Study of production of alpha amylase secreted from parent and mutant strain of Bacillus amyloliquefaciens and optimization of culture condition

Abstract

The present study, deals with the screening and selection of Bacillus amyloliquefaciens for the alpha amylase production. The selected strain was subjected to UV mutagenesis treatment in order to improve its amylolytic potential. Twenty wild strains were exposed to UV radiation for 1 to 20 minute. During the treatment, mutant strains were qualitatively and quantitatively screened by starch hydrolysis and enzyme activity respectively. Among these, Mutant 1 (M1) exhibited the highest enzyme activity (4.984 U/ml/min). This mutant showed 2.0 fold increased activity over the parental strain (Wild) in terms of enzyme production. RAPD- PCR (Random amplified polymorphic DNA) was done to detect DNA damages and mutation. Different six primers (decamer) were used for amplification. VAA 18 (decamer primer) amplified with DNA of Bacillus amyloliquefaciens and show polymorphism. After data analysis, 15 out of 9 bands were polymorphic. The result obtained from primer VAA 18 showed changes in band pattern which reflected DNA alternations or mutation. The optimization of cultural condition and nutritional requirements of the selected strains (wild (W) and mutant (M1, M2, M3, and M4)) were optimized in 250 ml Erlenmeyer flasks by shake flash fermentation. Effect of temperature, pH, incubation time, substrate concentration, carbon source, nitrogen source, different concentration of media s ingredients and metal ions were optimized by DNS method. The crude enzyme was characterized for different effects such as temperature 80°C was found to be optimum, pH 8, 72 hrs as optimum incubation time and 5% substrate concentration for (W and M1) strains. PEG and CaCl2 represented as good enhancer and EDTA as inhibitors in productivity. and#945;-amylase production was high in media containing lactose as carbon source, ammonium chloride as nitrogen source, low and high concentration of starch. The 79% desizing of cotton cloth was obtained with 50 mL enzyme units at pH 6.5 at 60°C in 1 hour.