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, Cu2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable after heat treatment at 80 degrees C for 30 min or 70 degrees 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 approximately (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.
Yoon
H G
HG
Graduate School of Biotechnology, Korea University, Seoul.
Kim
H Y
HY
Kim
H K
HK
Hong
B S
BS
Shin
D H
DH
Cho
H Y
HY
D000645:Ammonium Sulfate / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008670:Metals / Q000737:chemistry; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D008670:Metals; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C019868:chitosanase; D000645:Ammonium Sulfate