Bioresource Technology 125 (2012) 182187

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Overexpression of a Paenibacillus campinasensis xylanase in Bacillus megaterium

and its applications to biobleaching of cotton stalk pulp and saccharication
of recycled paper sludge
Hongchen Zheng a,d,1, Yihan liu a,c,d,1, Xiaoguang Liu b,d, Yang Han a,d, Jianling Wang c,d, Fuping Lu a,b,d,
a

Key Laboratory of Industrial Fermentation Microbiology, Education Ministry of China, Tianjin 300457, China
National Engineering Laboratory for Industrial Enzymes (NELIE), 300457 Tianjin, China
Tianjin Key Laboratory of Industrial Microbiology, 300457 Tianjin, China
d
College of Biotechnology, Tianjin University of Science & Technology, 300457 Tianjin, China
b
c

h i g h l i g h t s
" A Paenibacillus campinasensis xylanase gene was overexpressed in Bacillus megaterium.
" The recombinant strain produced 2.1-fold higher xylanase activity than the wild type.
" XynG1-1R can reduce chlorine consumption by 50% in biobleaching of cotton stalk pulp.
" Saccharication efciency of recycled paper sludge was enhanced by XynG1-1R.

a r t i c l e

i n f o

Article history:
Received 29 March 2012
Received in revised form 20 July 2012
Accepted 24 August 2012
Available online 5 September 2012
Keywords:
Recombinant xylanase
Paenibacillus campinasensis
Bacillus megaterium
Biobleaching
Saccharication

a b s t r a c t
A xylanase gene (xynG1-1) from Paenibacillus campinasensis G1-1 was expressed in Bacillus megaterium
MS941 and a high level of extracellular xylansae activity (304.26 IU/mL) was achieved after induction
with 0.5% xylose. The puried recombinant xylanase (XynG1-1R) revealed optimal activity at 60 C and
pH 7.0 and retained 79% and 81% activity after incubation without substrate at 60 C, pH 5.0 and pH
8.0 for 3 h, respectively. Application of XynG1-1R (15 IU/g pulp) to cotton stalk pulp bleaching increased
brightness by 3.65% over that of the control without the xylanase and reduced the need for chlorine compounds by 50% without loss of brightness and pulp ber qualities. When XynG1-1R (80 IU/g paper
sludge) was used in combination with mixed cellulolytic enzymes, the saccharication efciency of recycled paper sludge was increased by 10%. These results indicated that XynG1-1R is a promising candidate
for various industrial applications such as biobleaching and bioenergy conversion.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
As a major source of environmental pollution, the pulp and paper industry has to seek alternative environmentally friendly technologies to replace the use of harsh chemicals. Biobleaching with
microbial xylanases was identied as an effective process to decrease the amount of undesirable substances in the efuent and
to enhance the brightness of pulp without leading to a loss in its
viscosity and strength (Ziaie-Shirkolaee et al., 2008). Moreover, pa-

Corresponding author. Address: Industrial Microbiology Laboratory, College of

Biotechnology, Tianjin University of Science & Technology, No. 29, 13th Avenue,
Tianjin Economic and Technological Development Area, Tianjin 300457, China. Tel.:
+86 22 60600160; fax: +86 22 60602298.
E-mail addresses: lfp@tust.edu.cn, lfp001@yahoo.cn (F. Lu).
1
These authors contributed equally to this work.
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.08.101

per sludge, the solid residue arising from paper pulping and
bleaching, is currently disposed of in landlls or burned, resulting
in environmental pollution. However, the high lignocellulosic content of the sludge material makes it a promising feedstock for production of second-generation bioethanol (Peng and Chen, 2011).
Currently, a major challenge for bioethanol production is the yield
of monomer glucose from the cellulose fraction of the feedstock.
Therefore, attempts using xylanase to hydrolyze hemicellulose
which is wrapped around the cellulose were made to increase
the glucose yield. Endo-1,4-b- xylanase (EC 3.2.1.8), the key enzyme for the hydrolysis of the backbone of xylan, can degrade xylan in reprecipitated lignincarbohydrate complexes (LCC) on the
ber surface to facilitate extraction of cellulose and lignin (Beg
et al., 2001; Subramaniyan and Prema, 2002).
Based on amino acid sequence homologies and hydrophobic
cluster analysis, the majority of xylanases belong to glycosyl

H. Zheng et al. / Bioresource Technology 125 (2012) 182187

hydrolase (GH) families 10 and 11. A number of xylanase genes

have been cloned and expressed successfully in heterologous hosts
such as Escherichia coli, Pichia pastoris and Bacillus subtilis (Ahmed
et al., 2009). Gram-positive hosts such as B. subtilis or Bacillus
megaterium, in contrast to E. coli, have the capacity for protein export. B. megaterium appears to be an attractive host for recombinant protein production due to its intrinsic lack of alkaline
proteases as well as highly stable replication and maintenance of
recombinant plasmids (Malten et al., 2006; Rygus and Hillen,
1991; Vary, 1994).
P. campinasensis G1-1 (CGMCC NO.5023), isolated from a Chinese paper mill (Zheng et al., 2012), exhibits a strong capacity to
degrade xylan under high temperature and alkaline conditions;
however, the yield of the cellulose-free and thermostable xylanase
(XynG1-1) from P. campinasensis G1-1 or a recombinant E. coli BL21
(pET-G1-1) was relatively low. In the present study, the xylanase
gene (xynG1-1) from P. campinasensis G1-1 (GenBank Accession
No. JF830005.1) was expressed in B. megaterium MS941 and the recombinant xylanase was obtained at a high yield. The efcacy of
the recombinant xylanase on bleaching of cotton stalk pulp and
saccharication of recycled paper sludge was also evaluated.

183

2.3. Transformation

2. Methods

Transformation of E. coli DH5a was performed as described by

Sambrook and Russell (2001). Protoplasts of B. megaterium were
prepared according to the method of Akamatsu and Sekiguchi
(1982) with several modications. After B. megaterium MS941
was incubated in 50 mL of AB3 medium at 37 C to an optical density at 600 nm (OD600) of 1.0, the cells were centrifuged at 1350g
for 10 min and resuspended in 5 mL of SMMP. Protoplasts were
formed by treatment with 1 mg/mL of lysozyme for 60 min at
37 C. Transformation for B. megaterium MS941 was carried out
based on the protocol provided by the supplier. The protoplast suspension (500 lL) and 5 lg plasmid DNA (pSH-G1-1) were combined in a 12-mL tube, and the protoplast transformation was
induced by the addition of 1.5 mL PEG-P (40% (w/v) polyethylene
glycol 6000 in 1  SMM). After a 2-min incubation at room temperature, 5 mL of SMMP was added and the mixure was subjected
to centrifugation at 1350g for 10 min. The supernatant was decanted immediately after centrifugation, and 500 lL of SMMP were
added and the suspension was incubated at 37 C for 90 min with
gentle shaking. For regeneration of the protoplasts, 2.5-mL aliquots
of CR5-top agar (preheated to 43 C) and 50200 lL of transformed
cells were mixed gently in a sterile tube, poured onto an LB plate
containing tetracycline at 10 lg/mL and incubated overnight at
37 C.

2.1. Strains, plasmids and media

2.4. Expression and purication of recombinant xylanase

P. campinasensis G1-1 (CGMCC NO.5023) (Zheng et al., 2012)

was used for amplication of the xylanase gene. E. coli DH5a was
used for propagation of plasmids, and B. megaterium MS941 (MoBiTec GmbH, Germany) was used as a host for expression of the recombinant xylanase gene. An E. coli/B. megaterium shuttle vector,
pSTREPHIS1525 (MoBiTec GmbH, Germany), was used to achieve
secretory expression of xylanase in B. megaterium MS941. The plasmid contains a xylose-inducible promoter (PxylA), a signal peptide
sequence from the esterase LipA, a Strep II-tag sequence (upstream
of the multiple cloning sites) and a His6- tag sequence (downstream of the multiple cloning sites). SMMP containing equal volumes of 2  SMM (1 M sucrose, 40 mM maleic acid, 40 mM MgCl2,
pH 6.5) and 2  AB3 (Antibiotic Medium No. 3, DIFCO) was the
medium used for protoplast transformation. CR5-top agar containing 103 g/L sucrose, 10 g/L yeast extract, 0.2 g/L casamino acids,
10 g/L glucose, 6 g/L proline, 6.50 g/L MOPS, 0.66 g/L NaOH,
0.25 g/L K2SO4, 10 g/L MgCl2  6 H2O, 0.05 g/L KH2PO4, 2.2 g/L
CaCl2 and 4.0 g/L agar was used for regeneration of protoplasts. Recombinant strains were grown in LuriaBertani (LB) broth supplemented with ampicillin (Amp) or tetracycline (Tet) at nal
concentrations of 100 and 10 lg/mL, respectively.

One colony of the recombinant strain was inoculated into 5 mL

of LB medium (10 lg/mL Tet) and was cultured overnight at 37 C
in a rotary shaker. One milliliter of the overnight culture was transferred to 50 mL of LB medium (10 lg/mL Tet) in a 250-mL shake
ask and grown to an optical density at 600 nm (OD600) of 0.3 at
37 C. Recombinant xylanase production was induced by the addition of xylose to a nal concentration of 0.5% and samples were
withdrawn every 2 h for cell concentration measurement, xylanase
activity assay and protein analysis. The extracellular recombinant
xylanase was harvested by centrifugation at 2250g for 10 min
and the supernatant was loaded onto a Ni2+-NTA agarose
(Novagen, Madison, WI) column. Bound protein was eluted with
200 lL elution buffer (300 mM sodium chloride, 50 mM sodium
phosphate, 150 mM imidazole, pH 7.0). The protein concentration
was analyzed by the method of Bradford (1976).

2.2. Xylanase gene isolation and plasmid construction

The mature xylanase gene was amplied using genomic DNA of
P. campinasensis G1-1 as template and the following primers:
50 -CGAGCTCGTGCAACCACGATCACTTCTAACGAGA-30 and 50 -CGGGG
TACCTCACCGGATCTCCAAATAGTCAATG-30 (underlined letters indicate SacI and KpnI sites, respectively)(Zheng et al., 2012). PCR was
performed at 95 C for 5 min, followed by 30 cycles for 45 s at
94 C, 45 s at 55 C, and 90 s at 72 C, and a nal extension of
10 min at 72 C. The product was digested with SacI and KpnI, then
cloned into the corresponding sites of pSTREPHIS1525, resulting in
recombinant expression vector pSH-G1-1. The recombinant plasmid pSH-G1-1 was transformed into E. coli DH5a for propagation
and then was extracted and transformed into B. megaterium MS941.

2.5. SDSPAGE and zymogram analysis

SDSPAGE was performed on a 5% stacking and a 12% separating gel according to the method of Laemmli (1970), and protein
bands were visualized by staining with Coomassie Brilliant Blue
G250 (Sigma). Zymograms for recombinant xylanase were obtained by PAGE using a 0.1% (w/v) concentration of birchwood xylan incorporated into the polyacrylamide (Morag et al., 1990). After
electrophoresis, the enzyme was reactivated with 25% 2-propanol,
and the gel was incubated at 60 C for 30 min in 20 mM sodium
phosphate buffer (pH 7.0). After incubation, gels were stained for
residual carbohydrates with Congo red solution (1 mg/mL), destained with 1 M NaCl, and xed with 5% (v/v) acetic acid. Clear
areas in the zymograms indicated enzyme activity. Unstained protein molecular weight standards were obtained from Fermentas.
2.6. Xylanase assay
Xylanase activity was measured by determining the amount of
reducing sugars liberated from birchwood xylan (Sigma) using 3,5dinitrosalicylic acid (Miller, 1959). The reaction mixture consisting
of 100 ll of 1% birchwood xyan solution in sodium phosphate buf-

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H. Zheng et al. / Bioresource Technology 125 (2012) 182187

fer (20 mM, pH 7.0) and 100 ll of appropriately diluted enzyme extract was incubated at 60 C for 10 min. The reaction was terminated by adding 600 ll of 3,5-dinitrosalicylic acid and placing
the mixture in a boiling-water bath for 10 min. The control contained the same reagents, but the reaction was terminated before
adding enzyme. After cooling, the absorbance of the reaction mixtures, was measured against the control at 540 nm in a spectrophotometer. One international unit (IU) of xylanase activity was
dened as the amount of enzyme that liberated 1 lmol of reducing
sugars per minute under the above conditions.
2.7. Characterization of the recombinant xylanase
The optimum temperature and pH of the puried recombinant
xylanase were determined by assaying the xylanase activity at different temperatures (3080 C) and various pHs (pH 4.011.0). The
buffer used were acetic acid sodium buffer (pH 4.05.0), sodium
phosphate (pH 6.07.0), TrisHCl (pH 8.09.0) and glycineNaOH
(pH 10.011.0) at the same concentrations of 20 mM. Thermal stability was determined by incubating the recombinant xylanase in
20 mM sodium phosphate buffer (pH 7.0) at various temperatures
(3080 C) for 6 h. Similarly, to assess its pH stability, the recombinant xylanase was pre-incubated in buffers at various pH values
(pH 4.011.0) at 60 C for 6 h and the residual activities were
determined in 30-min intervals. The effects of various metal ions,
metal chelators and surfactants on recombinant xylanase activity
were evaluated by including these in the reaction mixtures at the
concentration of 1 and 5 mM. The kinetic parameters (Km and Vmax)
were estimated from a LineweaverBurk double reciprocal plot of
xylanase activity at various concentrations (0.11% w/v) of birchwood xylan under optimum reaction conditions for 10 min.
2.8. Biobleaching of cotton stalk pulp with the recombinant xylanase
Unbleached cotton stalk pulps consisting of 65.2% (w/w) cellulose, 15.4% (w/w) hemicellulose, 15.6% (w/w) lignin, 3.1% (w/w)
ash and 0.7% (w/w) other components were obtained from the
Tianjin Guangjuyuan paper mill, China. For biobleaching, pulp
samples equivalent to 20 g of air-dried weight (pH 8.0) in polyethylene (PE) bags at 10% consistency were pretreated with 125 IU/g
pulp of puried recombinant xylanase obtained from B. megaterium MS941 (pSH-G1-1) or commercial xylanase JT-G-M (Noao Sci&Tec Development Co., Ltd., Tianjin, China) in a water bath at 60 C
for 3 h with intermittent kneading. Control samples were treated
under the same conditions with inactivated (boiled) enzyme. After
pretreatment, the pulp slurry was ltered through a lter-paper on
a Buchner funnel and washed thoroughly with distilled water. The
pretreated pulps were subjected to chemical bleaching using a
three-stage sequence D0ED1 in which D indicates the chlorine dioxide stage and E the alkaline extraction stage (Ouyang et al., 2010).
Diminishing dosages (41.5%) of chlorine dioxide were used in D1
after pretreatment with a certain amount of xylanases. The properties of the pulps were analyzed with the TAPPI Test Methods,
(20002002): brightness (T452 om-83), kappa number (T236
om-85), tear index (T220 om-88), breaking length and tensile index (T494 om-88).

paper sludge at 10% (w/v) substrate concentration in 50 mM sodium acetate buffer (pH 5.0) were pretreated at 50 C on a rotatory
shaker at 180 rpm for 2 h. Thereafter, the slurry was hydrolyzed
with an enzyme mixture containing cellulase XWS-G-1 (25 FPU/g),
b-glucosidase (50 U/g), Tween-20 (0.1%) and recombinant xylanase/commercial xylanase MJT-G-NIS at dosages of 20120 IU/g at
50 C and 180 rpm for 48 h. Cellulase XWS-G-1, b-glucosidase
and xylanase MJT-G-NIS were purchased from Noao Sci&Tec
Development Co. Ltd., Tianjin, China. The enzymatic reaction without xylanase served as control. Cellulose, hemicellulose and lignin
contents of the dried paper sludge were assayed by means of
quantitative hydrolysis with sulphuric acid according to the method described by Browning (1967). Monomer glucose concentration
obtained by enzymatic saccharication was estimated by the DNS
method of Miller (1959) using glucose (Sigma) as standard and the
saccharication efciency was calculated according to Peng and
Chen (2011). The degree of saccharication was calculated as
follows:

Degree of saccharificatione%

Glucose concentration obtained

 100
Potential glucose concentration in the substrate

3. Results and discussion

3.1. Expression and activity of recombinant xylanase in B. megaterium
MS941
The level of recombinant xylanase XynG1-1R produced by B.
megaterium MS941 (pSH-G1-1) in the supernatant of the culture
medium reached a maximum of 304.26 IU/mL (Fig. 1) after 20 h
of induction, which was 2.1-fold higher than the extracellular
xylanase produced by P. campinasensis G1-1 after 72 h. No intracellular xylanase was detected. SDSPAGE (Fig. 2, lane 14) revealed
that recombinant XynG1-1R was mainly present in the growth
medium.
3.2. Purication and properties of the recombinant xylanase
Recombinant xylanase XynG1-1R was eluted from Ni2+-NTA
resins and observed as single band on 12% SDSPAGE with the
molecular weight of 41 KDa (Fig. 2, lane 5) which was very close
to that of the native xylanase of P. campinasensis G1-1. The zymogram analysis further conrmed the presence of active xylanase
(Fig. 2, lane 6). As shown in Fig. 2 (lane 3 and 4), very few extracel-

2.9. Application of recombinant xylanase in saccharication of

recycled paper sludge
Recycled paper sludge consisting of 39.2% (w/w) cellulose,
16.4% (w/w) hemicellulose, 12.6% (w/w) lignin, 22.1% (w/w) ash
and 9.7% (w/w) other components was obtained from the Tianjin
Guangjuyuan paper mill, China. Enzymatic saccharication of recycled paper sludge was carried out according to the method of
Kumar et al. (2012) with several modications. Ten g of recycled

Fig. 1. Growth and extracellular xylanase production of the recombinant Bacillus

megaterium MS941. B. megaterium MS941 (pSH-G1-1) was grown in LB medium
containing tetracycline (10 lg/mL). After OD60 nm of the culture reached 0.3, 0.5% w/
v xylose was added to the culture for inducing the expression of recombinant
xylanase. At intervals, the samples were withdrawn and analyzed for growth
(OD600nm) and xylanase activity (IU/mL). Data are presented as means SD (n = 3).

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H. Zheng et al. / Bioresource Technology 125 (2012) 182187

Fig. 2. SDSPAGE and zymogram analysis of recombinant xylanase from B.

megaterium MS941. Lane M: molecular weight of marker standard; lane 1: cellassociated proteins of recombinant B. megaterium MS941 (pSTREPHIS1525); lane 2:
cell-associated proteins of recombinant B. megaterium MS941 (pSH-G1-1); lane 3:
extracellular proteins of recombinant B. megaterium MS941 (pSTREPHIS1525); lane
4: extracellular proteins of recombinant B. megaterium MS941 (pSH-G1-1); lane 5:
puried XynG1-1R; lane 6: zymogram analysis of puried XynG1-1R.

Fig. 4. Stability of XynG1-1R at various temperatures (3080 C), pH 7.0 (a) and at
various pH (pH 4.011.0), 60 C (b) for 6 h. The residual activities were assayed
under optimal conditions and the original activity without incubation was taken as
100%. Data are presented as means SD (n = 3).

Table 1
Effects of metal ions and other additives on the activity of XynG1-1R.

lular proteins were secreted by B. megaterium MS941 which greatly

facilitates purication of the target protein (XynG1-1R). The specic activity of the puried XynG1-1R was up to 2856.5 IU/mg
which was 1.5-fold higher than that of the puried native xylnanase from P. campinasensis G1-1 (Zheng et al., 2012) as well as higher than that of other xylanases from Paenibacillus sp. (Ko et al.,
2010; Waeonukul et al., 2009; Watanabe et al., 2008). The higher
specic activity of the puried recombinant xylanase indicated a
higher level of purity than that of native xylanase puried from
P. campinasensis G1-1 by multistep purication (Zheng et al., 2012).
Puried XynG1-1R exhibited activity over a broad range of temperatures (3080 C) and pHs (pH 4.011.0) with an optimum at
60 C and pH 7.0, and retained 52% and 85% of its maximum activity assayed at the conditions of 50 C, pH 5.0 and 60 C, pH 8.0,
respectively (Fig. 3). XynG1-1R possesses the same optimum temperature and pH as the native xylanase (XynG1-1) from P. campinasensis G1-1 (Zheng et al., 2012), demonstrating that the main
catalytic properties of the enzyme were not altered as a result of
recombinant expression in B. megaterium MS941. In addition,
XynG1-1R retained at least 79% and 81% residual activity after
treatment at 60 C, pH 5.0 and pH 8.0 for 3 h, respectively
(Fig. 4). The stability of the recombinant xylanase at a wide range
of temperatures (3080 C) and pHs (pH 4.09.0) was better than
that of other bacterial xylanases (Beg et al., 2001; Zhao et al.,
2011). Puried XynG1-1R was activated by Ca2+ and Ba2+ and
was inhibited by Fe2+, Fe3+ and Zn2+ (Table 1). The reducing agents,
DTT and b- mercaptoethanol, stimulated the activity of XynG1-1R

(Table 1). The strong inhibition by SDS and EDTA (Table 1) indicating that hydrophobic interactions may be important in maintaining the structures of XynG1-1R and certain metal ions were
required for its activity. The Km and Vmax of XynG1-1R for birchwood xylan as the substrate were 6.08 mg mL1 and 1688.60
lmol min1 mg1 of protein, respectively, and thus were comparable to those of native xylanase from P. campinasensis G1-1(Zheng
et al., 2012). The kinetic parameters of XynG1-1R revealed a higher

Fig. 3. Xylanase activities of XynG1-1R assayed under various temperatures (30

80 C) and pH values (pH 4.011.0). The xylanase activities of puried XynG1-1R are
indicated as specic activities (IU/mg). Data are presented as means SD (n = 3).

Fig. 5. Effects of different xylanase dosages on the brightness of bleached cotton

stalk pulps. The brightness of cotton stalk pulps pretreated by inactivated (boiled)
enzyme under the same conditions served as a control. Data are presented as
means SD (n = 3).

Agents

1 mM

5 mM

Controla
KCl
CaCl2
BaCl2
MgCl2
MnCl2
FeCl2
FeCl3
ZnCl2
CuCl2
DTT
b-Mercaptoethanol
EDTA
SDS

100 4.32
95.62 1.90
124.21 8.24
102.33 6.86
95.56 5.69
94.76 4.36
52.44 2.62
26.77 3.41
38.54 3.66
85.49 6.92
121.24 3.44
143.26 2.23
44.08 2.32
24.51 3.11

100 3.51
96.35 2.44
125.44 6.22
109.24 4.31
95.89 6.55
96.24 3.77
51.69 2.54
21.52 3.35
32.42 3.43
83.55 7.98
127.33 3.45
145.52 2.45
42.76 2.38
21.45 3.21

Data are presented as means SD (n = 3).

a
The activity of the enzymes assayed under the optimum condition in the
absence of additives was dened as 100%.

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H. Zheng et al. / Bioresource Technology 125 (2012) 182187

Table 2
Effects of xylanase pretreatment (15 IU/g dry pulp) on chlorine dioxide consumption during D0ED1 bleaching of cotton stalk pulps.
Active
chlorine
on pulp
(w/w %)

Reduction
of chlorine
dioxide at
D1b (%)

Brightness (% ISO)

Pulp kappa number

Breaking length (km)

Tear index (mN m2 g1)

Burst index (kPa m2 g1)

Recombinant
xylanase

Xylanase
JT-G-M

Recombinant
xylanase

Xylanase
JT-G-M

Recombinant
xylanase

Xylanase
JT-G-M

Recombinant
xylanase

Xylanase
JT-G-M

Recombinant
xylanase

Xylanase
JT-G-M

Controla
4
3.5
3
2.5
2
1.5

0
20
30
40
50
60
70

78.05 3.41
80.97 2.43
79.65 2.41
79.11 2.44
78.06 2.54
75.90 2.51
73.29 2.21

78.0 2.42
81.0 2.11
79.8 2.22
79.1 2.46
78.0 2.47
76.0 2.46
74.0 2.44

2.9 0.21
1.6 0.33
1.6 0.09
1.8 0.41
2.2 0.78
2.8 0.34
4.1 0.56

2.9 0.13
1.3 0.32
1.4 0.11
1.7 0.48
2.0 0.66
2.5 0.25
3.9 0.47

5.19 0.22
5.38 0.12
5.38 0.45
5.34 0.88
5.33 0.08
5.29 0.45
5.28 0.66

5.19 0.32
5.30 0.11
5.29 0.41
5.29 0.76
5.22 0.11
5.21 0.34
5.20 0.45

5.86 0.21
6.98 0.23
6.98 0.44
6.95 0.22
6.59 0.34
6.10 0.81
6.09 0.99

5.86 0.21
6.64 0.31
6.56 0.41
6.55 0.31
6.34 0.47
6.06 0.43
5.98 1.13

3.10 0.34
3.87 0.48
3.86 0.21
3.75 0.44
3.70 0.14
3.59 0.61
3.56 0.91

3.10 0.16
3.62 0.45
3.62 0.23
3.44 0.56
3.36 0.54
3.21 0.55
3.14 0.67

Data are presented as means SD (n = 3).

a
D1, chlorine dioxide: 5.0% active chlorine on pulps (w/w), no xylanase pretreatment.
b
The percentage reduction of chlorine dioxide in D1 was converted to total reduction of chlorine dioxide.

afnity for substrate and catalytic efciency than most Bacillus spp.
xylanases reviewed by Beg et al. (2001).
3.3. Biobleaching of cotton stalk pulp with the recombinant xylanase
Compared with control pulps, both XynG1-1R and xylanase
JT-G-M pretreatments resulted in increasing brightness, and maximum increases of 3.65% ISO (XynG1-1R) and 3.81% ISO (xylanase
JT-G-M), at xylanase dosages of 15 IU/g pulp were observed (Fig. 5).
Therefore, 15 IU/g pulp of XynG1-1R and xylanase JT-G-M were
used to pretreat the cotton stalk pulp followed by chemical bleaching (D0ED1) with diminishing dosages (4%1.5%) of chlorine dioxide in D1. With decreasing chlorine dioxide consumption in D1,
the brightness of the bleached pulps decreased from 80.97% to
73.29% (Table 2). At 2.5% active chlorine consumption, XynG1-1
and xylanase JT-G-M pretreatment gained brightness levels of
78.06% and 78.09%, respectively which is close to that of the control (78.05%) (Table 2). A similar brightnesses of around 80% for
non-wood (bagasse, rice straw and wheat straw) pulps pretreated
with a xylanase produced by T. lanuginosus strain SSBP or a commercial xylanase, cartazyme (Sandoz), were reported by ZiaieShirkolaee et al. (2008). These results indicate that pretreatment
using XynG1-1R can cut down the amount of chlorine compounds
required by 50% (Table 2). The recombinant enzyme was as effective as commercial xylanase JT-G-M and more effective than most
other xylanases (Bissoon et al., 2002; Dhillon and Khanna, 2000;
Ziaie-Shirkolaee et al., 2008). The enzymatically pretreated pulps
had a lower kappa number and better strength properties than
the control (Table 2). At 2.5% active chlorine consumption,
XynG1-1R pretreatment resulted in a decreased kappa number of
0.7, increased breaking length of 0.14 km, increased tear index of
0.8 mN m2g1 and increased burst index of 0.6 kPa m2 g1, respectively. These values were more favorable than those obtained with
pretreatment by xylanase JT-G-M (Table 2). The decrease in kappa
number indicated that XynG1-1R was highly effective in delignication. In addition, XynG1-1R without cellulase activity protected
the pulp ber qualities better than xylanase JT-G-M.
3.4. Saccharication of recycled paper sludge
XynG1-1R was used in combination with mixed cellulolytic enzymes to enhance enzymatic saccharication of recycled paper
sludge. With 80 IU/g paper sludge of recombinant xylanase along
with cellulase XWS-G-1 (25 FPU/g), b-glucosidase (50 U/g) and
Tween-20 (0.1%) an increase in saccharication of 10% (w/w) over
that of the control without xylanase was achieved. This result
was similar to that obtained with the same dosage of xylanase
MJT-G-NIS (Fig. 6). The synergistic action of XynG1-1R and mixed
cellulolytic enzymes led to a total saccharication efciency of

Fig. 6. Effects of different xylanase dosages along with mixed cellulolytic enzymes
on the enzymatic saccharication of recycled paper sludge. The mixed cellulolytic
enzymes consisted of cellulase XWS-G-1 (25 FPU/g) and b-glucosidase (50 U/g)
supplemented with 0.1% Tween-20. Data are presented as means SD (n = 3).

90% (data not shown) which indicated XynG1-1R to be a promising

enzyme to enhance the production of secondary bioethanol from
recycled paper sludge.
4. Conclusion
Recombinant B. megaterium MS941 (pSH-G1-1) secreting a P.
campinasensis xylanase into the growth medium efciently has
potential for industrial production. The recombinant xylanase
(XynG1-1R) possessing high stability at wide range of temperatures (3080 C) and pHs (pH 4.09.0) is suitable for various
industrial applications such as pulp biobleaching and bioethanol ?tul?> production. Compared with commercial xylanase
JT-G-M, XynG1-1R without cellulase activity indicating better protection for the pulp ber qualities is more suitable for application
in pulp biobleaching.
Acknowledgements
Financial support from the National High Technology Research
and Development Program of China (Grant 2011AA100905-4),
the National Natural Science Fund (Grant 31101219), and Program
for Changjiang Scholars and Innovative Research Team in
University (Grant IRT1166) were gratefully acknowledged.
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