Journal
BIOTECHNOLOGY LETTERS
Volume 34, Issue 4, Pages 689-694Publisher
SPRINGER
DOI: 10.1007/s10529-011-0816-0
Keywords
Chitosanase; High-density fermentation; Pichia pastoris; Thermostability
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Funding
- Natural Foundiation of Hubei Province, P. R. China [2010CDB04501]
- Hubei Laboratory of Genetic Regulation and Integrative Biology [201004]
- National Key Scientific and Technological Project [2008ZX1000Z-009]
- National Sciences Foundation of China [30801008]
- National Sciences Foundation of Hubei Province, P. R. China [2008CDB110]
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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 similar to 3 mg/ml with a high-density fermentation in a 14 l fermenter and the enzyme activity was similar to 25,000 U/ml. The enzyme had half-lives of 2.5 h at 80A degrees C, 1 h at 90A degrees C and 32 min at 100A degrees 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 60A degrees C.
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