Biotechnol Lett DOI 10.1007/s10529-013-1392-2

ORIGINAL RESEARCH PAPER

Cloning, over-expression and characterization of a thermotolerant xylanase from Thermotoga thermarum Hao Shi • Yu Zhang • Hui Zhong • Yingjuan Huang • Xun Li • Fei Wang

Received: 9 July 2013 / Accepted: 17 October 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract The xyn10B gene, encoding the endo-1,4-bxylanase Xyn10B from Thermotoga thermarum, was cloned and expressed in Escherichia coli. The ORF of the xyn10B was 1,095 bp and encoded to mature peptide of 344 amino acids with a calculated MW of 40,531 Da. The recombinant xylanase was optimally active at 80 °C, pH 6.0 and retained approx. 60 % of its activity after 2 h at 75 °C. Apparent Km, kcat and kcat/Km values of the xylanase for beechwood xylan were 1.8 mg ml-1, 520 s-1 and 289 ml mg-1 s-1, respectively. The end products of the hydrolysis of beechwood xylan were mainly oligosaccharides but without xylose after 2 h hydrolysis.

Hao Shi and Yu Zhang contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s10529-013-1392-2) contains supplementary material, which is available to authorized users.

Keywords Thermotoga thermarum  Thermotolerant xylanase  Xylan  Xylanase  Xylooligosaccharides

Introduction Xylan, composed of both hetero- and homo-polysaccharides, is the main constituent of hemicellulosic biomass of plant polysaccharides and the second most abundant renewable carbon resources in the biosphere, comprising up to 39 % of the dry weight of terrestrial plants (Verma and Satyanarayana 2012). The hydrolysis of xylan involves the synergistic action of endo-b1,4-D-xylanases (EC 3.2.1.8), b-D-xylosidases (EC 3.2.1.37), a-L-arabinofuranosidases (EC 3.2.1.55), a-glucuronidases (EC 3.2.1.139), acetyl xylan esterases (EC 3.1.1.72) and feruloyl esterases (EC 3.1.1.73). Among these, xylanases and b-xylosidases are key ones for depolymerization of the backbone of xylan, while others cleave the side-chain residues (Zhang et al. 2011). Xylanases are a family of glycoside

H. Shi  Y. Zhang  H. Zhong  Y. Huang  X. Li  F. Wang (&) Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, China e-mail: [email protected]

Y. Zhang e-mail: [email protected]

H. Shi e-mail: [email protected]

X. Li e-mail: [email protected]

H. Zhong e-mail: [email protected] Y. Huang e-mail: [email protected]

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hydrolytic enzymes that cleave internal linkages of the b-1,4-glycosidically linked xylose backbone to yield oligosaccharides that are further hydrolyzed into xylose with b-xylosidases (Deesukon et al. 2011). Based on amino acid sequence similarities, xylanases are mainly grouped into glycoside hydrolase family (GHs) 10 and 11 (http://www.cazy.org/fam/ acc_GH.html). GH10 xylanases are found in plants, fungi and bacteria as well as archaea, whereas GH11 xylanases are only distributed in fungi and bacteria. Due to their catalytic activities in depolymerizing xylan and releasing lower XOs, xylanases have great potential and application in a wide range of industrial processes, including textile, paper and pulp, food and feed as well as biofuel industries (Pribowo et al. 2012). A large number of microbial xylanases have been reported (Deesukon et al. 2011; Khandeparker et al. 2011). In most industrial processes, however, a xylanase with high thermostability would be demanded as the consumption of the enzyme could be decreased. Therefore, it is essential to explore novel thermostable xylanases or to improve thermal tolerance of existing xylanases modified by gene engineering. In this study, we report the cloning, expression and functional characterization of the xyn10B gene encoding for Xyn10B, a novel GH10 xylanase from Thermotoga thermarum which shows a potential for producing xylooligosaccharides.

Materials and methods Materials Primers were synthesized by Sangon Bitech (Shanghai, China) Co. Ltd. Beechwood xylan, birchwood xylan, oat spelt xylan, Avicel and sodium carboxymethyl cellulose (CMC-Na) were purchased from Sigma. Mannan, arabinan, galactan were purchased from Megazyme.

Genomic DNA extraction, amplification Thermotoga thermarum was grown in medium as described by Windberger et al. (1989) and genomic DNA was extracted by the standard method (Mahuku 2004). The sequence of the xylanase gene (GenBank No. CP002351, Protein ID. AEH51686) based on the T. thermarum genomic DNA was amplified using primers 50 -GGAATTCCATATGGGACCACAGCCCATGTCGTC-30 , and 50 -CCGCTCGAGCTTGGAA CTGATTCTATTACACTC-30 (primers with the added compatible restriction sites of NdeI and XhoI, respectively). PCR was performed as follows: 94 °C, 5 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 50 s; and 72 °C, 10 min. Construction and sequencing of the expression vector To construct the recombinant vector, the amplified PCR products were purified using a PCR purification kit (Biomiga, Shanghai) and digested with NdeI and XhoI followed by the ligation into pET-20b vector at the corresponding sites. The ligation mixture was transformed into E. coli Top10 competent cells. The clones were screened for the positive recombinants using colony PCR. Positive clones were sequenced to obtain the correct clone of xylanase gene. Expression and purification of Xyn10B The processes of expression and purification of Xyn10B are shown in Table 1. Fractions containing Xyn10B xylanase were dialyzed overnight against storage buffer (20 mM Na2HPO4 buffer, pH 7.0, 50 mM NaCl, 10 % v/v glycerol) and kept at -80 °C until further use. SDS-PAGE analysis

Bacterial strains and plasmids Thermotoga thermarum DSM 5069 was used. Escherichia coli Top10 and E. coli BL21 (DE3) (Novagen) were used as hosts for DNA manipulations and gene expression, respectively. The vector pET-20b (Novagen) was used for the construction of the expression vector.

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The production, purity and molecular mass of the enzymes were determined by SDS-PAGE (Lammirato et al. 2011), using broad range molecular weight markers (MBI Fermentas) as standards. Gels were analyzed with image analysis system (Bio-Rad). The protein concentrations were determined using Bradford reagent with BSA as standard.

Biotechnol Lett Table 1 Purification of the recombinant Xyn10B xylanase Purification step

Total volume (ml)

Total activity (lmol min-1)

Total protein (mg)

Crude extracta

10

3500

125

10

3280

41

80

93.7

2.9

1

2880

15

192

82.3

6.9

Heat treatment

b

Ni affinity chromatographyc

Specific activity (lmol mg-1 min-1) 28

Recovery (%)

Purification (fold)

100

1

a

The recombinant strain was grown in LB medium (200 ml) with ampicillin (100 lg ml-1) at 37 °C to OD600 0.8 and was incubated further with IPTG for 5 h. The cells were harvested by centrifugation at 10,0009g for 15 min at 4 °C and resuspended in 10 ml imidazole buffer (10 ml 5 mM imidazole, 0.5 M NaCl, and 20 mM Tris/HCl buffer, pH 7.9), followed by sonication

b The cell extracts after sonication were heat treated at 50 °C for 30 min, and then cooled in an ice bath, centrifuged at 15,0009g for 20 min at 4 °C and the supernatant was kept c

The obtained supernatants were loaded on to an immobilized Ni2? affinity column (Novagen, USA), and eluted with 1 M imidazole, 0.5 M NaCl, and 20 mM Tris/HCl buffer (pH 7.9)

Enzyme assay

Biochemical characterization of Xyn10B

The reaction mixture containing 50 ll 1 % (w/v) beechwood xylan and 10 ll diluted enzyme solution was incubated in 50 mM imidazole buffer (pH 6.0) at 80 °C for 10 min. The total reaction system was 200 ll. The reaction was stopped by adding 300 ll 3,5-dinitrosalicylic acid (DNS) reagent. The amount of reducing sugar liberated was determined using DNS with xylose as the standard. One unit of xylanase activity was defined as the amount of enzyme that produced 1 lmol xylose equivalent per min.

Optimum temperature and pH of xylanase activity were determined using xylan from 1 to 10 mg ml-1. Determination of substrate specificity and kinetic properties Substrate specificity was determined using oat spelt xylan, beechwood xylan, birchwood xylan, mannan, arabinan, galactan, Avicel and CMC-Na. The reaction was carried out as above. Vmax, Km and kcat were

Fig. 1 Homology modeling of the Xyn10B. The distance of active amino acid residues between Glu145 and Glu252 was ˚ 5.5 A

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determined under optimum conditions and calculated from Michaelis–Menten equation. Analysis of hydrolysis product Beechwood xylanbirchwood xylan or oat spelt xylan was treated with purified Xyn10B, and the hydrolysis products were analyzed using an ion chromatography system (ICS; Dionex, USA) and TLC. A xylooligosaccharide mixture (Sigma) consisting of xylose, xylobiose, xylotriose, xylotetraose and xylohexaose was used as the standard. Bioinformatic analysis Phylogenetic relationships were deduced using the Neighbor-joining (NJ) and Maximum-parsimony (MP) methods as performed in Paup 4.0. Homology modeling was performed using Swiss Model. The crystal structure of a GH10 xylanase from Geobacillus stearothermophilus (EXPDB entry code: 2X8T: A) was used as three-dimensional template for restraint-based modeling as implemented in the SPDBV_4.04_PC.

revealed there was a close relationship with the GH10 xylanases from the same genus Thermotoga and Petrotoga mobilis (GenBank No: YP_001567298) (Supplementary Fig. 1). Expression and purification For functional analysis of the recombinant protein, the ORF region of the xyn10B gene was expressed in E. coli BL21 (DE3). The recombinant protein was extracted from the soluble fraction of the bacterial crude and purified (Table 1). Extracts from the bacteria harboring the construct Xyn10B xylanase exhibited a single band at approx. 40 kDa according to the SDSPAGE analysis (Fig. 2). Consistent with the theoretical molecular weight of the mature protein (40,531 Da) without the signal peptide, the target protein Xyn10B was successfully expressed and purified.

Results and discussion PCR amplification, construction, sequencing and bioinformatics analysis of xylanase The targeted xyn10B gene was amplified with specific primers xyn10B1/xyn10B2 and transformed into competent cells of E. coli BL21 (DE3) by heat-shock method, and then confirmed by colony PCR and gene sequencing. An ORF of 1,032 bp was identified to encode a mature polypeptide of 344 amino acid residues (without signal peptides) which only includes an amino acid catalytic domain. Deduced molecular mass and pI of Xyn10B were 40,531 Da and 6.39, respectively. Its amino sequence showed high homology to GH10 endo-1,4-b-xylanases. Homology modeling revealed that Xyn10B had the same eightfold (b/a) structure as the G. stearothermophilus T-6 endo-1,4-xylanase (Fig. 1). Glu145 and Glu252 were the catalytic nucleophile and proton ˚, donor, respectively. The calculated distance (5.5 A Fig. 1) between their functional catalytic clusters is consistent with the retaining hydrolytic mechanism (Davies and Henrissat 1995). The phylogenetic trees

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Fig. 2 SDS-PAGE analysis of recombinant Xyn10B in E. coli BL21 (DE3). Lane M: protein marker, lane 1: the total protein of BL21 (DE3) harboring pET-20b-xyn10B, lane 2: the purified Xyn10B by heat treatment (50 °C, 30 min), lane 3: the purified Xyn10B by Ni2? affinity column chromatography (eluted with 400 mM imidazole)

Biotechnol Lett

Biochemical characterization The recombinant xylanase was active from pH 4.5 to 7.5 and from 60 to 95 °C with optima at pH 6.0 and 80 °C (Fig. 3a, c). Similar to other thermophilic xylanases, the maximum activity of Xyn10B occurred at somewhat neutral pH and temperatures higher than 50 °C (Khandeparker et al. 2011). It was stable at pH 5.5–7.5 at 70 °C for 1 h and more than 70 % of its initial activity remained at this range (Fig. 3b). Xyn10B also retained [60 % of its initial activity for 2 h between 60 and 75 °C (Fig. 3d). The thermostability of Xyn10B is better than the commercial xylanase from Thermomyces lanuginosus, whose activity is only stable at 60 °C for 1 h (Gaffney et al. 2009).

Fig. 3 Temperature and pH dependence on activity of the Xyn10B. a Optimum pH (pH 4.0–8.5, 80 °C for 10 min). b pH stability (pH 4.0–8.5, 70 °C for 1 h). c Optimum temperature (pH 6.0, 60–85 °C for 10 min). d Thermostability (pH 6.0, 60–80 °C for 0, 30, 60, 90 and 120 min). The residual activity

Of various metal ions, only Cu2? completely inhibited enzyme activity (Table 2). Cu2? inhibition of the activity of xylanases was also found with that from Geobacillus thermoleovorans (Verma and Satyanarayana 2012. Unlike the xylanase from Streptomyces sp. SWU10, Mg2? and Al3? impaired the enzyme activity apparently (Deesukon et al. 2011). Furthermore, chemical reagents only slightly affected Xyn10B activity. The kcat/Km value of Xyn10B was more than tenfold higher than that from Trichoderma reesei and Chaetomium sp. (Jiang et al. 2010; Jun et al. 2009). The specific activity of Xyn10B was 192 lmol mg-1 min-1 while commercial xylanases from Trichoderma viride (Sigma) is 100–300 lmol mg-1 min-1 (Table 3).

was monitored, and the maximum activity was defined as 100 % (a, c) or initial activity was defined as 100 % (b, d). Values shown were the mean of triplicate experiments, and the variation about the mean was below 5 %

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Biotechnol Lett Table 2 Effects of cations and chemical reagents on the activity of purified Xyn10B

Table 4 Hydrolysis products of beechwood xylan, as mg l-1, detected by ICS

Cationa

Residual activity (%)

Control

100 ± 2

Xylose

10.2

Mg

2?

1h

46 ± 1.5

Xylobiose

22.2

Zn2?

80 ± 3.2

Xylotriose

63.9

Mn2?

133 ± 3

Xylotriose

39.7

Ba2?

72 ± 1.6

Xylopentaose

26.2

Ca2?

76 ± 0.6

Xylohexaose

47.3

3?

Al

46 ± 1.1 0

Conversion percentage, %

10.5

Cu2? Co2?

120 ± 4

2?

Ni

2h 11.5 64.2

3h 14.4

14.9

5h 15.3

6h 20.5

111

165

182

215

287

377

446

86.8

140

187

268

325

89.9

144

209

282

322

160

200

260

289

148

115 25.8

92.0

4h

38.3

50.4

68.4

79.2

Samples of beechwood xylan (2 g l-1) were incubated with Xyn10B xylanase (0.15 U) for 1–6 h

81 ± 0.4

Chemical reagentsb Tween 60

78 ± 1.2

Tris

71 ± 2.1

SDS

82 ± 0.9

Values shown are the mean of triplicate experiments, and the variations about the mean are below 5 %. The enzyme was incubated with each reagent for 2 h at 80 °C before the addition of xylan to initiate the enzyme reaction. The activity of the enzyme without adding chemical reagent or metal ion was defined as 100 % = 192 lmol mg-1 min-1 a

Final concentration, 1 mM

b

Final concentration, 0.05 % Tween 60 and Tris, 0.1 % SDS

Xylan hydrolysis There was no activity detected on starch, CMC-Na and Avicel, suggesting that this enzyme is a cellulase-free xylanase. Therefore, it has potential applications in the paper and pulp industry. The hydrolysis products were analyzed by ICS (Table 4) and TLC (Supplementary Fig. 2). Xylooligosaccharides (XOs), such as xylobiose, xylotriose, xylotetraose and xylopentaose, were the major products of beechwood xylan degradation after 2 or 3 h hydrolysis, and xylose was barely detectable by ICS or on a TLC plate. Therefore, the Xyn10B is considered as a suitable candidate for

production of XOs, which are recognized as prebiotics for both human and animal health (Chapla et al. 2012). Since XOs can be further degraded into xylose by bxylosidase, the Xyn10B may have other important applications in biofuels, food and other industries. As far as we know, the xylanases from Streptomyces thermocyaneoviolaceus and Streptomyces matensis possess a potential in generating XOs from abundantly-available lignocellulosic biomasses (Chapla et al. 2012; Shin et al. 2009). However, these xylanases are not suitable for the hydrolysis of xylan above 60 °C.

Conclusion The cloning, expression and characterization of Xyn10B xylanase from T. thermarum is described. Compared to the enzymatic properties from other microorganisms, the Xyn10B has a higher thermostability upto 75 °C and is more efficient in xylan hydrolysis. Therefore, the Xyn10B is a useful hydrolytic enzyme for hemicellulose hydrolysis in industrial application.

Table 3 Kinetic parameters for the xylanases using beechwood xylan as substrate Species

Vmax (lmol mg-1 min-1)

Km (mg ml-1)

kcat (s-1)

kcat/Km (ml mg-1 s-1)

T. thermarum

769

1.8

520

289

Trichoderma reesei

ND

2.7

75.4

27.9

Paecilomyces themophila Chaetomium sp.

1,290 ND

2.4 0.9

555 22.7

231 25.2

ND not determined

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Reference This study Jun et al. (2009) Li et al. (2006) Jiang et al. (2010)

Biotechnol Lett Acknowledgments This work was financially supported by the Jiangsu Education (11KJA48001, CXZZ11_0526), the National Natural Science Foundation of China (Nos. 31370572, 31200564) and the Doctorate Fellowship Foundation of Nanjing Forestry University as well as the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

References Chapla D, Pandit P, Shah A (2012) Production of xylooligosaccharides from corncob xylan by fungal xylanase and their utilization by probiotics. Bioresour Technol 115: 215–221 Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure (London, England: 1993) 3:853–859 Deesukon W, Nishimura Y, Harada N, Sakamoto T, Sukhumsirichart W (2011) Purification, characterization and gene cloning of two forms of a thermostable endo-xylanase from Streptomyces sp. SWU10. Process Biochem 46:2255–2262 Gaffney M, Carberry S, Doyle S, Murphy R (2009) Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris. Enzym Microb Technol 45:348–354 Jiang Z, Cong Q, Yan Q, Kumar N, Du X (2010) Characterisation of a thermostable xylanase from Chaetomium sp. and its application in Chinese steamed bread. Food Chem 120:457–462 Jun H, Bing Y, Keying Z, Daiwen C (2009) Functional characterization of a recombinant thermostable xylanase from Pichia pastoris: a hybrid enzyme being suitable for xylooligosaccharides production. Biochem Eng J 48:87–92

Khandeparker R, Verma P, Deobagkar D (2011) A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing. New Biotechnol 28:814–821 Lammirato C, Miltner A, Kaestner M (2011) Effects of wood char and activated carbon on the hydrolysis of cellobiose by b-glucosidase from Aspergillus niger. Soil Biol Biochem 43:1936–1942 Li L, Tian H, Cheng Y, Jiang Z, Yang S (2006) Purification and characterization of a thermostable cellulase-free xylanase from the newly isolated Paecilomyces themophila. Enzym Microb Technol 38:780–787 Mahuku GS (2004) A simple extraction method suitable for PCR-based analysis of plant, fungal, and bacterial DNA. Plant Mol Biol Rep 22:71–81 Pribowo A, Arantes V, Saddler JN (2012) The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover. Enzym Microb Technol 50:195–203 Shin JH, Choi JH, Lee OS, Kim YM, Lee DS, Kwak YY, Kim WC, Rhee IK (2009) Thermostable xylanase from Streptomyces thermocyaneoviolaceus for optimal production of xylooligosaccharides. Biotech Bioprocess Eng 14:391–399 Verma D, Satyanarayana T (2012) Cloning, expression and applicability of thermo-alkali-stable xylanase of Geobacillus thermoleovorans in generating xylooligosaccharides from agro-residues. Bioresour Technol 107:333–338 Windberger E, Huber R, Trincone A, Fricke H, Stetter KO (1989) Thermotoga thermarum sp.nov and Thermotoga neapolitana ocurring in African continental solfataric springs. Arch Microbiol 151:506–512 Zhang J, Siika-aho M, Puranen T, Tang M, Tenkanen M, Viikari L (2011) Thermostable recombinant xylanases from Nonomuraea flexuosa and Thermoascus aurantiacus show distinct properties in the hydrolysis of xylans and pretreated wheat straw. Biotechnol Biofuels 4:12

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