotide
primers were designed based on the DNA sequence data. The each PCR prod-
uct was cloned into pSTV28 plasmid vector (Takara Bio Inc.) and transformed to
E. coli JM109 (Promega), and the xylanase activity of each recombinant E. coli was
examined by xylan-overlaid plate and DNS assay in liquid. The most xylanase-
active plasmid inserted with xy1 was selected and designated as KRICT PX3. For
high expression of the gene, KRICT PX3 was amplified with generated primers,
5 -CTCGAGATGGATACATTGAAGTTGTATGTG-3 (sense, PX3F with XhoI site as under-
lined) and 5 -GGATCCCTATTCGTTGCTCCCC-3 (antisence, PX3R with BamHI site as
underlined) and inserted into the pIVEX GST fusion expression vector (Roche Applied
Science) using XhoI and BamHI site. The constructed Plasmid, pIVEX GST-PX3, was
transformed into E. coli BL21 (Promega) to produce the recombinant GST fusion
protein.
2.2. Expression and purification of xylanase
The E. coli BL21 containing a recombinant plasmid (IVEX-GST-PX3) cell were
harvested from an overnight in 1 ml LB medium containing ampicillin (100 ␮g/ml)
at 37 ◦ C and 200 rpm in a shaking incubator. The overnight culture was diluted
with 200 ml of new LB medium and grown until the optical density at 600 nm
was 0.6. Expression was induced by adding 1 mM IPTG and incubation continued
under the same conditions for 3 h longer. The cells were collected by centrifugation
at 6000 × g for 10 min, washed in 10 ml of ice-cold 1X phosphate-buffered saline
(PBS, Sigma–Aldrich), repeated triple times. The cells from the final washing pro-
cess was resuspended in the lysis buffer (pH 7.0, 200 mM Tris–HCl, 10 mM NaCl,
10 mM ␤-mercaptoethanol, 1 mM EDTA), and disrupted by sonication with sonic
disruptor (CosmoBio Co., LTD). After cell disruption, the cell debris was removed
by centrifuged at 10,000 × g for 20 min at 4 ◦ C, the GST fusion xylanase was purified
from the crude extract using GST binding resin column (Novagen, Madison WI, USA).
The column was previously equilibrated with the washing buffer (pH 7.0, 50 mM
Tris–HCl, 100 mM NaCl), then the lysate was applied to the equilibrated column
with the flow rate at 10–15 cm/h. After washing the GST resin with 20 bed volumes
of cold washing buffer, treated with restriction protease Factor Xa (Roche Applied
Science) for removed GST domain from the fusion protein, and the fusion protein was
eluted with elution buffer. Then the extract was loaded on p-aminobenzamidine-
agarose column (Sigma–Aldrich) according to the manufacturer’s instruction for
removed protease Factor Xa. Only KRICT PX3 xylanase was eluted and fraction-
ated with freshly made elution buffer (pH 7.0, 50 mM Tris–HCl, 150 mM NaCl). All
fractions were examined with Bradford’s protein determination (Bradford reagent,
Sigma–Aldrich)), Sodium dodecyl sulfate-polyacrylamide gel (15% polyacrylamide)
electrophoresis (SDS-PAGE) analysis, and DNS assay [16,17].
2.3. Properties of recombinant xylanase
Xylanase activity was measured according to the method slightly modified from
Saha using 50 ␮l of 1% (w/v) solution of birchwood xylan (Fluka) and 200 mM of each
pH buffer incubated with 30 ␮l of an appropriately diluted enzyme (3.3 mg/ml) for
20 min at different temperatures. The released reducing sugars were assayed using
the DNS method [16]. One unit of xylanase activity was defined as the amount of
the enzyme that liberated reducing sugar ends equivalent to 1 ␮mol of xylose per
minute under the assay conditions. The optimal temperature and pH condition for
the xylanase activity of recombinant KRICT PX3 protein were examined in 96-well
micro plates with DNS assay at various temperatures (ranging from 10 to 80 ◦ C)
and pH conditions (ranging from pH 2.0 to 12.0). The effect of birchwood xylan
concentration on xylanase activity was evaluated under optimal assay conditions.
Diluted enzyme solution (100 ␮g protein in 30 ␮l) was incubated with 0.5 ml of var-
ious concentrations (0–10 mg/ml) of xylan in 50 mM glycine buffer (pH 9.0) at 50 ◦ C
for 20 min. Xylanase activity was measured with DNS assay at 540 nm as described
above. The kinetic parameters (Michaelis–Menten constant, Km and maximal reac-
tion velocity, Vmax ) were estimated by linear regression from double-reciprocal plots.
Effect of metallic ions and other chemicals on the xylanase activity of KRICT PX3
protein was studied as described above at pH 7 with addition of 1 mM NaCl, LiCl,
KCl, NH4 Cl, CaCl2 , MgCl2 , MnCl2 , CuSO4 , ZnSO4 , FeCl3 , CsCl2 , ethylenediamine tetra-
acetic acid (EDTA), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), phenylmethane
sulphonyl fluoride (PMSF), acetate, and furfural, respectively.
2.4. Analysis of hydrolytic products
Xylan hydrolysis was measured as follows: 1% of birchwood xylan, wheat ara-
binoxylan, and rye arabinoxylan were hydrolyzed with 50 ␮g of purified xylanase
KRICT PX3 in 10 ml vial under constant temperature of 50 ◦ C, with a stirring rate
of 200 rpm in a shaking indubator for 24 h reaction. Each portion of 10 ␮l, xylo-
oligomer standard, and enzyme blank was analyzed after clean up with boiling 5 min
and filtering with 0.2 ␮m syringe filter. HPLC (Younglin Instrument Acme 9000,
Korea) analysis was conducted under conditions of Shodex Asahipak column NH2 P-
50 4E, fluent of water/CH3 CN (1:3), flow rate of 1 ml/min., detector of RI (refractive
index), column temperature of 30 ◦ C.
2.5. Nucleotide sequence analysis and 3D structure modeling
Nucleotide and deduced amino acid sequences were analyzed with CLC Free
Workbench, Ver. 3.2.1 (CLC bio A/S, www.clcbio.com). Related sequences were
obtained from database searches (SwissPort and GenBank). The biomolecular 3D
structure of KRICT PX3 xylanase was predicted with a deduced amino acid sequence
as a homology model structure comparing to Xyn10B (PDB ID: 3emz) from Crystal
structure of xylanase B from Clostridium stercorarium F9 and Xylan-binding domain
from Carbohydrate-binding module(CBM) 22, formerly x6B domain with Discovery
Studio 2.5 (accelrys® ).
3. Results and discussions
3.1. Purification of recombinant xylanase KRICT PX3
Based on the screening of the bacteria for their ability to
hydrolyze xylan, the strain HPL-003 exhibited high xylanase activ-
ity. Strain HPL-003 was identified as P. terrae and designated
as P. terrae HPL-003 because of 99.4% similarity score. Seven
putative xylanase-encoding genes (xy1–xy7) were detected by
sequence similarity search with referred to xylanase within the
 H.Y. Song et al. / Enzyme and Microbial Technology 54 (2014) 1–7 3

Table 1
ORF for xylanase-encoding genes within the full genome sequence of the P. terrae HPL-003.

ORF ORF start ORF end Length AC GENE [SOURCE] BLAST P

Identities (%)

Pae1223 1238186 1236042 2145 ZP 09776461 Endo-1,4-beta-xylanase [Paenibacillus sp. Aloe-11] 82

Pae1433 1474357 1473401 957 YP 003949542.1 Xylanase b [Paenibacillus peoriae KCTC 3763] 93
Pae2662 2747462 2748100 639 YP 003948895.1 Endo-1,4-beta-xylanase [Paenibacillus polymyxa SC2] 96
Pae3487 3588445 3589467 1023 YP 003870032.1 Xylanase [Paenibacillus polymyxa E681] 95
Pae3491 3593213 3591603 1611 YP 003973307.1 Endo-1,4-beta-xylanase [Bacillus atrophaeus 1942] 88
Pae4427 4566882 4565344 1539 YP 003973307.1 Endo-1,4-beta-xylanase [Bacillus atrophaeus 1942] 81
Pae4446 4587823 4587503 321 ZP 08191157.1 Endo-1,4-beta-xylanase [Clostridium papyrosolvens DSM 2782] 50

full genome sequence of the P. terrae HPL-003 at the National M 1 2

Center for Biotechnology Information (NCBI) from the BLAST kDa
software. The information of putative xylanase-encoding genes
and similarities were endo-1,4-beta-xylanase [Paenibacillus sp.
Aloe-11] of 82%, xylanase b [Paenibacillus peoriae KCTC 3763] of
116 -
93%, endo-1,4-beta-xylanase [Paenibacillus polymyxa SC2] of 96%, 97 -
xylanase [Paenibacillus polymyxa E681] of 95%, endo-1,4-beta-
xylanase [Bacillus atrophaeus 1942] of 88%, endo-1,4-beta-xylanase 66 -
[Bacillus atrophaeus 1942] of 81%, and endo-1,4-beta-xylanase
[Clostridium papyrosolvens DSM 2782] of 50% (Table 1). All seven
55 -
sets of genes were cloned into pSTV28 plasmid vector (Takara Bio 45 -
Inc.) and transformed to E. coli JM109 (Promega). Among seven
genes a bacterial recombinant carrying xy1 was selected as the
36 -
most xylanase-active plasmid by measuring the reducing sugars
with xylan-overlaid plate and DNS assay in liquid and designated
as xylanase KRICT PX3. The genome sequence of KRICT PX3 was sub- 29 -
mitted to GenBank, and assigned as Accession Number JF320814.
24 -

3.2. Characterization of recombinant xylanase KRICT PX3

The final preparation of KRICT PX3 xylanase gave a major single

band on SDS-PAGE (Fig. 1), with a molecular weight (MW) of 65 kDa,
which appeared just below the 66 kDa MW marker (Fig. 1, lane 2).
Also, the GST-fused KRICT PX3 protein of MW 91 kDa was observed
SDS-PAGE analysis. M : Marker,
slightly below the 97 kDa MW markers (Fig. 1, lane 1). The optimal
lane 1 : PX3 cell lysate,
pH, showing maximal xylanase activity, was measured at a pH of
lane 2 : Only PX3
7.5, which was considered 100% activity, and retaining about 98% of
Fig. 1. SDS PAGE analysis of the purified KRICT PX3 (MW 65 kDa, lane 2) from cell
its activity at a pH range of 7–8, and about 90% of its activity at a pH lysate (lane 1) over-expressed with GST-fused xylanase in E. coli BL21 (91 kDa, fused
range of 6–9, respectively (Fig. 2A). About 50% of the activity was with GST 26 kDa and KRICT PX3 65 kDa), and molecular weight markers (lane M).
maintained even at pH of 4 and 11. The optimal temperature for xylanase.
the KRICT PX3 xylanase activity appeared to be 50–60 ◦ C (Fig. 2B).
The KRICT PX3 xylanase was very stable for 24 h at 50 ◦ C; however,
the activity of the KRICT PX3 xylanase sharply decreased at 60 ◦ C determined to be 0.2 mg/mL and 153.8 U/mg proteins, respectively
after incubation for 10 min (Fig. 3). Kinetic analysis of this xylanase (Fig. 4).
KRICT PX3 with birchwood xylan was performed at 50 ◦ C in pH 7.0 There are various xylanase produced by a number of bacte-
(Fig. 3). Km and Vmax values of the protein for birchwood xylan were ria and fungi [7,13], so far, there have been a rare reports of

A 150 B
150
Xylanase activity (Unit)

Xylanase activity (Unit)

100
100

50 50

0 0
0 3 4 5 6 7 8 9 10 11 0 40 50 60 70
pH Temperature (oC)

Fig. 2. The xylanase activity of KRICT PX3 at various pH (A) and temperature (B) conditions. The enzyme was assayed at 50 ◦ C in 50 mM citric buffer (pH 2∼6.5), phosphate
buffer (pH 7∼9), and glycine buffer (pH 9.5∼12), and also assayed at the temperature range of 40∼70 ◦ C in 50 mM phosphate buffer (pH 7.0), respectively. activity.
4 H.Y. Song et al. / Enzyme and Microbial Technology 54 (2014) 1–7

Table 2
100 Effect of metallic ions and other chemicals on the xylanase KRICT PX3 activity.

Additives (1 mM) Relative Additives (1 mM) Relative

xylanase activity (%)

80 activity (%) activity (%)

50oC None 100 CuSO4 26

NaCl 105 ZnSO4 72
60 60oC LiCl 101 FeCl3 88
KCl 101 EDTA 54
NH4Cl 99 2-ME 89
40 CaCl2 102 DTT 95
MgCl2 93 PMSF 95
MnCl2 98 Acetate 101
20 CsCl2 97 Furfural 98

0
10 20 30 40 50
Time (min)
Fig. 3. Stability of KRICT PX3 at two temperatures. The activity was assayed at sev-
eral intervals in 50 mM phosphate buffer (pH 7.0) keeping under 50 or 60 ◦ C water
bath.
xylanases having wide pH range (pH 4.0–11.0), highly activity at
50 ◦ C exhibited specific activity of 153.8 ␮mole/min/mg proteins
on birchwood xylan and stability for at least 24 h at 50 ◦ C (data not
shown) from records of the genus Paenibacillus [8,9,11,18–29].
Most salts, such as NaCl, LiCl, KCl, NH4 Cl, CaCl2 , MgCl2 , MnCl2 ,
FeCl2 , and CsCl2 , did not significantly change the enzyme activity
at 1 mM. Also, 1 mM of ethylenediamine tetra-acetic acid, 2-
mercaptoethanol, phenylmethanesulphonyl fluoride, and furfural
were not effective on the enzyme activity (Table 2). Chemi-
cal hydrolysis of lignocelluloses results in hazardous byproducts,
phenolic compounds from lignin degradation, furan derivatives
(furfural and HMF) from sugar degradation, and aliphatic acids
(acetic acid, formic acid, and levulinic acid) are considered to be
fermentation inhibitors generated from chemical conversion of lig-
nocellulosic biomass [10]. However, most common ions did not
affect this enzyme activity at 1 mM concentration.
The degradation profile of birchwood xylan, wheat arabinoxy-
lan, and rye arabino xylan by KRICT PX3 xylanase, monitored
through the HPLC analysis, revealed that the major hydrolysis prod-
uct was xylobiose with smaller amounts of xylose, and xylotriose
(Fig. 5). The amount of xylobiose especially continued to increase
during the entire reaction. This indicates that KRICT PX3 is an
0.10 Y = 0.0013x + 0.0066
r2 = 0.9913, Km = 0.2, Vmax = 153.8
0.08
endo-type xylanase with similar catalytic property of most GH10
60 xylanases [7,30,31].
Based on primary structure comparisons of the catalytic
domains, most xylanases have been classified as family 10 or 11
of glycoside hydrolases, whereas a few have been classified as
family 5, 7, 8, or 43 (http://afmb.cnrs-mrs.fr/CAZY/) [7]. However,
a few xylanases that exhibit homology to enzymes belonging to
other glycoside hydrolase families have also been described [7,37].
Among them, three xylanases of family 30 (GH30), previously clas-
sified in family 5, have been biochemically characterized. These
enzymes have an absolute requirement of methyl-glucuronosyl
(MeGlcA) side residues for hydrolysis of xylan, releasing as products
xylooligosaccharides substituted in the penultimate xylose from
the reducing end [39,40]. The distinctive trait is their specificity for
glucuronoxylans; they show high activity on glucuronoxylans but
none on arabinoxylans.
The KRICT PX3 xylanase from the Paenibacillus terrae strain HPL-
003 seems to be an extracellular enzyme. There is some evidence
supporting this assumption. First, the xylan-degrading activity was
observed to have a significantly larger-sized halo in the xylan-
overlaid agar plate. When the gene was transformed to E. coli, its
activity was monitored in both detection methods using the xylan-
overlaid agar plate and the DNS assay of culture media, respectively.
Also, the results of location prediction of KRICT PX3 xylanase by
PSORT-B and ProtCompB computer analysis programs indicate that
KRICT PX3 is an extracellular enzyme [32]. This character of the
xylanase is different from intracellular or extracellular xylanases,
such as xylanase B from Paenibacillus sp. BP-23 and xylanase 5 from
Paenibacillus sp. strain W-61 [33] were intracellular xylanase, but
the XynA1 xylanase from Paenibacillus sp. strain JDR-2 is similar
to extracellular xylanase. Extracellular xylanases are considered
important from an industrial viewpoint, as they ease the extraction
procedure during mass production with a simple and cost-effective
bioprocess [34].
3.3. Nucleotide sequence analysis and 3D structure modeling

0.06 Analysis of KRICT PX3 with CDD (Conserved Domain Database)

at NCBI indicated that the xylanase KRICT PX3 contains N-terminal,
1/V

carbohydrate binding domain 22 (CBM 22) domain and C-terminal,

0.04 a catalytic domain of glycosyl hydrolase family 10 (GH10). From the
amino acid sequence analysis and alignment with other microbial
xylanase, it was confirmed that KRICT PX3 xylanase is composed
0.02 of two domains, the larger domain contains the catalytic site and
displays a (␤/␣)8 barrel fold while the small domain probably func-
tions as a xylan binding domain. The two domains are connected
0 by a flexible linker peptide (PPLAIEKDIPSL). Catalytic domain is
0 10 20 30 40 50 60 70 belongs to the GH10 family with many highly conserved regions
1/S and almost similar essential amino acids for the catalytic activ-
ity [7], [32]. However, sequence alignment between xylanse KRICT
Fig. 4. Lineweaver–Burk double reciprocal analysis of KRICT xylanase with birch-
wood xylan as the substrate. The enzyme activity was measured with various PX3 and template of crystal structure of xylanase B from Clostrid-
concentrations of birchwood xylan in 50 mM phosphate buffer (pH 7.5) at 50 ◦ C. ium stercorarium F9 reveals 56% identity and 75% similarity at 1.8 Å
 H.Y. Song et al. / Enzyme and Microbial Technology 54 (2014) 1–7 5

Fig. 5. HPLC analysis of the products after hydrolysis of glucurono- and (1.0%) by KRICT PX3 xylanase for 24 h in 50 mM sodium phosphate buffer (pH 7.0) at 50 ◦ C, respectively.
arabino-xylans.

Fig. 6. Xylanase KRICT PX3 homology model structure (Dark: identical/similarity region, Bright: no match) simulated the crystal structure of xylanase B from Clostridium
stercorarium F9 as a template showing the identity and similarity of 56 and 75 percent with 340 amino acids, respectively.
6 H.Y. Song et al. / Enzyme and Microbial Technology 54 (2014) 1–7
resolutions (Fig. 6). On the basis of amino acid sequence similarity,
sequence alignment between xylanse KRICT PX3 and template of
xylan-binding domain from Carbohydrate-Binding Module (CBM)
22, formerly x6B domain is revealed 21% identity and 35% similar-
ity at 2.1 Å resolutions. This xylanase structure showed a distinctive
group of enzyme with low identity and similarity that cluster sep-
arately from the rest of GH10 xylanases, and seem to constitute a
new type of xylanases [35].
To date, six high-resolution X-ray structures of the catalytic
domain of the family 10 xylanases have been available, and
substrate-binding domains of xylanases and cellulases are classi-
fied into more than ten cellulose binding domain (CBD) families on
the basis of amino acid sequence. Some substrate-binding domains
in the xylan degrading enzymes are specific for xylan and therefore
they are referred to as xylan-binding domains (XBDs). Among them,
XBDs of XlnA and arabinofuranosidase B from S. lividans are clas-
sified as a new family of substrate-binding domains in cellulases
and hemicellulases [36,41,42]. The biochemical characterization of
a new GH10 xylanases having new xylan-binding domains (KRICT
PX-3) will be required to compare with the rest of GH10 xylanases,
in order to understand their role in xylan hydrolysis and their con-
tribution to biomass degradation.
4. Conclusions
In conclusion, xylanase KRICT PX3 cloned from Paenibacillus ter-
rae HPL-003 was found to be a novel xylanase. The enzyme was
stable over a wide pH range and good stability for at least 24 h at
50 ◦ C. This xylanase allows the potential application in saccharifica-
tion processes essential for bio-ethanol and bio-based production,
bleaching pulp and paper industry, fiber refining and deinking, food
and pharmaceutical industries to save time and costs during the
xylan-related process. From this perspective, KRICT PX3 xylanase
is attractive for further development and application through the
modification by rational design of proteins or by directed molecular
evolution of proteins [24], which are ongoing in our further studies.
Maybe this domain represents a new type of carbohydrate-binding
module (CBM) or stabilizing domain. To confirm that the small
domain plays a functional role in the xylan binding, the authors
should study xylan and/or insoluble polysaccharide-binding ability
of this domain and analyze its function [41,42].
Acknowledgements
This study was supported by a grant 10035574 funded by the
Ministry of Knowledge Economy, Republic of Korea.
Added and rearranged
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