title: High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris.
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.
authors.affiliation: Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, Hubei Province, People's Republic of China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Chen
authors.forename: Chao
authors.initials: C
authors.lastname: Zhai
authors.forename: Lixin
authors.initials: L
authors.lastname: Kang
authors.forename: Chunhua
authors.initials: C
authors.lastname: Li
authors.forename: Hong
authors.initials: H
authors.lastname: Yan
authors.forename: Yuling
authors.initials: Y
authors.lastname: Zhou
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yu
authors.forename: Lixin
authors.initials: L
authors.lastname: Ma
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D048271:Chitosan / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D009844:Oligosaccharides / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D009844:Oligosaccharides; D011994:Recombinant Proteins; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase