Preparation of Chitooligosaccharides from Chitosan using Crude Enzyme of Bacillus cereus D-11

In order to enzymatically produce chitooligosaccharide using the crude enzyme preparation from Bacillus cereus D-11, we first studied the optimal reaction conditions. It was found that the optimal temperature for hydrolysis of chitosan was 55℃. The ratio of enzyme/substrate should not be lower than 0.13 U/mg in the reaction mixture. The enzyme activity was stable below 50℃. The products of enzymatic reaction were analyzed by both thin layer chromatography and high performance liquid chromatography. Under the appropriate condition, chitosan was hydrolyzed using the enzyme preparation. The resulting chitooligosaccharides were purified and separated by Dowex (H+) ion exchange chromatography. From 4 g soluble chitosan, 0.95 g (GlcN)₂, 1.43 g (GlcN)₃, and 1.18 g (GlcN)₄ were recovered.