Study On The Relation Of The Function And α-Amylase Structure

This paper studied the Relation of the Function and α-Amylase Sequence Motifs .Alpha-amylase are very important enzymes in industry field,and study on it is very active nowadays. They are widely applied in starch processing, ethanol producing, spinning, weaving and other industrial field. Many kinds of alpha-amylase were cloned from different microbiology. Truncated a gene open up a new way to study functional a-amylase and makes it possible to improve their activity and speciality rapidly.It is significant for both theory study and application to study the cloned genes as well as expression in E.coli.In this study the a-am ylase derived from Xanthomonas campestrispv.campestris 8004, consists of an N-terminal catalytic domain and C-terminal domain,was cloned by divided four segments and inserted into a plasmid which was transferred into Escherichia coli. Then the expression productions were analyzed and the functional gene was researched.A truncated Xanthomonas campestris pv. campestris 8004 a-amylase (KSXC 2) lacking the N-terminal signal sequence was constructed and expressed in Escherichia coli. It is interesting that the no-signal a-amylase was fully active toward soluble starch. These results indicate that the enzyme was correctly folded and retained a tertiary structure,allowing a normal substrate recognition and catalytic activity. In our case, we found that the signal sequence was not essential for the periplasmic production of active amy2.To investigate whether the non-catalysists have an effect on the hydrolytic capability and speciality of the enzymes,three truncated a-amylase were cloned. The recombinant expression vector KSXC1, KSXC3 and KSXC4 were transformated into E.coli DH5αcompetent cell and induced by 0.5% soluble starch.The transformed cells produced substantial amylolytic activity. Their molecular mass (14 kDa, 25.7kDa,35.8 kDa), as estimated in sodium dodecyl sulfate polyacrylamide gel electrophoresis, were consistent with the molecular mass values calculated from the derived amino acid sequences of the truncated a-amylase. Analyzed the truncated enzyme activity of supernatant in the same expression amount, the result indicated that the enzyme activity of mutant gene recombinant expression vector KSXC1 was higher than the original gene recombinant expression vector KSXC. So, we can...