High-level Expression of an Acidic and Thermostable Chitosanase in Pichia pastoris Using Multi-copy Expression Strains and High-celldensity Cultivation

• Published in: Biotechnology and bioprocess engineering pp. 562 - 570
• Main Authors: Zhou Ronghua, Liao Xianqing, Liu Fang, Dong Qing, Chen Wei, Wang YaPing, Rao Ben
• Format: Journal Article
• Language: English
• Published: 한국생물공학회 01.08.2020
• Subjects: 생물공학
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-019-0445-3

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. KCI Citation Count: 0