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

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 beta -linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co2+ and 1.4-fold by Mn2+. However, Co2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60 degreesC and 6.5, respectively. The enzyme was stable after heat treatment at 86 degreesC for 30 min or 70 degreesC 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 (ClcN)(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) similar to (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 mater-salt free system.