Probing the role of asparagine mutation in thermostability of Bacillus KR-8104 α-amylase.

Abstract Asparagine deamidation is one of the important determinants of protein thermostability. Here, structure based mutagenesis has been done in order to probe the role of Asn residues in thermostability of a Ca independent Bacillus sp. KR-8104 α-amylase (BKA). Residues involved in potential deamidation processes have been selected and replaced using a site directed mutagenesis. Fourteen different variants were tested for thermostability by measuring residual activities after incubation at high temperature. In comparison to the wild-type enzyme, four mutated variants are able to increase the half life of the protein at high temperatures. The highest stabilization resulted from the substitution of asparatate in place of asparagine at position 112, leading to a nearly fivefold increase of the enzyme's half-life at 70 °C. Also replacement of Asn129 to aspartic acid and Asn312 to serine markedly increased the half-life of the enzyme at 70 °C indicating that the deamination of these residues may have a deleterious effect on BKA.