Cloning and overexpression of a Paenibacillus beta-glucanase in Pichia pastoris: Purification and characterization of the recombinant enzyme

文献类型： 外文期刊

第一作者： Yang, Peilong

作者： Yang, Peilong;Shi, Pengjun;Wang, Yaru;Bai, Yingguo;Meng, Kun;Luo, Huiying;Yuan, Tiezheng;Yao, Bin

作者机构：

关键词： Paenibacillus;endo-beta-1,33(4)-D-glucanase;gene;cloning;expression;characterization;Pichia pastoris

期刊名称：JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：2.351； 五年影响因子：2.65 ）

ISSN： 1017-7825

年卷期： 2007 年 17 卷 1 期

页码：

收录情况： SCI

摘要： Isolation, expression, and characterization of a novel endo-beta-1,3(4)-D-glucanase with high specific activity and homology to Bacillus lichenases is described. One clone was screened from a genomic library of Paenibacillus sp. F-40, using lichenan-containing plates. The nucleotide sequence of the clone contains an ORF consisting of 717 nucleotides, encoding a beta-glucanase protein of 238 amino acids and 26 residues of a putative signal peptide at its N-terminus. The amino acid sequence showed the highest similarity of 87% to other beta-1,3-1,4-glucanases of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 34 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley beta-glucan, lichenan, and laminarin. The gene encodes an endo-beta-1,3(4)-D-glucanase (E.C.3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was 60 degrees C. The K-m, V-max, and k(cat) values for lichenan are 2.96 mg/ml, 6,951 mu mol/min center dot mg, and 3,131 s(-1), respectively. For barley beta-glucan the values are 3.73 mg/ml, 8,939 mu mol/min center dot mg, and 4,026 s(-1), respectively. The recombinant Bg1 had resistance to pepsin and trypsin. Other features of recombinant Bg1 including temperature and pH stability, and sensitivity to some metal ions and chemical reagents were also characterized.

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