title: Cloning and expression of chitin deacetylase gene from a Deuteromycete, Colletotrichum lindemuthianum.
abstract: The chitin deacetylase gene was cloned from cDNA of Colletotrichum lindemuthianum ATCC 56676, and the open reading frame consisted of a possible prepro-sequence of 27 amino acids at the N-terminus and a mature chitin deacetylase. The deduced amino acid sequence of the mature enzyme revealed 26% identity and 46% similarity with a chitin deacetylase from Mucor rouxii. The molecular mass of the protein estimated from the amino acid sequence data was 24.3 kDa, which was in good agreement with the MALDI-TOF MS analysis data of the purified protein (24.17-24.36 kDa). The gene product was overexpressed in Escherichia coli cells as a fusion protein with six histidine residues at its C-terminus. The fusion protein formed inclusion bodies, but chitin deacetylase activity was restored from the inclusion bodies by a simple renaturation step with 8 M urea treatment. The recombinant enzyme was purified by affinity chromatography and gel filtration steps, and had a final specific activity of 4.22 units mg(-1) of protein. Trypsin digestion of the recombinant enzyme resulted in 2.1-fold increase in activity, suggesting that the removal of the prepro-domain from the recombinant enzyme resulted in an increase in its activity.
authors.affiliation: National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tokuyasu
authors.forename: M
authors.initials: M
authors.lastname: Ohnishi-Kameyama
authors.forename: K
authors.initials: K
authors.lastname: Hayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Mori