Thermostabilization of Bacillus amyloliquefaciens alpha-amylase by computational mutation

Nowadays, thermostable enzymes are used in various biotechnological processes . Increased stability of enzymes to extreme temperatures allows the amount of enzyme in the reaction to be reduced. Increasing the reaction temperature improves the reaction yield and reduces the time taken for product accumulation. α-amylases (EC 3.2.1.1) are used in industrial processing of starch-containing materials. The majority of these reactions are conducted at high temperatures, resulting in high demand for thermostable α-amylases. Several scientific groups have identified amino acid substitutions that lead to enhanced thermostability of α-amylases.In this study some amino acids from Bacillus amyloliquefaciens alpha-amylase were substituted with other amino acids using in silico approaches to finally achieve mutations that can improve the thermostability of the enzyme. The I-Mutant2.0 web server results showed that one of the selected mutations improves the thermostability of the enzyme . Therefore, by I-Tasser, cph model and Swiss model web servers the mutations were exerted on the 3D structure of the enzyme and mutant 3D models from enzyme structure were achieved. The models were refined by 3Drefine web server. The models quality assessment was performed by ModFOLD4 server. The best model was selected and then docked with partial amylose polymer to found the effect of the mutation on the binding of enzyme with it substrate. Docking technique was performed by AutoDock software. The I-mutant2.0 results showed that one of the mutation enhanced thermostability. This is S335K mutation. after exerting the mutation on the 3D structure, and refining the models, docking of best model with partial amylose polymer showed that the binding of enzyme and its substrate was not negatively affected. In conclusion, we suggest this substitution mutation for achieving alpha-amylases with high thermostability for applying in industrial processes that need high temperature.

Keywords:

Alpha-amylase , Thermostability , Mutation , AutoDock