1. High-level Expression of an Acidic and Thermostable Chitosanase in Pichia pastoris Using Multi-copy Expression Strains and High-cell-density Cultivation
- Author
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Liu Fang, Rao Ben, Liao Xianqing, Zhou Ronghua, Wang YaPing, Dong Qing, and Chen Wei
- Subjects
0106 biological sciences ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Biomedical Engineering ,Bioengineering ,biology.organism_classification ,Polysaccharide ,01 natural sciences ,Applied Microbiology and Biotechnology ,Pichia pastoris ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme ,Aspergillus oryzae ,Chitin ,Biochemistry ,Glucosamine ,010608 biotechnology ,Fermentation ,Chitosanase ,030304 developmental biology ,Biotechnology - 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.
- Published
- 2020