Abstract: In order to determine the relationship between amino acid residue at the special site and the activity of Thermococcus. siculi HJ21 α-amylase, pEt-28a-His6 plasmid with TSA gene was used as a template for the site mutagenesis from Asp186 to Asn186. The activity of α-amylase with and without mutation was measured. According to the difference between per-mutant and mutant cDNA sequences, the secondary structure and three-dimensional structure of TSA protein were also predicted using DNAStar and Deepview protein analysis software. The results showed that Asp186 was of critical significance for maintaining the activity of T. siculi HJ21 α-amylase. The site-directed mutagenesis of T. siculi HJ21 α-amylase at position 186 changed the properties of secondary structure as well as hydrogen-bonding networks, thus resulting in the loss of its activity.

Key words: α-amylase, site-directed mutagenesis, TSA gene, position 186, enzymatic activity