| The thermostable amylases in this study were special starch modifiers.The differences between them and general amylases were that they had a variety of glycosyl transfer and degradation on starch, amylopectin andβ-cyclic dextrin, and hydroly them into maltose and panose which had health effects. This subject studied enzymic expression conditions of thermostable amylase of genetic engineering bacterium when it was induced by lactose and IPTG, respectively.Then, enzymatic purification and properties were studied. Finally, we studied the expression conditions optimization of temperature-based thermostable amylase of genetic engineering bacterium.Firstly, culture conditions of thermostable amylase of genetic engineering bacterium were studied.The optimal medium used in enzymatic expression of this genetic engineering bacterium was LB medium through optimization and integration analysis. Its optimal expression conditions were bacteria concentration (OD600) 0.6, the concentration of IPTG 0.6mmol/l, pH 6.0; induction temperature 37°C and induction time 5h.Secondly, inducer components of thermostable amylase of genetic engineering bacterium were studied.When lactose was inducer, optimal expression conditions of this recombinant thermostable amylase were lactose concentration(g/100ml)0.5%; bacteria concentration (OD600)3.0; induction temperature37°C,induction time5h and pH5.0.However,enzymatic activity was only 10% of its activity induced by IPTG. So, lactose could not take the place of IPTG in inducement.The optimal expression conditions of maltose-based amylase when lactose and IPTG induced together were lactose concentration(g/100ml)0.5%; IPTG concentration 0.2 mmol/l; bacteria concentration(OD600)2.0, induction temperature 37°C, induction time 4h and pH7.0.The amount of IPTG was only 1/3 in the induction of IPTG alone. Enzymatic activity reached 80% of its activity induced by IPTG. Therefore, IPTG induced alone could be completed replaced by lactose and IPTG induced together.Thirdly, The thermostable amylases were purified with GST.The activity of the purified thermostable amylases were 12000U/g. The molecular weight of it was 90kDa.The optimization reaction temperature is 60～70℃. We kept enzyme at 4℃and 35℃,and then measured the remaining enzyme activity in regularity.We found the enzyme lost 50% activity after stored for three weeks at 4℃and lost 50% activity after stored for three days at 35℃. The enzyme had an optimum pH of 6.6. It was relatively stable in the range of pH 5.4 to pH 7.4, but the stability declined rapidly as soon as pH lowered 5.4 or above 7.4. These results indicate the enzyme have a wide stable range of pH.Fourthly, the catalysis products by recombinant thermostable amylase were also studied.With the help of HPLCluna NH2 column detection, we found the catalysis products of thermostable amylase were all maltose and glucose when starch andβ-cyclic dextrin as the substrate, respectively.The hydrolysis mechanism ofβ-cyclic dextrin was that recombinant thermostable amylase opened the ring ofβ-cyclic dextrin firstly, then, hydrlyed with maltose as a unit.The final products were maltose and the rest of a glucose, its ratio was 3:1.Eventually, we optimized culture conditions for the production of recombinant maltogenic amylase by Escherichia coli which had the plasmid induced by temperature. The optimal culture temperature was 30-32℃. The optimal pH 6.0. The optimal cell density OD600 1.2. The optimal temperature and induced time 38℃1h,39℃1h,40℃1h and 41℃1h. The plasmid kept 11 generation in stability. |
PDF Full Text Request