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KMID : 0545120070170010058
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 1 p.58 ~ p.66
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Cloning and Overexpression of a Paenibacillus β-Glucanase in Pichia pastoris: Purification and Characterization of the Recombinant Enzyme
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Yang Peilong
Shi Peng-Jun
Wang Yaru
Bai Ying-Guo
Meng Kun
Luo Hui-Ying
Yuan Tie-Zheng
Yao Bin
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Abstract
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Isolation, expression, and characterization of a novel endo-β-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 β-glucanase protein of 238 amino acids and 26 residues of a putative signal peptide at its Nterminus. The amino acid sequence showed the highest similarity of 87% to other β-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 3-l high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley β- glucan, lichenan, and laminarin. The gene encodes an endo-β- 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 60oC. The Km, Vmax, and kcat values for lichenan are 2.96 mg/ml, 6,951 μmol/min·mg, and 3,131 s-1, respectively. For barley β-glucan the values are 3.73 mg/ml, 8,939 μmol/min·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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KEYWORD
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Paenibacillus, endo-β-1, 3(4)-D-glucanase, gene cloning, expression, characterization, Pichia pastoris
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