The Study On High-Level Expression And Enzyme Properties Of Mutant α-Amylase Gene

Based on alpha-amylase gene and mutant gene of Xanthomonas campestris k8004,pET-30a(+) plasmid ,The recombinant expression vector pXC8004 and pXCH21 wasconstructed by gene cloning technology. The mutant gene was gotten for the originalalpha-amylase by gene-directde mutagenesis method. The activity of enzyme which wasexpressed by the mutant gene was higher than the original gene. We have realized the tworecombinent vector pXC8004 and pXCH21 high level expression in E.coli BL21(DE3)PlusS.1.The recombinant expression vector pXC8004 and pXCH21 wan transformated into E.coliBL21 competent cell and induced by IPTG . We got the high-level expression of alpha-amylasegene and its mutant. From the study of references and experiment, we got the optimum inducedconditions. When the OD600 of bacterial solution reached to 0.4~0.6 and the dose of IPTGwas 1mmol/l , the induced result was the best. The target proteins amount to 11% of the totalproteins in cell.2.The induced cultivated E.coli BL21 was fragmentated by ultrasonic, then centrifugated andkept in the supernatant. Analyzed the enzyme activity of supernatant in the same expressionamount , the result indicated that the enzyme activity of mutant gene recombinant expressionvector pXCH21 was higher than the original gene recombinant expression vector pXC8004 by30U/L. So, we can conclude that it was the enzyme activity center that had been changed ,not theincreasing of enzyme expression amount which improved the activity of mutant alpha-amylasegene.3.We had designed a pair of pcr primers, but there was an extra base in its sense primer, whichresulted in terminating of translation of the target protein ahead of schedual. The molecular oferror-reading protein was 24 KDa. Although it had very low identity by comparing with theoriginal gene, the mutant gene had still the function of hydrolysis of α-1,4 glucosidicanlinkages.