Molecular cloning, sequence analysis and expression of a GHF 43 xylanase from Aspergillus niger in Escherichia coli

被引:2
|
作者
Zhou, Chen-Yan [1 ]
Wang, Yong-Tao [2 ]
Zhu, Tie-Chui [2 ]
Fu, Guan-Hua [1 ]
Wang, Dan-Dan [1 ]
机构
[1] Xinxiang Med Univ, Sch Life Sci & Technol, Xinxiang 453003, Peoples R China
[2] Xinxiang Med Univ, Affiliated Hosp 1, Weihui 453100, Peoples R China
来源
关键词
Aspergillus niger; cloning; enzymatic properties; Escherichia coli; expression; xylanase; GENE CLONING; THERMOSTABLE XYLANASE; BACILLUS; USAMII; PURIFICATION;
D O I
10.2323/jgam.60.234
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A new xylanase gene (xyn43A) from Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL. The coding region of the gene was separated by only one intron 86 bp in length. It encoded 318 amino acid residues of a protein with a calculated molecular weight (MW) of 33.47 kDa plus a signal peptide of 19 amino acids. The amino acid sequence of the xyn43A gene showed 77.56% amino acid identity to A. nidulans xylanase, and the phylogenetic tree analysis revealed that xyn43A had close relationships with those of family 43 of glycosyl hydrolases reported from other microorganisms. Three-dimensional structure modeling showed that Xyn43A had a typical five-blade beta-propeller fold. The mature peptide encoding cDNA was subcloned into pET-28a (+) expression vector. The resultant recombinant plasmid pET-28a-xyn43A was transformed into Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. A maximum activity of 61.43 U/mg was obtained from the cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn43A. The recombinant xylanase had optimal activity at pH5.0 and 45 degrees C. Fe3+, Cu2+ and EDTA had an obvious active effect on the enzyme.
引用
收藏
页码:234 / 240
页数:7
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