J. Microbiol. Biotechnol.
(2013), 23(3), 397–404
http://dx.doi.org/10.4014/jmb.1212.12017
First published online January 19, 2013
pISSN 1017-7825 eISSN 1738-8872
An β-1,4-Xylanase with Exo-Enzyme Activity Produced by Paenibacillus
xylanilyticus KJ-03 and Its Cloning and Characterization
Park, Dong-Ju†, Yong-Suk Lee†, Jie Chang, Shu-Jun Fang, and Yong-Lark Choi*
Department of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea
Received: December 6, 2012 / Revised: December 27, 2012 / Accepted: December 28, 2012
Paenibacillus xylanilyticus KJ-03 was isolated from soil
samples obtained from a field with Amorphophallus
konjac plants. A gene encoding xylanase was isolated from
KJ-03 and cloned using a fosmid library. The xynA gene
encodes xylanase; it consists of 1,035 bp and encodes 345
amino acids. The amino acid sequence deduced from the
P. xylanilyticus KJ-03 xylanase showed 81% and 69%
identities with those deduced from the P. polymyxa E681
and Paenibacillus sp. HPL-001 xylanases, respectively.
The xynA gene comprises a single domain, consisting of a
catalytic domain of the glycosyl hydrolase (GH) 10 family.
The xynA gene was expressed in Escherichia coli BL21
(trxB), and the recombinant xylanase was purified by Ni-
affinity chromatography. The purified xylanase showed
optimum activity with birchwood xylan as a substrate at
40oC and pH 7.4. Treatment with Mg2+ and Li+ showed a
slight decrease in XynA activity; however, treatment with
5 mM Cu2+ completely inhibited its activity. The results of
the thin layer chromatography analysis indicated that the
major hydrolysis product was xylobiose and small amounts
of xylose and xylotriose. XynA showed increased activity
with oat spelt xylan and birchwood xylan, but showed
only slight activity with locust bean gum.
Keywords: Paenibacillus xylanilyticus KJ-03, xylanase,
genome library, purification, transxylosylation
Hemicelluloses are non-cellulosic polysaccharides including
xylan, galactan, and arabinan, and are found in plant cell
walls [1, 3]. Xylan is the major hemicellulose in plant cell
walls and the most abundant polysaccharide in nature next
to cellulose [8, 17-19]. It is a heterogeneous polysaccharide,
consisting of a backbone structure of β-1,4-linked D-
*Corresponding author
Phone: +82-51-200-7585; Fax: +82-51-200-6536;
E-mail: ylchoi@dau.ac.kr
†
These authors contributed equally to this work.
xylopyranose residues with O-acetyl, 4-O-methyl-D-
glucuronosyl, and α-L-arabinofuranosyl residues in side
chains [12, 27]. Recently, xylan has generated considerable
interest as a renewable biomass that is capable of producing
biofuels and chemicals [1]. Biodegradation of xylan was
carried out by several enzymes such as β-1,4-endoxylanase,
β-xylosidase, α-L-arabinofuranosidase, α-glucuronidase,
acetyl xylan esterase, and phenolic acid esterase [23, 29].
Among these enzymes, β-1,4-xylanases are the primary
enzymes for the degradation of xylan, because they can
randomly hydrolyze the backbone structure of β-1,4-xylans
to degrade polysaccharides. Finally, β-xylosidases catalyze the
hydrolysis of xylooligosaccharides to smaller oligosaccharides,
and the side chains in xylan are released by α-L-
arabinofuranosidases, α-glucuronidases, and acetyl xylan
esterases [8].
Based on the amino acid sequences similarity, xylanases
are divided into the glycosyl hydrolase (GH) 10 and 11
families. GH10 family xylanases [2, 7, 10] are acidic pI
and high molecular mass (>30 kDa) enzymes, and have
specificity towards substituted xylans, whereas GH11
family xylanases [14, 16, 19] are alkaline or acidic pI and
low molecular mass enzymes, and have high specificity
towards xylan substrates. Most of the GH10 family xylanases
consist of a catalytic domain, carbohydrate-binding module,
and other functional domains with link sequences, whereas
GH11 family xylanases have a single catalytic domain and
a high similarity of amino acids [22].
Xylanases from microorganisms have attracted considerable
interest recently because of their biotechnological potential
applications in various industries [3]. The xylanase
pretreatment for bleaching of pulp and paper can reduce
the consumption of chlorine-containing bleaching chemicals
and prevent wastewater pollution in the environment.
However, paper and pulp industries prefer cellulase-free
xylanases with an optimal activity at broad pH and
temperature values [1]. It also plays an important in clarifying
juices and improving the agricultural silage production. In
398 Park et al.
the bakery industry, xylanase makes the doughs soft, and
sugars such as xylose and xylooligosaccharides are used
by the enzymatic hydrolysis of xylan [3]. Undigested fibers
from hemicelluloses increase the viscosity of the feed in the
intestine of animals and decrease the nutritive value of the
feed [26]. Xylanase can degrade hemicellulose in feed to
improve animal nutrition and also enhance the digestibility
of animal feed. Recent studies on xylanases have also been
focused on production of fermentable xylose that is essential
for a cost-effective conversion of lignocellulosic biomass
to bioethnol [9]. Therefore, microbial strains hydrolyzing
lignocelluloses are generating more considerable interest
in the bioethanol industry.
Many xylanases from bacteria and fungi have been
cloned and the enzymes obtained have been characterized
[15, 24]. There have been reports on the production of
cellulase-free xylanases in Bacillus and Streptomyces [19,
29]. Specifically, Paenibacillus species are able to produce
a variety of polysaccharide-degrading enzymes that are
related to the metabolism of plant carbohydrate polymers
[5]. Paenibacillus that produces useful industrial enzymes
has potential applications in various industries.
In a previous report, the isolation of P. xylanilyticus KJ-
03 from soil samples was described [21]. In this study, we
describe the cloning and expression of the xylanase gene
from P. xylanilyticus KJ-03 in E. coli. The enzyme was
purified and characterized from the recombinant strain.
MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Culture Media
P. xylanilyticus KJ-03 was identified by its 16S rDNA nucleotide
sequence, which has been assigned as the GenBank accession
number HQ258920.1 in the previous study [21]. P. xylanilyticus KJ-
03 was used as a source of genomic DNA for XynA gene cloning.
The strain KJ-03 was grown on tryptic soy medium supplemented
with 0.2% birchwood xylan. pUC118 (Takara, Japan) and pET-32a(+)
(Novagen, Germany) were used as cloning and expression vectors,
respectively. Escherichia coli (E. coli) JM109 and E. coli BL21 (trxB)
were used as host cells for gene cloning. These strains were cultured
in Luria-Bertani medium (LB) supplemented with ampicillin
(100 µg/ml) or kanamycin (50 µg/ml) at 37oC, where appropriate.
Construction and Screening of Genome Library
The genomic DNA from P. xylanilyticus KJ-03 was prepared as
described by Sambrook and Russel [25] and sheared approximately
into 23-40 kb fragments using a pipette. The sheared DNA was
blunt-end repaired and then electrophoresed to collect 23-40 kb
DNA fragments. The prepared DNA fragments were ligated into the
pCC1FOS fosmid vector (Epicentre) and packaged using the Lamda
Packaging Extracts (Epicentre). The packaged library was transformed
into E. coli EPI300-T1R. The library clones were plated on the LB
agar plate supplemented with 0.2% Remazol Brilliant Blue R-xylan
(RBB-xylan) and 12.5 µg/ml chloramphenicol for xylanase activity
[4]. The clones were incubated for 2 days at 37oC, and the positive
clone (Fosxyl-2) showed a clear zone around colonies against a blue
background. The fosmid DNA of Fosxyl-2 was purified by a fosmid
purification kit (Epicentre). The DNA partially digested with Sau3AI,
and 2-10 kb DNA fragments were ligated into the BamHI-digested
pUC118 and transformed into E. coli JM109. A clone, pXyl-14
forming clear zone around colonies, was selected on LB agar plate
supplemented with 0.2% RBB-xylan. Homology searches of nucleotide
sequences obtained were carried out by using the BLAST at the
National Center for Biotechnology Information (NCBI). A phylogenetic
tree was conducted using the ClustalX program.
Expression of the Recombinant Xylanase
The xylanase gene from KJ-03 was amplified by PCR using the forward
and reverse primers as follows: forward primer, 5'-AGTGAATTC
ATGGAATCCAGGCGTGAA-3'; reverse primer, 5'-GTAGTCGAC
TCACTTTTTCTCCATTGCAGC-3'. EcoRI and SalI sites were added
to the forward and reverse primers, respectively. The PCR was
performed with Ex Taq DNA polymerase (Takara, Japan) (30 s at
95oC, 30 s at 64oC, 60 s at 72oC). The amplified DNA fragment was
cloned into the pET-32a(+) vector. The recombinant plasmid pET-
XynA was transformed into E. coli BL21 (trxB). Transformed E. coli
BL21 (trxB) cells were grown at 37oC in LB medium with 100 µg/ml
ampicillin and 50 µg/ml kanamycin to an OD600 of 0.5. Expression was
induced by adding 0.05 mM isopropyl-β-D-thiogalactopyranoside
(IPTG) and the cells were cultured again at 4 h.
Purification of Xylanase and Zymogram Analysis
The cells were harvested by centrifugation at 6,000 rpm for 10 min,
suspended in 20 mM sodium phosphate (pH 7.4), and then disrupted
by sonication. The clear supernatants were applied to a HisTrap HP
column (Amersharm Phamacia Biotech.) equilibrated with 20 mM
sodium phosphate (pH 7.4), 0.3 M NaCl, and 5 mM imidazole. The
bound proteins were eluted with 20 mM sodium phosphate (pH 7.4),
0.3 M NaCl, and 0.5 M imidazole. The fractions exhibiting xylanase
activity were desalted by dialysis against 20 mM sodium phosphate
buffer (pH 7.4) and concentrated using Amicon Ultra-4 (Millipore,
Ireland). The purified proteins were treated with enterokinase and
collected using a HisTrap HP column. The purified proteins were
analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE). The zymogram analysis was performed using the method
of Ratanakhanokchai et al. [23]. The enzyme was electrophoresed
on a 10% gel containing 0.1% birchwood xylan. The gel was
soaked in 25% isopropanol and then in 0.1 M sodium phosphate
(pH 7.4) for 20 min at 4oC. After incubation for 1 h at 40oC, the gel
was stained with 0.5% Congo red and washed with 1 M NaCl.
Enzyme Assays
Xylanase activity was determined by measuring the amount of
reducing sugars released from xylans using the dinitrosalicylic acid
(DNS) method [20]. The reaction mixture containing 50 µl of 0.1%
xylan, 198 µl of sodium phosphate (pH 7.4), and 2 µl of enzyme
solution was incubated at 40oC for 10 min. The reaction was stopped
by adding 0.75 ml of DNS. One unit of xylanase activity was defined
as the amount of enzyme required to produce 1 µmol of reducing
sugar per minute. To estimate the optimal temperature and pH, the
activity of the enzyme was determined by the standard assay conditions
at different temperatures (ranging from 10oC to 80oC) and pH (3.0-
10.6). For determination of the optimal pH, 50 mM of various buffers
[citrate buffer (pH 3.0-6.0), sodium phosphate buffer (pH 6.0-8.0),
 GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF XYLANASE FROM PAENIBACILLUS XYLANILYTICUS KJ-03
Tris-HCl buffer (pH 8.0-9.0), and glycine-NaOH buffer (pH 9.0-
10.6)] were used. For determining the temperature stability, the enzyme
was pre-incubated at different temperatures, 40oC, 45oC, and 50oC,
for 1 h without substrates. For the pH stability, the enzyme was pre-
incubated with various buffers at 4oC for 3 h. The remaining activity
was assayed at 40oC for 10 min. The effects of different metal ions
and chemical reagents on the enzyme activity were determined under
the standard assay conditions using various reagents such as 5 mM
Ca2+, Cu2+, Fe2+, Li+, Mg2+, Mn2+, Zn2+, Na+, dithiothreitol (DTT),
and ethylene diamine tetraacetic acid (EDTA).
Activities against 1% birchwood xylan, oat spelt xylan, locust
bean gum, carboxymethylcellulose (CMC), lichenan, and laminarin
were determined under the standard assay conditions using the DNS
method. When p-nitrophenyl-β-D-xylopyranoside (p-NP xyloside)
and p-nitrophenyl-β-D-glucopyranoside (p-NP glucoside) were used
as substrates, the enzyme activity was assayed colorimetrically by
measuring the absorbance at 410 nm. One unit of enzyme activity
was defined as the amount of enzyme that released 1 µmol of p-NP
per minute under the standard assay conditions.
TLC Analysis of Hydrolysis Products
The hydrolysis products of xylan and xylobiose by XynA were
analyzed by thin layer chromatography (TLC). The purified xylanase
was incubated with birchwood xylan for 3 h at 40oC. The hydrolysis
of xylobiose was carried out with the purified enzyme and xylobiose
in 20 mM sodium phosphate (pH 7.4) at 37oC for 24 h. An aliquot
(1 µl) of each sample was spotted on TLC silica gel 60 F254 plates
Fig. 1. Nucleotide and deduced amino acid sequences of the xynA gene.
The putative ribosomal binding site (SD) is underlined. The conserved Glu residues that are essential for GH10 family xylanase are boxed. The stop codon
is indicated by an asterisk (*).
399
(Merck, Germany). The plates were developed with a solvent of n-
butanol, acetic acid, and water (6:3:2), sprayed with aniline-diphenylamine
reagent (4 ml of aniline, 4 g of diphenylamine, 200 ml of acetone, and
30 ml of 85% phosphoric acid), and followed by heating at 150oC
for 3 min. Xylooligosaccharides such as xylose, xylobiose, xylotriose,
and xylotetraose (Megazyme, Ireland) were used as standards.
GenBank Accession Number
The nucleotide sequence of the xylanase studied in this work has
been deposited in the GenBank database under the accession number
HQ258919.
RESULTS
Cloning of the xynA Gene
P. xylanilyticus KJ-03 was isolated from soil samples
obtained from a field with Amorphophallus kojac plants.
When these microbes were cultured, they formed large
yellow zones with Congo red on each tryptic soy agar
(TSA) plate containing various polysaccharides such as
birchwood xylan, oat spelt xylan, CMC, glucomannan,
locust bean gum, and starch, respectively [21]. A fosmid
library of P. xylanilyticus KJ-03 genomic DNA was
constructed for screening the xynA gene. Approximately
2,000 fosmid clones were transferred to LB agar plates
400 Park et al.
Fig. 2. Phylogenetic tree of XynA and GH10 family xylanases
based on amino acid sequence homology.
The tree was implemented using the neighbor-joining method in ClustalX.
containing 0.2% RBB-xylan, of which 45 fosmid clones
formed clear zones around the colonies after incubation for
2 days. Among these, 1 fosmid clone was selected for
further study and was identified as Fosxyl-2. Sau3AI was
used for digesting 2-10 kb DNA fragments of Fosxyl-2;
these fragments were subcloned into pUC118. A single
clone, pXyl14, that hydrolyzed RBB-xylan was selected;
the hydrolysis was indicated by clear zones around the
colonies. The recombinant plasmid pXyl14 was found to
contain a 5 kb insert DNA.
The sequence analysis of the 5 kb DNA fragment obtained
from pXyl14 showed 4 complete open reading frames
(ORFs). ORF4 showed 81% homology with the endo-1,4-
β-xylanase isolated from P. polymyxa E681.The ORF4
was found to encode a protein of 345 aa with a molecular
mass of approximately 40 kDa and pI of 5.25. A putative
ribosomal binding site (AGGAGG) located 6 bp upstream
from the xynA start codon (ATG) is shown in Fig. 1.
SignalP did not detect any signal peptide at the N-terminal.
The XynA showed 2 conserved regions similar to those
found in xylanases of the GH10 family; these regions are
WDVVNE and TELD, each containing 2 Glu residues,
Glu-136 and Glu-245. A phylogenetic tree of 10 xylanases
in the GH10 family was conducted with the ClustalX
Table 1. Summary of purification steps of the recombinant XynA expressed in E. coli BL21 (trxB).
Purification step Total activity (U) Total protein (mg)
Cell extract 202 45.6
Ni-affinity chromatography 118 3.6
Fig. 3. SDS-PAGE and zymogram of XynA.
Lane 1, molecular weight markers; lane 2, cell-free lysate; lane 3, the
purified XynA by Ni-affinity chromatography; lane 4, the purified XynA
digested by enterokinase; lane 5, zymogram of the purified XynA for
xylanase activity.
program (Fig. 2). This result showed that the XynA was a non-
secretory enzyme, which was similar to signal peptide-less
xylanases in family GH10.
Purification and SDS-PAGE of Xylanase
The xynA gene was cloned into an expression vector, and
the recombinant xylanase was expressed and purified from
E. coli. The purification of XynA was 7.5-fold, with a
recovery yield of 58% (Table 1). The purified His-tagged
XynA was a single protein with a molecular mass of
68 kDa (Fig. 3). After removing the His-tag, XynA was
analyzed using SDS-PAGE, and the gel obtained showed
the presence of a 40 kDa molecule, which is similar to the
molecular mass obtained using nucleotide sequencing.
The purified enzyme had a specific activity of 33 U/mg.
XynA showed a strong active band in the SDS-PAGE gel
containing 0.1% birchwood xylan. This result indicated
that the apparent purified enzyme was KJ-03 xylanase.
Characterization of Xylanase
The purified xylanase showed optimum activity with
birchwood xylan at 40oC and was the most active between
10oC and 80oC (Fig. 4A). Xylanase showed up to 50% of
its optimal activity between 20oC and 60oC. The optimal
pH of XynA was determined to be 7.4 (Fig. 4B). Xylanase
showed activity at a broad pH range of 4.0-9.6 and 43%
maximum activity at pH 9.0. The thermal stability of XynA
was determined at various temperatures, 40oC, 45oC, and
Specific activity (U/mg) Purification fold Yield (%)
4.4 1 100
33 7.5 58
 GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF XYLANASE FROM PAENIBACILLUS XYLANILYTICUS KJ-03 401

Fig. 4. Effects of temperature and pH on the activity of the Fig. 5. Effects of temperature and pH on the stability of the
purified XynA. purified XynA.
(A) The activity of the enzyme was measured at pH 7.4 and temperature (A) The temperature stability of the enzyme was determined at 40oC ( ● ),
ranging from 10oC to 80oC. (B) The activity of the enzyme was measured 45oC ( ■ ), and 50oC ( ▲ ). (B) The pH stability of the enzyme was
at 40oC in citrate buffer (pH 3.0-6.0), sodium phosphate buffer (pH 6.0- determined by incubating the enzyme for 3 h at various pHs.
8.0), Tris-HCl buffer (pH 8.0-9.0), and glycine-NaOH buffer (pH 9.0-
10.6).
Table 2. Effect of metal ions and chemicals on the activity of
o
XynA.
50 C (Fig. 5A). After 1 h, xylanase showed 95% and 32%
Metal ions and chemicals Relative activity (%)
of its original activity at 40oC and 45oC, respectively, but
its stability dramatically decreased at 50oC. After incubating None 100
for 3 h at a pH of 7.0-9.6, xylanase retained more than CaCl2 34
50% of its activity (Fig. 5B). XynA was found to be stable at CuCl2 0
an alkaline pH range. The effect of treatment with various FeSO4 17
metal ions and other reagents on XynA activity was detected LiCl 94
(Table 2). Treatment with Cu2+ strongly inhibited the xylanase MgCl2 97
activity, whereas treatment with Zn2+ showed a 7% decrease MnCl2 68
in activity of the control sample. Treatment with DTT and ZnCl2 7
EDTA did not have much effect on the activity of the DTT 100
purified enzyme. The substrate specificity of the purified EDTA 96
enzyme was determined using various substrates. The enzyme
activity was detected by measuring the concentration of
reducing sugars or p-NP when various polysaccharides the enzyme also showed activity towards locust bean gum
and p-NP derivatives were used as substrates (data not (13%). XynA did not hydrolyze other polysaccharides such
shown). The enzyme was found to hydrolyze xylans such as CMC, lichenan, and laminarin. The enzyme had the
as birchwood xylan (70%) and oat spelt xylan (100%), and ability to hydrolyze p-NP xyloside, but not p-NP glucoside.
402 Park et al.
Fig. 6. Degradation of birchwood xylan (A) and transxylosylation
of xylobiose (B) by XynA.
(A) The reaction mixture was incubated at 40oC for 15 min (lane 2),
30 min (lane 3), 1 h (lane 4), and 3 h (lane 5). Lane 1, xylose (X1) to
xylotetraose (X4). (B) TLC of hydrolysis products from xylobiose by
XynA. Lane 1, xylobiose (X2) to xylotetraose (X4); lane 2, xylobiose was
incubated with XynA.
Hydrolysis Products of Xylan and Xylobiose
The results of the TLC analysis showed that XynA
degraded birchwood xylan and xylooligosaccharides to a
series of oligosaccharides and xylose, xylobiose, xylotriose,
and xylotetraose. The results particularly showed that the
major hydrolysis products of XynA activity on birchwood
xylan were xylobiose, xylotriose, xylotetraose, and small
amounts of xylose (Fig. 6A). Furthermore, the results
showed that the enzyme for xylooligosaccharide degraded
xylotriose and xylotetraose more efficiently than xylobiose.
Xylobiose and xylose were the major products released
from xylotriose and xylotetraose (data not shown). The
hydrolysis of xylobiose resulted in the synthesis of xylose
and xylotriose as the final products (Fig. 6B).
DISCUSSION
In this report, the xylanase gene was isolated from the
fosmid library of P. xylanilyticus KJ-03 cells. Although
many xylanases have been isolated from Paenibacillus sp.
[6-9, 12, 17, 24, 26], the present study is the first to report
the isolation of a xylanase gene cloned from P. xylanilyticus.
Henrissat [10] reported that most of the known xylanases
are classified into GH10 and GH11 families. Using the
BLAST search program, the amino acid sequence deduced
from the xylanase of KJ-03 was compared with that deduced
from the xylanase of other species. The results showed that
XynA has a single domain and is homologous to xylanases
belonging to the GH10 family. The amino acid sequence
deduced from XynA isolated from P. xylanilyticus was
81%, 69%, 68%, and 64% identical to that of xylanase
isolated from P. polymyxa E681, Paenibacillus sp. HPL-
001, Cohnella laeviribosi strain HY-21, and Geobacillus
thermodenitrificans NG80-2 in the GH10 family, respectively.
The molecular mass of the purified xylanase was approximately
40 kDa, and the calculated pI was found to be acidic. The 2
glutamic acid residues in XynA are conserved for catalytic
activity like in all the members of the GH10 family. XynA
does not have a signal peptide and has only a single
catalytic domain. These results indicate that the XynA
isolated in this study can belong to the GH10 family and
that it seems to be an intracellular enzyme. However, most
of the xylanases in the GH10 family consist of 2 or more
functional domains such as the catalytic domain and
carbohydrate-binding modules [8, 17, 19, 24]. A few xylanases
isolated from Paenibacillus sp. BP-23 and C. laeviribosi
HY-21 are intracellular and non-modular enzymes that
consist of a single catalytic domain without accessory
domains [7, 13]. Unlike most extracellular xylanases in the
GH10 family, XynA of KJ-03 is similar to a few xylanases
from Paenibacillus sp. BP-23 and C. laeviribosi HY-21.
The molecular mass of the purified enzyme was higher
than that of the enzyme isolated from Paenibacillus sp.
BP-23 (38 kDa), but was found to be lower than that of C.
laeviribosi HY-21 (42 kDa). The optimal activity of XynA
was reported at 40oC, which is a temperature similar to that
observed for the xylanases isolated from B. licheniformis
and Paenibacillus sp. HPL-001 and lower than that
observed for the xylanases isolated from P. barcinonensis
and P. curdlanolyticus B-6 [9, 16, 24, 26]. The optimal pH
of XynA was found to be similar to that of xylanases
isolated from P. curdlanolyticus B-6 (pH 7.0) and Cohnella
laeviribosi HY-21 (pH 7.5) [13, 24]. Xylanases from several
Paenibacillus sp. were reported to show optimal activity at
a pH between 5.5 and 6.5, except for xylanase isolated
from P. curdlanolyticus B-6. The metal ions Cu2+ and Zn2+
showed inhibition of the enzyme activity. Similarly, the
xylanases of Geobacillus sp. MT-1, Bacillus sp. strain K1,
and Paenibacillus sp. HPL-001 in family GH10 exhibited
a decrease in activity with Cu2+ and Zn2+ [9, 11, 23, 28].
These results suggested that metal ions may affect the
active site of the catalytic region and cause a structure
change of the xylanase.
XynA effectively hydrolyzed insoluble xylan. Oat spelt
xylan was hydrolyzed to a greater extent than birchwood
xylan. This may be because oat spelt xylan is not acetylated
and has few branches. However, unlike XynA, most of the
xylanases from the GH10 family show high substrate
specificity to birchwood xylan [1, 24, 26]. The hydrolysis
 GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF XYLANASE FROM PAENIBACILLUS XYLANILYTICUS KJ-03
of xylan by XynA was similar to that by GH10 family
xylanases isolated from P. barcinonensis, Paenibacillus sp.
HPL-001, and Clostridium acetobutylicum ATCC 824 [1,
9, 26]. However, the hydrolysis products from xylan and
xylooligosaccharides in some of the GH10 family xylanases
isolated from C. laeviribosi HY-21, P. curdlanolyticus B-6,
and P. thermophila [13, 24, 30] do not include xylose.
Although KJ-03 xylanase is an endo-β-1,4 xylanase, it
showed high activity with p-NPX, which indicates that
xylanase shows exo-type activity. Furthermore, XynA
hydrolyzed xylobiose to xylose and yielded a longer
oligosaccharide than xylobiose. These results indicate that
XynA has both transxylosylation and exo-type activities.
Studies have reported that some of the GH10 family
xylanases have either exo-type activity or transxylosylation
activity [7, 12]. Therefore, hydrolysis pattern of XynA is a
unique intracellular xylanase that differs from other known
GH10 family xylanases [12, 22, 27]. In a previous study,
the P. xylanilyticus KJ-03 isolated from a nearby plant
degraded various polysaccharides [5, 21]. These results
showed that XynA degrades carbohydrates, thereby providing
sugars to bacteria.
In this study, a xylanase isolated from P. xylanilyticus
KJ-03 was cloned and expressed in E. coli, and the purified
enzyme obtained was characterized. XynA showed high
specificity towards birchwood and oat spelt xylan, and was
found to hydrolyze xylobiose to xylose. XynA is an endo-
type enzyme with activity similar to xylosidase. Xylanase
degrades lignocellulosic biomass to fermentable sugars.
Therefore, in the bioethanol industry, xylanase can be used
for degrading xylans present in lignocelluloses to produce
sugars that can be used to synthesize ethanol in a cost-
effective manner. The xylanase from KJ-03 also enhances
the digestibility of animal feed by preventing their viscosity
in the intestine. Thus, XynA may have industrial applications
in the bioethanol, feed, and food industries.
Acknowledgments
This research was supported by Basic Science Research
Program through the NRF of Korea funded by the Ministry
of Education, Science and Technology.
REFERENCES
1. Ali, M. K., F. B. Rudolph, and G. N. Bennett. 2005.
Characterization of thermostable Xyn10A enzyme from mesophilic
Clostridium acetobutylicum ATCC 824. J. Ind. Microbiol.
Biotechnol. 32: 12-18.
2. Amaya-Delgado, L., T. Mejia-Castillo, A. Santiago-Hernandez,
J. Vega-Estrada, F. G. S. Amelia, B. Xoconostle-Cazares, et al.
2010. Cloning and expression of a novel, moderately thermostable
403
xylanase-encoding gene (Cfl xyn11A) from Cellulomonas
flavigena. Bioresour. Technol. 101: 5539-5545.
3. Beg, Q. K., M. Kapoor, L. Mahajan, and G. S. Hoondal. 2001.
Microbial xylanase and their industrial applications: A review.
Appl. Microbiol. Biotechnol. 56: 326-338.
4. Biely, P., D. Mislovicova, and R. Toman, 1988. Remazol
brilliant blue-xylan: A soluble chromogenic substrate for xylanases.
Methods Enzymol. 160: 536-542.
5. Cho, K. M., S. J. Hong, R. K. Math, S. M. A. Islam, J. O. Kim,
Y. H. Lee, et al. 2008. Cloning of two cellulase genes from
endophytic Paenibacillus polymyxa GS01 and comparison with
cel44C-man26A. J. Basic Microbiol. 48: 464-472.
6. Chow, V., G. Nong, and J. F. Preston. 2007. Structure, function,
and regulation of the aldouronate utilization gene cluster from
Paenibacillus sp. strain JDR-2. J. Bacteriol. 189: 8863-8870.
7. Gallardo, O., P. Diaz, and F. I. J. Pastor. 2003. Characterization
of a Paenibacillus cell-associated xylanase with high activity on
aryl-xylosides: A new subclass of family 10 xylanases. Appl.
Microbiol. Biotechnol. 61: 226-233.
8. Heo, S., J. Kwak, H. W. Oh, D. S. Park, K. S. Bae, D. H. Shin,
and H. Y. Park. 2006. Characterization of an extracellular
xylanase in Paenibacillus sp. HY-8 isolated from an herbivorous
longicorn beetle. J. Microbiol. Biotechnol. 16: 1753-1759.
9. Hwang, I. T., H. K. Lim, H. Y. Song, S. J. Cho, J. S. Chang,
and N. J. Park. 2010. Cloning and characterization of a
xylanase, KRICT PX1 from the strain Paenibacillus sp. HPL-
001. Biotechnol. Adv. 28: 594-601.
10. Henrissat, B. 1991. A classification of glycosyl hydrolases
based on amino acid sequence similarities. Biochem. J. 280:
309-316.
11. Hu, Y., G. Zhang, A. Li, J. Chen, and L. Ma. 2008. Cloning and
enzymatic characterization of a xylanase gene from a soil-
derived metagenomic library with an efficient approach. Appl.
Microbiol. Biotechnol. 80: 823-830.
12. Ito, Y., T. Tomita, N. Roy, A. Nakano, N. Sugawara-Tomita, S.
Watanabe, et al. 2003. Cloning, expression, and cell surface
localization of Paenibacillus sp. strain W-61 xylanase 5, a
multidomain xylanase. Appl. Environ. Microbiol. 69: 6969-6978.
13. Kim, D. Y., M. K. Han, H. W. Oh, K. S. Bae, T. S. Jeong, S. U.
Kim, et al. 2010. Novel intracellular GH10 xylanase from Cohnella
laeviribosi HY-21: Biocatalytic properties and alterations of
substrate specificities by site-directed mutagenesis of Trp residues.
Bioresour. Technol. 22: 8814-8821.
14. Kim, D. Y., M. K. Han, D. S. Park, J. S. Lee, H. W. Oh, D. H.
Shin, et al. 2009. Novel GH10 xylanase, with a fibronectin type 3
domain, from Cellulosimicrobium sp. strain HY-13, a bacterium
in the gut of Eisenia fetida. Appl. Environ. Microbiol. 75:
7275-7279.
15. Kim, H., K. H. Jung, and M. Y. Pack. 2000. Molecular
characterization of xynX, a gene encoding a multidomain
xylanase with a thermostabilizing domain from Clostridium
thermocellum. Appl. Microbiol. Biotechnol. 54: 521-527.
16. Lee, C. C., R. E. Kibblewhite-accinelli, M. R. Smith, K.
Wagschal, W. J. Orts, and D. W. S. Wong. 2008. Cloning of
Bacillus licheniformis xylanase gene and characterization of
recombinant enzyme. Curr. Microbiol. 57: 301-305.
17. Lee, T. H., O. L. Pyung, and Y. E. Lee. 2007. Cloning,
characterization, and expression of xylanase A gene from
404 Park et al.
Paenibacillus sp. DG-22 in Escherichia coli. J. Microbiol.
Biotechnol. 17: 29-36.
18. Lee, Y. E. and P. O. Lim. 2004. Purification and characterization
of two thermostable xylanases from Paenibacillus sp. DG-22. J.
Microbiol. Biotechnol. 14: 1014-1021.
19. Li, N., P. Yang, Y. Wang, H. Luo, K. Meng, N. Wu, et al. 2008.
Cloning, expression, and characterization of protease-resistant
xylanase from Streptomyces fradiae var. k11. J. Microbiol.
Biotechnol. 18: 410-416.
20. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for
determination of reducing sugar. Anal. Chem. 31: 426-428.
21. Park, I. H., J. Chang, Y. S. Lee, S. J. Fang, and Y. L. Choi.
2012. Gene cloning of endoglucanase Cel5A from cellulose-
degrading Paenibacillus xylanilyticus KJ-03 and purification
and characterization of the recombinant enzyme. Protein J. 31:
238-245.
22. Raha, A. R., L. Y. Chang, A. Sipat, K. Yusoff, and T. Haryanti.
2006. Expression of a thermostable xylanase gene from Bacillus
coagulans ST-6 in Lactococcus lactis. Lett. Appl. Microbiol. 42:
210-214.
23. Ratanakhanokchai, K., K. L. Kyu, and M. Tanticharoen. 1999.
Purification and properties of a xylan-binding endoxylanase from
alkaliphilic Bacillus sp. strain K-1. Appl. Environ. Microbiol.
65: 694-697.
24. Rattiya, W., P. Pason, K. L. Kyu, K. Sakka, A. Kosugi, Y. Mori, and
K. Ratanakhanokchai. 2009. Cloning, sequencing, and expression
of the gene encoding a multidomain endo-β-1,4-xylanase from
Paenibacillus curdlanolyticus B-6, and characterization of the
recombinant enzyme. J. Microbiol. Biotechnol. 19: 277-285.
25. Sambrook, J. and D. W. Russel. 2001. Molecular Cloning: A
Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, USA
26. Valenzuela, S. V., P. Daiz, and F. I. J. Pastor. 2010. Recombinant
expression of an alkali stable GH 10 xylanase from Paenibacillus
barcinonensis. J. Agric. Food Chem. 58: 4814-4818.
27. Watanabe, S., D. N. Viet, J. Kaneko, Y. Kamio, and S. Yoshida.
2008. Cloning, expression and transglycosylation reaction of
Paenibacillus sp. strain W-61 xylanase1. Biosci. Biotechnol.
Biochem. 72: 951-958.
28. Wu, S., B. Liu, and X. Zhang. 2006. Characterization of a
recombinant thermostable xylanase from deep-sea thermophilic
Geobacillus sp. MT-1 in East Pacific. Appl. Microbiol.
Biotechnol. 72: 1210-1216.
29. Yoon, K. H. 2009. Cloning of the Bacillus subtilis AMX-4
xylanase gene and characterization of the gene product. J.
Microbiol. Biotechnol. 19: 1514-1519.
30. Zhang, M., Z. Jiang, S. Yang, C. Hau, and L. Li. 2010. Cloning
and expression of a Paecilomyces thermophila xylanase gene in
E. coli and characterization of the recombinant xylanase.
Bioresour. Technol. 101: 688-695.