Screening High Yield Bacillus Cereus Of Amylase-Producing Strain And Studying The Characterization Of The Amylase

Alpha amylase is one of the most widely enzyme preparation used in industry, which can be produced by bacteria, fungi, yeast amylase, and also can be extracted from the animals and plants. Alpha amylase were used in starch processing industry, brewing fermentation industry, the fabric of desizing process, pharmaceutical, medical, industrial detergent, paper industry and feed industry etc.Bacillus cereus sww106screened and identificated by laboratory as starting strains, we used five single mutation methods on sww106including ultraviolet, the fast neutron irradiation,sodium nitrite, Diethyl sulfate(DES) and nitrosoguanidine(NTG) and composite methods of UV and NTG to screen high enzymatic activity mutations. The results showed that UV is the best in physical mutation. We selected UV254-6by irradiated120s over20cm,with ultraviolet wavelength254nm.The enzymatic activity was increased2.9times than sww106. Then selected UV254-6as initial strain, when the NTG concentration was0.5mg/ml, treated40min, obtained a high enzyme activity strain UV-NTG-3, the enzymatic activity was3.59times than sww106.Use UV-NTG-3strains as starting strain again with NTG treat it,The concentration was0.4mg/ml of NTG,60min, screened a mutations UN2-2, increased4.22times than sww106.After10times spread, the genetic stability is good, alpha amylase activity is still97%.The enzymatic properties of UN2-2was studied, including specificity of the substrate,optimal temperature, heat stability, optimal pH, pH stability, activation and inhibitors,etc.The results showed that The product were maltose and glucose catalysis, this proof that the enzyme was alpha amylase.The optimal temperature is60℃,between50℃-70℃,the amylase was stability,it was conformed to middle-temperature alpha amylase. The optimal pH was6.0, when the pH was5.0-8.0,the enzyme was stability. Ca2+played a significant role on amylase activity, when Ca2+was5mmol/L,The relative activity was the highest,reached120.92%.Na+Mg2+, K+also had certain activation function, but Fe3+, Mn2+had not apparent activation function. Zn2+,Cu2+had certain inhibition. When EDTA existed, The enzyme activity was inhibited and dropped quickly.Fermentation conditions were studied of UN2-2.The results showed that The optimal cultural medium was corn starch1.2%,soybean protein powder0.8%, CaC121.2mmol/L. The best cultivation condition was initial pH6.5,37℃, inoculation amount5%,200r/min.The cultural medium composition and conditions were optimized, enzyme activity up to5.341U/mL, improved1.29times, increased5.45times than the initial strains sww106.