Proceedings of the Korean Society for Applied Microbiology Conference (한국미생물생명공학회:학술대회논문집)
- 2000.04a
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- Pages.157-167
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- 2000
GENETIC AND BIOCHEMICAL ANALYSIS OF A THERMOSTABLE CHITOSANASE FROM Bacillus sp. CK4
- Yoon, Ho-Geun (Graduate School of Biotechnology, Korea University) ;
- Cho, Hong-Yon (Graduate School of Biotechnology, Korea University)
- Published : 2000.04.01
Abstract
A thermostable chitosanase gene from the isolated strain, Bacillus sp. CK4, was cloned, and its complete DNA sequence was determined. The thermostable chitosanase gene was composed of an 822-bp open reading frame which encodes a protein of 242 amino acids and a signal peptide corresponding to a 30 kDa enzyme in size. The deduced amino acid sequence of the chitosanase from Bacillus sp. CK4 exhibits 76.6%, 15.3%, and 14.2% similarities to those from Bacillus subtilis, Bacillus ehemensis, and Bacillus circulans, respectively. C-terminal homology analysis shows that Bacillus sp. CK4 belongs to the Cluster III group with Bacillus subtilis. The size of the gene was similar to that of a mesophile, Bacillus subtilis showing a higher preference for codons ending in G or C. The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues were changed to E50D/Q, E62D/Q, and D66N/E by site-directed mutagenesis. The D66N/E mutants enzymes had remarkably decreased kinetic parameters such as
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