High-level Expression of an Acidic and Thermostable Chitosanase in Pichia pastoris Using Multi-copy Expression Strains a
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pISSN 1226-8372 eISSN 1976-3816
RESEARCH PAPER
High-level Expression of an Acidic and Thermostable Chitosanase in Pichia pastoris Using Multi-copy Expression Strains and High-celldensity Cultivation Zhou Ronghua, Liao Xianqing, Liu Fang, Dong Qing, Chen Wei, Wang YaPing, and Rao Ben
Received: 26 November 2019 / Revised: 27 February 2020 / Accepted: 27 February 2020 © The Korean Society for Biotechnology and Bioengineering and Springer 2020
Abstract Chitin is a linear homopolymer of acetylated β(1,4)-linked glucosamine residues and among the most abundant polysaccharides in the world. Here, we identified and purified a novel chitosanase (CCHA) from Aspergillus oryzae NKY2017 obtained from Hu’bei province in China. Construction of a cDNA library from this strain revealed the gene sequence subsequently expressed in Pichia pastoris and subsequent construction of multi-copy expression plasmids (CCHA1/2/3/4). The results demonstrated elevated levels of CCHA expression 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°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°C. These results demonstrated abundant CCHA expression in P. pastoris and suggested the efficacy of CCHA for use in industrial applications.
Zhou Ronghua†, Liao Xianqing†, Liu Fang, Dong Qing, Chen Wei, Rao Ben* National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan, China Tel/Fax: +86-27-59101911 E-mail: 623253512 @qq.com Wang YaPing State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of BioResources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province 430062, China †
These authors contributed equally to this work.
Keywords: Chitosanase, high-density fermentation, multicopy strains, Pichia pastoris
1. Introduction Chitin is a linear homopolymer of acetylated β-(1,4)-linked glucosamine residues and among the most abundant polysaccharides in the world [1]. Chitin is found mainly in insects, crustaceans, nematodes, and fungi, and following deacetylation, chitosan can be obtained in a soluble form suitable for multiple applications. The oligosaccharides derived from chitosan are water soluble and exhibit multiple useful biological activities, including antitumor, antibacterial, and antifungal activities. These features make chitosan and its associated oligosaccharides widely useful in the agriculture, food, and pharmaceutical industries. Chemical treatment and enzymatic hydrolysis are the two primary met
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