1. High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris
- Author
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Yuling Zhou, Lixin Kang, Hong Yan, Lixin Ma, Xiaolan Yu, Chunhua Li, Chao Zhai, and Xiaomei Chen
- Subjects
Glycoside Hydrolases ,Gene Expression ,Oligosaccharides ,Bioengineering ,Applied Microbiology and Biotechnology ,Pichia ,Aspergillus fumigatus ,Pichia pastoris ,Enzyme Stability ,Chitosanase ,chemistry.chemical_classification ,Chitosan ,Expression vector ,biology ,Protein Stability ,Hydrolysis ,Temperature ,General Medicine ,biology.organism_classification ,Recombinant Proteins ,Yeast ,Enzyme assay ,Enzyme ,chemistry ,Biochemistry ,biology.protein ,Fermentation ,Biotechnology - Abstract
The sequence of an endo-chitosanase gene (CSN) from Aspergillus fumigatus was optimized based on the preferred codons of Pichia pastoris and synthesized in vitro through overlapping PCR (CSN-P). The gene was cloned into a yeast expression vector, pHBM905A, and secretorily expressed in Pichia pastoris GS115. The yield of CSN-P reached ~3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was ~25,000 U/ml. The enzyme had half-lives of 2.5 h at 80°C, 1 h at 90°C and 32 min at 100°C. It retained 70% activity after incubation with 10 M urea at room temperature for 30 min. This enzyme was used for a large-scale preparation of oligosaccharides: 3 g enzyme converted 200 kg chitosan into oligosaccharides in 24 h at 60°C.
- Published
- 2011