Purification and properties of chitosanase from Bacillus circulans MH-K1.

Abstract

A chitosanase was concentrated from the culture broth of Bacillus circulans MH-K 1 and was purified to homogeneity by CM-cellulose and gel permeation chromatography. The enzyme has a molecular weight of about 30,000, its Km is 0.63 mg chitosan/ml and its pus 9.2. The maximum velocity of chitosan degradation by the enzyme was obtained at 50°C when pH was maintained at 6.5. The enzyme was stable within the range of 0-40°C and pH 4.0-9.0. p-Chloromercuribenzoate and the metal ions of Cue +, Hg2 +, Nit +, and Zn2 + inhibited the enzyme activity. The enzyme degraded chitosan, glycolchitosan and CM-chitosan, but f-1,4-glucans such as chitin or its derivatives and CM-cellulose were not susceptible to the enzyme. The degree of deacetylation of chitosan significantly affected its susceptibility to the enzyme action. The most susceptible substrate was 80 deacetylated chitosan, and the substrates with less than 40% deacetylation were not affected by the enzyme. It is suggested that the presence of Nacetylglucosamine residues in the molecule of chitosan play an important role in the recognition of the substrate by the enzyme. The enzyme showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of the dimer and trimer of glucosamine. The smallest of the substrates was a tetramer of glucosamine.Chitosan is a polymer with 18-1,4-linked glucosamine residues. It is usually obtained by artificial deacetylation of chitin, a polymer of N-acetyl;/3-Dglucosamine, with a concentrated NaOH solution. Recently, as the medical uses of chitosan, its derivatives or its partially degraded oligosaccharides have been developed, the demands are growing for chitosanases, needed for mild degradation of chitosan (1).

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

“…In order to determine the carboxylic residues that were possible candidates for such function in chitosanase, we profited from the rather unusual homology between the first two chitosanases for which the primary sequences were determined. The chitosanases from B. circulans MH-K1 (27) and from Streptomyces sp. N174 (23) are similar in several ways.…”

Purification and some properties of a novel chitosanase from Bacillus subtilis KH1.

“…1993; Yabuki et al, 1998;Yoshihara et al, 1992). Most purified chitosanases have been characterized as endo-type enzymes, which cleave chitosans at random, their reaction velocities being highly dependent on the degree of acetylation of chitosan.…”

Uncoupling chitosanase production from chitosan

“…As the expression of genes encoding extracellular enzymes is strictly controlled by various mechanisms, the addition of chitosan to the culture medium is often necessary to induce chitosanase production. Examples of well studied chitosanases purified directly from the culture supernatants of the native strains include the GH46 chitosanase from Bacillus circulans MH-K1 8 and the GH8 chitosanase from Paenibacillus fukuinensis D2. 9 Addition of chitosan to the culture medium was required in both cases.…”