Thermostable chitosanase from Bacillus sp. strain CK4: Its purification, characterization, and reaction patterns

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2001-04, Vol.65 (4), p.802-809
• Main Authors: Yoon, H.G. (Korea Univ., Seoul (Korea R.)), Kim, H.Y, Kim, H.K, Hong, B.S, Shin, D.H, Cho, H.Y
• Format: Article
• Language: English
• Subjects: Ammonium Sulfate - chemistry; AMMONIUM SULPHATE; BACILLUS; Bacillus - enzymology; Bacillus sp. strain CK4; BIOCHEMICAL REACTIONS; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; CHITOSAN; Chitosan oligosaccharide; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Thin Layer; ENZYMIC ACTIVITY; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - chemistry; Hydrogen-Ion Concentration; HYDROLASES; Isoelectric Focusing; Metals - chemistry; Miscellaneous; Mission oriented research; Molecular Weight; OLIGOSACCHARIDES; Oligosaccharides - chemistry; PURIFICATION; Substrate Specificity; Temperature; TEMPERATURE RESISTANCE; Thermostable chitosanase
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.65.802

A thermostable chitosanase, purified 156-fold to homogeneity in an overall yield of 12.4%, has a molecular weight of about 29,000±2,000, and is composed of monomer. The enzyme degraded soluble chitosan, colloidal chitosan, and glycol chitosan, but did not degrade chitin or other β-linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co 2+ and 1.4-fold by Mn 2+ . However, Cu 2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60°C and 6.5, respectively. The enzyme was stable after heat treatment at 80°C for 30 min or 70°C for 60 min and fairly stable in protein denaturants as well. Chitosan was hydrolyzed to (GlcN) 4 as a major product, by incubation with the purified enzyme. The effects of ammonium sulfate and organic solvents on the action pattern of the thermostable chitosanase were investigated. The amounts of (GlcN) 3 -(GlcN) 6 were increased about 30% (w/w) in DAC 99 soluble chitosan containing 10% ammonium sulfate, and (GlcN) 1 was not produced. The monophasic reaction system consisted of DAC 72 soluble chitosan in 10% EtOH also showed no formation of (GlcN) 1 , however, the yield of (GlcN) 3 ∼ (GlcN) 6 was lower than DAC 99 soluble chitosan-10% ammonium sulfate. The optimal concentration of ammonium sulfate to be added was 20%. At this concentration, the amount of hexamer was increased by over 12% compared to the water-salt free system.