Purification and characterization of chitinase produced by bacillus amyloliquefaciens

ABSTRACT

The crude chitinase preparation obtained from Bacillus amloliquefaciens cultures was partially purified by fractional precipitation with ethanol, acetone or ammonium sulphate. The 65% ammonium sulphate fraction was the most active and was further purified by gel filtration on Sephadex G-200 followed by ion exchange chromatography on Ecteola ET-11 cellulose yielding 4 chitinolytic enzyme components. Two enzyme components, CHII and CHIII, showed high activity and protein recovery. The purity of both enzymes was checked by polyacrylamide gel electrophoresis. CHII enzyme was more specifid for chitin as substrate and showed a higher thermostability than CHIII. Both enzymes showed a temperature optimum of 45°C and pH optimum of 5.5 to 5.9. They did not show specific cationic requirements and were partially inhibited by zinc, cupric, silver and cobalt ions. Their amino acid compositions were different. SDS electrophoresis revealed that each enzyme was formed of 2 subunits of different molecular weights, CHII subunit had a M.W. of 26.5 and 23.5 KD while CHIII was 31.6 and 27.5 KD