Primary structure of chitosanase produced by Bacillus circulans MH-K1.

Abstract

To determine the primary structure of chitosanase, which was produced by Bacillus circulans MH-K 1, 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 by 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 by containing 259 amino acids and a signal peptide) was coded by the fragments. There was no meaningful homology to other enzymes including chitinase.The difference between chitin and chitosan is the degree of acetylation of D-glucosamine residues. Usually those of chitin are acetylated more than 60% and those of chitosan are acetylated less than 40%. The enzyme which hydrolyzes chitin is chitinase, and for chitosan is chitosanase.

Crystal Structure of Chitosanase from Bacillus circulans MH-K1 at 1.6-Å Resolution and Its Substrate Recognition Mechanism

“…EAG1 (90% identity with MH-K1 chitosanase) (deposited in GenBank TM , accession number AB008788 (Akiyama, K., Fujita, T., Kuroshima, K., Sakane, T., Yokota, A., and Takata, R.). Most of the reported amino acid sequence of MH-K1 chitosanase was determined from the protein sequence, but several regions were partially deduced from the nucleotide sequence (16). The amino acids corrected in the present study were located in the regions deduced from the nucleotide sequence.…”

“…Most chitosanases are found in microorganisms (8 -11), and a few are found in plants (12)(13)(14)(15). The complete amino acid sequences have been reported for procaryotic chitosanases from Bacillus circulans MH-K1 (MH-K1 chitosanase) 1 (16), Streptomyces sp. N174 (N174 chitosanase) (17), and Nocardioides sp.…”

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

“…While all the known chitinases fell into two classes (classes I and II as defined in Ref. 13 or, respectively, families 19 and 18 of glycosyl hydrolases (14)) no homologies were found between the chitosanases and either class I or class II chitinases (10,12,14). On the other hand, both chitosanases showed extensive homologies in their N-terminal segments (12).…”

“…The cloning and sequencing of the first two chitosanase genes from Bacillus circulans MH-K1 (10) and from Streptomyces sp. N174 (11,12) allowed the comparison of their deduced amino acid sequences with those of known chitinases of plant, fungal, or bacterial origin (reviewed in Refs.…”

Molecular characterization and antifungal activity of a family 46 chitosanase fromAmycolatopsissp. CsO-2

“…To investigate active centers within the CtoA protein, its amino acid sequence was aligned with that of N174 chitosanase (Masson et al, 1994), together with other family 46 chitosanases from Nocardioides sp. N106 (Masson et al, 1995), Bacillus circulans MH-K1 (Ando et al, 1992), and Pseudomonas sp. A-01 (Ando et al, 2008).…”