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Cloning and Overexpression of a Paenibacillus ${eta}-Glucanase$ in Pichia pastoris: Purification and Characterization of the Recombinant Enzyme
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  • Cloning and Overexpression of a Paenibacillus ${eta}-Glucanase$ in Pichia pastoris: Purification and Characterization of the Recombinant Enzyme
  • Cloning and Overexpression of a Paenibacillus ${eta}-Glucanase$ in Pichia pastoris: Purification and Characterization of the Recombinant Enzyme
저자명
Yang. Peilong,Shi. Pengjun,Wang. Yaru,Bai. Yingguo,Meng. Kun,Luo. Huiying,Yuan. Tiezheng,Yao. Bin
간행물명
Journal of microbiology and biotechnology
권/호정보
2007년|17권 1호|pp.58-66 (9 pages)
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한국미생물생명공학회
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정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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기타언어초록

Isolation, expression, and characterization of a novel $endo-{eta}-1,3(4)-D-glucanase$ 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 ${eta}-glucanase$ 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 ${eta}-1,3-1,4-glucanases$ of Bacillus. The gene fragment Bg1 containing the mature glucanase protein was expressed in Pichia pastoris at high expression level in a 3-1 high-cell-density fermenter. The purified recombinant enzyme Bg1 showed activity against barley ${eta}-glucan$, lichenan, and laminarin. The gene encodes an $endo-{eta}-1,3(4)-D-glucanase$ (E. C. 3.2.1.6). When lichenan was used as substrate, the optimal pH was 6.5, and the optimal temperature was $60^{circ}C$. The $K_m,;V_{max},;and;k_{cat}$ values for lichenan are 2.96mg/ml, $6,951{mu}mol/min{cdot}mg,;and;3,131s^{-1}$, respectively. For barley ${eta}-glucan$ the values are 3.73mg/ml, $8,939{mu}mol/min{cdot}mg,;and;4,026s^{-1}$, 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.