Kinetics of Hydrolysis Reaction of Chitin Using Chitinase Purified from Marine Bacterium Alteromonas marina

摘要

Chitinase with a molecular weight of 45 kDa was purified from a marine bacterium, Alteromonas marina. It was obtained after cation exchange chromatographic isolation of crude enzymes from a culture solution where A.marina was incubated for 4 days using colloidal chitin as a carbon source. The two chitinases obtained were named Chi-A and Chi-B. Chi-A and Chi-B were purified to 6.73-fold, 1.32-fold from the crude enzyme, respectively. The optimum pH of Chi-A was found to be 7.0, and the enzyme was stable in the pH range of 4.0 ~ 9.0. The optimum temperature of Chi-A was 45 °C, and the enzyme was stable till 45 °C, but beyond this temperature the enzyme was deactivated. The enzyme hydrolyzed (GlcNAc)5 to (GlcNAc)3 and (GlcNAc)2, (GlcNAc)4 to two molecules of (GlcNAc)2 and to (GlcNAc)3 and (GlcNAc)1, and (GlcNAc)3 to (GlcNAc)2 and (GlcNAc)1, (GlcNAc)2 was not hydrolyzed by Chi-A. The reaction rate constants were calculated according to the reaction mechanism just mentioned above. As a result, the reaction rate constant k5 for (GlcNAc) 5 hydrolysis was found to be larger than k4 for (GlcNAc) 4 and k3 for (GlcNAc)3.