Enzyme xylanase

8/11/2019 Enzyme xylanase

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High-level soluble expression of a thermostable xylanase

from thermophilic fungus Thermomyces lanuginosus

in Escherichia coli via fusion with OsmY protein

Yilin Le , Huilei Wang

School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, Jiangsu 212013, PR China

a r t i c l e i n f o

Article history:

Received 11 February 2014

and in revised form 7 March 2014

Available online 18 March 2014

Keywords:

Xylanase

Fusion protein

Soluble expression

Escherichia coli

Thermomyces lanuginosus

a b s t r a c t

A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem

emerged from overexpression of xynA inEscherichia colihas been the formation of inclusion bodies. Here

we describe the xynA was fused with the hyperosmotically inducible periplasmic protein ofE. coli, OsmY.

The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of

fusion protein reached 98 6 U/ml when cells containing pET-OsmY-xynA were expressed without IPTG

induction at 37 C. The induction is probably due to auto-induction due to lactose in the medium (Studier

(2005)[21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than

that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36 1.3 U/ml)

and the periplasmic (42 4 U/ml) when cells containing pET-OsmY-xynA were induced without IPTG

addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified

fusion protein showed a single band on SDSPAGE gel with a molecular mass of 44 kDa. The purified

fusion enzyme exhibited the highest activity at 65 C and pH 6.0.

2014 Elsevier Inc. All rights reserved.

Introduction

Thermomyces lanuginosus produces a thermostable GF11 endo-

xylanase encoded by xynA gene. This xylanase is free of cellulase

activity, and hydrolyses xylan to produce xylooligosaccharides

with little xylose[1]. These properties make the enzyme attractive

for the industrial application. Escherichia coli is one of the most

extensively used prokaryotic organisms for the industrial produc-

tion of enzyme because of its well-characterized genetics, and its

ability to grow rapidly and at high density on inexpensive sub-

strates[2].

The xylanase gene xynA has been sequenced and cloned into

E. coli as a LacZ fusion protein, but efficient expression was not

obtained[3]. Recently, the DNA sequence of xynA has been opti-

mized, and the expression of the enzyme in E. coli has reached

a high level by using recombinant plasmid pET-20b-xynA. How-

ever, the recombinant enzyme was mainly found in inclusion

bodies, and only a small proportion was soluble and active [4].

It is a common problem that some recombinant proteins will

aggregate to form inclusion bodies in the cytoplasm and/or

periplasm [5]. Inclusion body formation of eukaryotic proteins

inE. coliwith many contributing factors: insolubility of the prod-

uct at the concentrations being produced, inability to fold cor-

rectly in the bacterial environment, or lack of appropriate

bacterial chaperone proteins[6]. Many attempts have been made

to improve the soluble expression of recombinant proteins in

E. coli. The formation of inclusion bodies could be decreased by

changing the promoter to regulate the level of expression, con-

trolling the growth conditions (especially the pH of the culture),

controlling fermentation medium, changing the temperature of

induction and enabling secretion into the periplasm, fusing the

target gene to another gene [7].

OsmY has been used as a fusion partner to excrete target pro-

teins into the medium[810]. When fused to OsmY, E. colialkaline

phosphatase,Bacillus subtilisa-amylase, and human leptin could be

secreted into the medium at high levels [11].

Here we report the construction of vector pET-OsmY-xynA, and

the overexpression of soluble fusion protein OsmY-xynA in E. coli.

Materials and methods

Bacterial strains, plasmids and growth media

E. coliDH5a(TaKaRa, Dalian, China) was used as hosts for genecloning.E. coliBL21(DE3) (TaKaRa, Dalian, China) was used as hosts

http://dx.doi.org/10.1016/j.pep.2014.03.004

1046-5928/ 2014 Elsevier Inc. All rights reserved.

Corresponding author. Tel.: +86 511 88796122.

E-mail address:[email protected](Y. Le).

Protein Expression and Purification 99 (2014) 15

Contents lists available at ScienceDirect

Protein Expression and Purification

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http://dx.doi.org/10.1016/j.pep.2014.03.004mailto:[email protected]://dx.doi.org/10.1016/j.pep.2014.03.004http://www.sciencedirect.com/science/journal/10465928http://www.elsevier.com/locate/yprephttp://www.elsevier.com/locate/yprephttp://www.sciencedirect.com/science/journal/10465928http://dx.doi.org/10.1016/j.pep.2014.03.004mailto:[email protected]://dx.doi.org/10.1016/j.pep.2014.03.004http://-/?-http://-/?-http://-/?-http://-/?-http://crossmark.crossref.org/dialog/?doi=10.1016/j.pep.2014.03.004&domain=pdfhttp://-/?-


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for the expression of fusion protein. The cells harboring expression

plasmids were cultured in LuriaBertani (LB)1 medium supple-

mented with ampicillin (100 lg/ml).

Construction of fusion expression plasmid pET-OsmY-xynA

Based on the nucleotide sequences coding for the OsmY protein

(GenBank accession No. NC_000913.3), the PCR amplification wascarried out by usingE. coliDH5a genome as template, with the fol-lowing primers: 50-GCCGAATTCATGACTATGACAAGACTGAAG-30

and 50-GGCGGATCCCTTAGTTTTCAGATCATTTTTAAC-30. PCR was

carried out using the high-fidelityPyrobestDNA polymerase (TaKa-

Ra, Dalian, China). PCR products were purified using the QIAquick

PCR purification kitand followed by digestionwith BamHIa n d EcoR

I restriction enzyme(s). The reverse PCR amplification was carried

out by using plasmid pET-20b-xynA[4] as template, with primers

X1 (50-GCCGAATTCTATATCTCCTTCTTAAAGTTAAAC-30) and X2

(50-GGCGGATCCCAGACTACCCCGAACTCTGAAG-30). PCR products

were purified using the QIAquick PCR purification kit and followed

by digestion with corresponding restriction enzyme(s). The di-

gested PCR products were ligated to OsmY at BamH I/EcoR I sites.

Expression and purification of fusion protein

The plasmid pET-OsmY-xynA was transformed into the E. coli

BL21(DE3) by electroporation. The cells carrying pET-OsmY-xynA

were grown at 37 C, and induced for gene expression by addition

IPTG (isopropyl-b-D-thio galactopyranoside). The recombinant en-

zyme was isolated from the periplasm by cold osmotic shock

according to a published protocol [12]. The cells (wet weight

1.5 g) harvested by centrifugation at 6000g for 5 min were re-

suspended in 12 ml of 100 mM TrisHCl containing 20% sucrose

and 1 mM EDTA (pH 8.0), and then pelleted by centrifugation at

8000g for 5 min followed by re-suspension in 5 ml of ice-cold

water for 10 min. After the addition of MgCl2to a final concentra-

tion of 1 mM, the cell suspension was incubated on ice for a further

10 min before being pelleted by centrifugation at 8000g for

10 min at 4C. The supernatant (5 ml) was loaded onto a 1 ml Hi-

sTrap HP columns (GE Healthcare), washed with 60 mM imidazole

and 0.5 M NaCl in 20 mM TrisHCl buffer (pH 7.9), and eluted with

1 M imidazole and 0.5 M NaCl in 20 mM TrisHCl buffer (pH 7.9).

The pooled fractions were dialyzed into storage buffer containing

1 mM EDTA, and 20% (v/v) glycerol before the enzyme was stored

at 20 C. The SDSPAGE was performed according to standard

procedures. Protein concentration was determined by the Bradford

method using BSA as a standard[13].

Enzyme assays

Xylanase activity was determined by the 4-hydroxybenzoic acid

hydrazide method [14]. Xylan from birch wood (Sigma Aldrich,

Munich, Germany) was used as the substrate. The reaction mixture

comprised of 100 ll 1% (w/v) birch wood xylan in water, 90 llphosphate buffer (50 mM, pH 6.0) and 10 ll properly diluted en-zyme. The reaction was conducted at 65 C for 10 min, and stopped

when 600 ll of 4-hydroxybenzoic acid hydrazide solution wereadded into the reaction mixture. The reducing sugar was deter-

mined by reading the absorbance at 410 nm after the test tubes

were incubated for 10 min in boiling water bath and cooled down

on ice. One unit of xylanase activity was defined as the amount of

enzyme releasing 1 lmol reducing sugar per min.

Results

Construction of expression plasmids

The gene encoding the OsmY (including the signal sequence)

was amplified from the genomic DNA ofE. coliDH5a, and insertedinto the plasmid pET-20b-xynA (pelB signal sequence was deleted)

[4]at BamH I/EcoR I sites. Newly generated plasmid is designatedas pET-OsmY-xynA (carried an N-terminal OsmY signal sequence).

Target protein xylanase from fungus T. lanuginosus was linked to

the C-terminus of OsmY by aBamH I site sequence. The fusion pro-

tein OsmY-xynA was expressed with a C-terminal His-tag.

Expression level and solubility of the fusion protein OsmY-xynA

The recombinant plasmid pET-OsmY-xynA was isolated, which

was then transformed into E. coli BL21(DE3) for the production of

fusion protein OsmY-xynA using IPTG induction. The xylanase

activity of fusion protein was obtained after induction at different

IPTG concentrations (Fig. 1). Interestingly, xylanase activity pro-

duced by cells containing pET-OsmY-xynA with 0 mM, 0.1 mM,

0.3 mM, 0.5 mM IPTG, was 98 6 U/ml, 6.4 0.2 U/ml, 6.3 0.15U/ml, 6 0.1 U/ml, respectively. There was a gradual decrease in

xylanase activity upon increasing the IPTG concentration (Fig. 1).

The recombinant cells induced without IPTG addition produced

an activity level 16 times higher than that expressed with

0.5 mM IPTG induction.

Previously, intracellular expression of the xylanase was im-

proved by sequence optimization by site-directed mutagenesis

without changing the protein sequence [4]. But the recombinant

xylanase mainly appeared as inclusion bodies [4]. In the current

study, the expression levels and solubility of the fusion protein ex-

pressed from pET-OsmY-xynA were shown in Fig. 2. The fusion

protein expression level is very high when the fusion gene expres-

sion was induced by the addition of IPTG at 37 C (Fig. 2, line 35),

and only a small proportion was soluble and active (Fig. 2line 79).However, the fusion protein was expressed as almost soluble form

when the fusion gene expression was induced without IPTG addi-

tion (Fig. 2line 2 and 6).

Secretion of fusion protein in E. coli

When pET-OsmY-xynA vector was used to express fusion pro-

tein, the xylanase activities in the extracellular, periplasmic and

cytoplasmic fractions were monitored (Fig. 3). Protein expression

was initiated by the addition of IPTG to a final concentration of

0.5 mM when the optical density at 600 nm (OD600) reached 0.8.

Fig. 1. Total expresion levels of fusion protein OsmY-xynA in cytoplasm, periplasmand culture medium were observed after induction at different IPTG concentrations.1 Abbreviations used:LB, LuriaBertani; IPTG, isopropyl-b-D-thio galactopyranoside.

2 Y. Le, H. Wang/ Protein Expression and Purification 99 (2014) 15


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WhenE. coliBL21(DE3) was used as host to express fusion pro-

tein in LB medium, a maximal cell density (OD6005.3 0.1) was ob-

tained without IPTG addition (Fig. 3). In comparison, a significantly

higher level of xylanase activity was observed in the extracellular,

periplasmic and cytoplasmic fractions when cells induced withoutIPTG addition (Fig. 3).

As shown inFig. 3a, when the fusion protein was expressed by

the addition of IPTG to a final concentration of 0.5 mM, most of the

xylanase activity (5.7 0.2 U/ml) was observed in the extracellular

fraction at 24 h post-induction. However, when the fusion protein

was expressed without IPTG induction, most of the activity was ob-

served in the extracellular (36 1.3 U/ml) and the periplasmic

(42 4 U/ml) (Fig. 3b).

Purification of the fusion protein

The fusion protein was purified in two steps: cold osmotic

shock and nickel affinity chromatography (Table 1). The periplasm

fusion proteins were extracted by the cold osmotic shock proce-dure and furthermore purified with nickel affinity chromatogra-

phy. An analysis by SDSPAGE showed that the recombinant

fusion OsmY-xynA had a molecular mass of about 44 kDa (Fig. 4).

After the enzymes were purified, the specific activity of fusion pro-

tein OsmY-xynA was 739 45 U/mg. In comparison, the purified

xylanase from Aspergillus niger exhibited a specific activity of

808.5 U/mg towards birch wood xylan [15]. The specific enzyme

activity of xylanase from Thermotoga thermarum was up to145.8 U/mg [16]. XynA from Clostridium cellulovorans had a high

specific activity with birch wood xylan (825 U/mg) [17].

Effects of pH and temperature on enzyme activity and stability

When birch wood xylan was used as substrate, the optimal

reaction of fusion protein occurred at 65 C, pH 6.0 (Fig. 5a and

b). The purified enzyme retained over 80% of its activity after hold-

ing at a pH ranging from 5.8 to 7.8 for 1 h at 60 C(Fig. 5c). The

thermostability was evaluated by determination of the residual

ligating activity after incubating the mixtures (0.042 mg/ml

OsmY-xynA, 50 mM pH 6.0) for various times at 60C, 65C,70C.Fig. 5d shows the residual activity assayed under standard

reaction conditions.

Table 1

Purification of fusion protein OsmY-xynA from E. coliharboring pET-OsmY-xynA.

Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Purification fold

Periplasmic fraction 3.3 442.2 134 1.0

Nickel affinity chromatography 0.27 199.5 739 5.5

Fig. 4. SDSPAGE analysis for the expression of fusion protein. lanes: M, protein

markers; 1, total protein of E. coli containing pET-20b(+); 2, total protein and 3,

soluble protein of E. coli containing pET-OsmY-xynA; 4, periplasmic protein

obtained by cold osmotic shock; 5, fusion protein purified after a His-tagged

affinity chromatography.

Fig. 3. The effect of IPTG induction on the cell density and the production of recombinant protein in the E. colicells harboring pET-OsmY-xynA. When the OD600 reached 0.8

(denoted as time zero), IPTG was added to cell cultures. (a) with 0.5 mM IPTG induction, (b) without IPTG addition. Symbols: -j- cell density; -D- xylanase activities in the

extracellular; -h- xylanase activities in the periplasmic; -s- xylanase activities in the cytoplasmic fractions.

Fig. 2. SDSPAGE analysis for the expression of fusion protein in pET-OsmY-xynA

after induction at different IPTG concentrations. Lanes: M, protein markers; 1, total

protein ofE. colicontaining pET-20b(+); 25, total protein ofE. colicontaining pET-

OsmY-xynA induction with 0 mM, 0.1 mM, 0.3 mM, 0.5 mM IPTG, respectively; 69,

soluble protein ofE. colicontaining pET-OsmY-xynA induction with 0 mM, 0.1 mM,

0.3 mM, 0.5 mM IPTG, respectively.

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Discussion

To facilitate enzyme production and the industrial use of the

thermophilic xylanase preparations, several xylanase genes from

thermophilic fungi have been cloned and expressed in different

heterologous systems[18,19]. The mature xylanase from T. lanug-

inosushas 196 amino acids, with 31 amino acids encoded by the

codons rarely used in E. coli [3]. In previous study, the xylanase

gene fromT. lanuginosushad been optimized for high level expres-

sion in the cytoplasm ofE. coli by using pET-20b-xyA, but the en-

zyme was produced at a largely insoluble state, the soluble

expression level of recombinant enzyme was very poor, with ahighest activity of 4.1 U/ml[4].

OsmY of E. coli is the hyperosmotically inducible periplasmic

protein. Many studies have been focused on the excretion of target

proteins into the medium. When fused to OsmY, many recombi-

nant proteins of different origin could be excreted into the medium

[811].

Here, we constructed a vector pET-OsmY-xynA to export fusion

protein. OsmY-xynA was successfully expressed at a high level in a

soluble form when the fusion gene expression was induced with-

out IPTG addition. Interestingly, the majority of the recombinant

proteins were in the insoluble fraction when cells were induced

by the addition of different IPTG concentrations (Fig. 2). SDSPAGE

analysis revealed that the fusion proteins expressed from pET-

OsmY-xynA after induction at different IPTG concentrations were

separated into two adjacent bands, suggesting that some of the en-

zyme molecules were still carrying signal peptide in the periplasm

(Fig. 2). These observations suggested that recombinant fusion

xylanase was not correctly folded when the heterogeneous pro-

teins were expressed in a very high rate after induction with IPTG.

The pET system is the most powerful system used for the cloning

and expression of recombinant proteins in E. coli[20].

Induction is probably due to auto-induction due to lactose in

the medium[21]. It is known that LB medium may be contami-

nated with lactose, and lactose contamination increases back-

ground level of protein expression. Supplementing culture media

with glucose could maintain very low basal expression levels ofT7 RNA polymerase in the kDE3 lysogenic expression hosts used

in the pET System [22,23]. Much lower expression of the target

protein was observed (4.5 0.5 U/ml) when the strain harboring

pET-OsmY-xynA was grown in LB medium supplemented with

1% glucose by incubation overnight at 37 C without IPTG.

In our previous study, the xylanase gene from T. lanuginosus was

overexpressed in the cytoplasm, but the enzyme was produced at a

largely insoluble state[4]. The improvement of soluble protein is

primarily attributed to the fact that the periplasmic spac provides

a more oxidative environment than the cytoplasm[24]. The fusion

proteins were translocated into the periplasmic space, and enzyme

activity analysis shown that about 43% of enzyme activity was

localized in the periplasmic space (Fig. 3a). A simple osmotic shock

was used to obtain the products (Fig. 4).

Fig. 5. The effects of temperature and pH on the xylanase activity and enzyme stability of OsmY-xynA. (a) The optimal temperature determined by standard assay at various

temperatures (pH 6.0). (b) The optimal pH determined at 65 C in citrate buffer buffer, or phosphate buffer. (c) The pH stability of OsmY-xynA. The remaining activity was

determined after purified enzyme (0.042 mg/ml) was incubated at 60 C for 1 h in 50 mM in citrate buffer buffer, or phosphate buffer. (d) The thermostability of the OsmY-

xynA. The purified enzyme (0.042 mg/ml) in 50 mM phosphate buffer (pH 6.0) was incubated for various durations at 60 C (-N-), 65 C (-d-), 70 C (-j-). Residual activities

were assayed at 65C in 50 mM phosphate buffer (pH 6.0). The initial activity (739 45 U/mg) was defined as 100%. Data are means standard deviations from three

replications.

4 Y. Le, H. Wang/ Protein Expression and Purification 99 (2014) 15


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Previous results have shown that the signal peptide of OsmY is

responsible for the secretion into the periplasm, while OsmY is

responsible for the excretion from the periplasm to the medium

[11]. On the basis of xylanase activity, about 37% of enzyme activ-

ity was in the medium when cells were induced without IPTG addi-

tion (Fig. 3).

In summary, while the mechanism for the pET-OsmY-xynA

mediated high-level soluble expression of an aggregation-pronexylanase in E. coli is not completely understood, this paper offers

an alternative protocol to prevent the inclusion body formation

of a thermostable xylanase from thermophilic fungus.

Acknowledgments

This work was supported by the National Natural Science Foun-

dation of China (Grant No. 31300088), the Natural Science Founda-

tion of the Jiangsu Higher Education Institutions of China (Grant

No. 12KJB180002) and Doctoral Scientific Research Foundation of

Jiangsu University (Grant No. 10JDG117).

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