Bioquimica Cromatografia De Proteinas

J. Microbiol. Biotechnol. (2012), 22(7), 930–938 http://dx.doi.org/10.4014/jmb.1110.10060 First published online March 31, 2012 pISSN 1017-7825 eISSN 1738-8872

Isolation, Purification, and Characterization of a Thermostable Xylanase from a Novel Strain, Paenibacillus campinasensis G1-1
Zheng, Hongchen1,4†, Yihan Liu1,3,4†, Xiaoguang Liu2,4, Jianling Wang3,4, Ying Han4, and Fuping Lu1,2,4*
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Key Laboratory of Industrial Fermentation Microbiology, Education Ministry of China, 300457 Tianjin, China National Engineering Laboratory for Industrial Enzymes (NELIE), 300457 Tianjin, China 3 Tianjin Key Laboratory of Industrial Microbiology, 300457 Tianjin, China 4 College of Biotechnology, Tianjin University of Science and Technology, 300457 Tianjin, China Received: November 18, 2011 /Revised: January 13, 2012 / Accepted: February 27, 2012

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, SephadexG75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by Ca2+, Ba2+, DTT, and βmercaptoethanol, but was inhibited by Ni2+, Fe2+, Fe3+, Zn2+, SDS, and EDTA. The purifiedXynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of 60oC and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of 70oC~80oC), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.Keywords: Paenibacillus campinasensis, thermostable xylanase, isolation, purification, characterization
*Corresponding author Phone: +86-22-60600160; Fax: +86-22-60602298; E-mail: lfp@tust.edu.cn
† Hongchen Zheng and Yihan Liu, who contributed equally to this work, are both nominated as the first author.

Xylanases (endo-1,4-β-D-xylanohydrolase; E.C. 3.2.1.8) are important enzymes for the degradationof the major hemicellulosic polysaccharide of hardwood, since they hydrolyze xylopyranosyl linkages of β-1,4-xylan [6]. According to the similarity of amino acid sequences of their catalytic domain, xylanases have been grouped commonly into glycoside hydrolase (GH) families 10 and 11. These two families differ in substrate specificity, enzyme action, molecular weight, and net electric charge [2,18, 22]. As the key xylan-degrading enzymes, xylanases have attracted considerable attentions because of their widespread application in various industrial processes such as pulp bleaching, animal feeding, baking and brewing, wastetreating, bioenergy converting, textiles, and production of xylooligosaccharides [2, 11, 18]. A number of xylanases have been shown to be produced by fungi, bacteria, andactinomycetes [2, 18, 20]. These xylanases from various microorganisms may have diverse physicochemical properties and structures. Thus, studies of microorganisms that utilize xylan and the enzyme systems involved are becoming increasingly relevant in ecological and economic terms [14, 18]. In recent years, major efforts have been focused on the isolation of thermophilic and alkalophilic...