Abstract

• The present research work focuses on the extraction of chitosanase enzyme from soil bacteria (Bacillus megaterium). In this research, the soil sample was collected from Htauk-kyant Township, Yangon Region for the isolation and cultivation of bacteria. In the isolation process, bacteria were isolated from the soil sample by serial dilution plate method, followed by culture in nutrient agar medium. Ten bacterial strains (A1 to A10) were isolated from the soil sample and were characterized by microscopic examination and gram staining methods. Among these bacterial strains, A1, A2, A3, A4, A6, A8, A9 and A10 were found to be gram positive, whereas only A5 and A7 were gram negative. According to the biochemical tests, out of eight gram positive bacterial strains, only A2 was observed to give the positive results on motility tests, methyl red tests, citrate utilization tests, starch hydrolysis tests, sugar fermentation tests and negative results on indole tests, nitrate tests, (VP) Voges-Proskauer tests that similar to the characteristics of chitosanase enzyme producing bacteria (Bacillus megaterium). Hence, A2 might be identified as Bacillus megaterium. For extraction of chitosanase enzyme, the isolated bacterial strain (A2) was cultured on chitosanase producing medium of 0.6 % poly peptone, 0.1 % K2HPO4, 0.05 % MgSO4.7H2O, 0.6 % yeast extract, 0.1 % glucose and 0.5 % colloidal chitosan and incubated at 30 .C. The optimum incubation time (3 days) of enzyme forming bacteria, inoculum sizes of bacteria (10 %), age of culture of bacteria (3 days), temperature of enzyme-catalyzed reaction (55 .C) and pH (7.0) of chitosanase producing medium were determined based on the chitosanase activities. Turbid enzyme bacterial solution was so prepared under above conditions for preparation of enzyme bacterial solution. The enzyme bacterial solution was centrifuged with 3000 rpm at room temperature and the supernatant enzyme solution was collected. The crude chitosanase solution was obtained and then partially purified by successive precipitation method by using 30 % and 70 % saturation of ammonium sulphate. Finally the total enzyme activity, protein contents and specific activity of crude enzymes obtained from each purification step were determined.