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High-level Expression of an Acidic and Thermostable Chitosanase inPichia pastorisUsing Multi-copy Expression Strains and High-cell-density Cultivation

文献类型： 外文期刊

作者： Zhou Ronghua 1 ; Liao Xianqing 1 ; Liu Fang 1 ; Dong Qing 1 ; Chen Wei 1 ; Wang YaPing 2 ; Rao, Ben 1 ;

作者机构： 1.Hubei Acad Agr Sci, Natl Biopesticide Engn Technol Res Ctr, Hubei Innovat Ctr Agr Sci & Technol, Biopesticide Branch,Hubei Biopesticide Engn Res C, Wuhan, Peoples R China

2.Hubei Univ, State Key Lab Biocatalysis & Enzyme, Engn Hubei Collaborat Innovat Ctr Green Transform, Hubei Key Lab Ind Biotechnol,Biol Fac, Wuhan 430062, Hubei, Peoples R China

关键词： Chitosanase; high-density fermentation; multicopy strains; Pichia pastoris

期刊名称：BIOTECHNOLOGY AND BIOPROCESS ENGINEERING （ 影响因子：2.836； 五年影响因子：2.281 ）

ISSN： 1226-8372

年卷期： 2020 年 25 卷 4 期

页码：

收录情况： SCI

摘要： Chitin is a linear homopolymer of acetylated beta-(1,4)-linked glucosamine residues and among the most abundant polysaccharides in the world. Here, we identified and purified a novel chitosanase (CCHA) fromAspergillus oryzaeNKY2017 obtained from Hu'bei province in China. Construction of a cDNA library from this strain revealed the gene sequence subsequently expressed inPichia pastorisand subsequent construction of multi-copy expression plasmids (CCHA1/2/3/4). The results demonstrated elevated levels ofCCHAexpression in multi-copy strains, with strain CCHA4 chosen for high-density fermentation and enzyme-activity experiments. High-density fermentation achieved a CCHA yield of 22,500 U/mL, and temperature and pH optimization resulted in the highest CCHA activity at 40 degrees C and 4.0, respectively. We used this enzyme for a large-scale preparation of oligosaccharides: 4 g enzyme could convert 150 kg chitosan into oligosaccharides in 24 h at 40 degrees C. These results demonstrated abundant CCHA expression inP. pastorisand suggested the efficacy of CCHA for use in industrial applications.

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