title: Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization.
abstract: The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step purification based on specific binding of the histidine residues was achieved. The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 degrees C and 8.0, respectively. The specific activity of the pure protein is 72 U/mg. One unit of enzyme activity is defined as the amount of enzyme that produces 1 micromol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.
authors.affiliation: Food Engineering and Bioprocess Technology Program, Asian Institute of Technology, Klong Luang, Pathumthani 12120, Thailand.
authors.forename: Binesh
authors.initials: B
authors.lastname: Shrestha
authors.forename: Karine
authors.initials: K
authors.lastname: Blondeau
authors.forename: Willem F
authors.initials: WF
authors.lastname: Stevens
authors.forename: Françoise L
authors.initials: FL
authors.lastname: Hegarat
mesh_terms: D000581:Amidohydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D020231:Colletotrichum / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
chemical_list: D011993:Recombinant Fusion Proteins; D000581:Amidohydrolases; C019866:chitin deacetylase