Improving Thermostability Of Acidic ?-amylase Through Molecular Modification

The work aimed to improve the thermostability of an a-amylase, Gs4j-AmyA, from a deep-sea thermophile Geobacillus sp.4j. Based on the research of thermostability in BLA(Bacillus lecheniformis a-amylase) and BStA(Bacillus stearothermophilus a-amylase),11 amino acid sites were selected to mutate. Expressed into the supernatant of E. coli, four mutant a-amylases RGI179-181Q, IG181-182*, C363G, and N463T have been found more thermostable through preliminary selection by comparing the residual relative enzyme activity after incubation at 100?, pH 5.5 for 10 min. And combination mutant a-amylases IG181-182*/C363G and IG181-182*/N463T have also been obtained, predicted to improve thermostability.Ultra-filtration and Ni2+-column affinity chromatography were used to purify the wild-type and mutant a-amylases. In order to study the thermostability of mutant a-amylases, half-lives of the wild-type and these mutant amylases at three different temperatures were determined. Among them, the mutants RGI179-181Q and IG181-182* (IG181-182 deletion) showed extreme improvement in thermostability, whose half-lives at 70? were 59 and 63 times longer than the wild-type, respectively. Single mutant amylases C363G and N463T showed no enhancement on thermostability. The half-life of the mutant C363G only improved at 70? by 20.9%, while the half-lives of the mutant N463T at 85? and 95? decreased by 6.2% and 34%, respectively. However, the half-lives of the combination mutant amylases IG181-182*/C363G and IG181-182*/N463T at 70? further increased by 17% and 39%, compared with the mutant IG181-182*. The optimum temperature (65?70?) and pH (5.5?5.6) of the mutants were almost the same with the wild-type.The catalytic efficiencies of four mutant a-amylases C363G, N463T, IG181-182*/C363G and IG181-182*/N463T decreased by 59%,50%,37% and 16%, respectively, compared with that of the wild-type Gs4j-AmyA. However, the efficiency of the mutants IG182-182* and RGI179-181Q showed almost the same with the wild type, as the kcat value was roughly 40×105 min-1. The structure model indicated that the Ca2+-binding network enhanced because of the deletion of which was an important reason why the thermostability of mutant IG181-182* improved. Substitution of N463 with T led to decreased number of H-bonds (from 5 to 3), which may probably lead to the decreasement in thermostability of single-mutant N463T.