PRIMARY STRUCTURE OF CHITOSANASE PRODUCED BY BACILLUS-CIRCULANS MH-K1

To determine the primary structure of chitosanase, which was produced by Bacillus circulans MH-K1, its amino acid sequence was analyzed. Total 183 amino acids were determined. Two kinds of primer were synthesized according to the obtained amino acid sequence, and were used for PCR amplification of chitosanase gene. A 620 bp fragment was amplified, and was used for a probe for Southern hybridization of the genomic DNA which was cut by some restriction enzymes. A 5.6 kb PstI fragment was isolated and introduced into pUC 19 vector. Colonies which harbored chitosanase gene containing pUC 19 were detected by colony hybridization with the probe. HindIII/HindIII fragment (1.2 kb) and HindIII/SacI fragment (0.7 kb) were sub-cloned and sequenced. The chitosanase gene (open reading frame is 900 bp containing 259 amino acids and a signal peptide) was coded by the fragments. There was no meaningful homology to other enzymes including chitinase.