Single-site mutation of C363G or N463T strengthens thermostability improvement of IG181–182 deleted acidic α-amylase from deep-sea thermophile Geobacillus sp.

Abstract

ABSTRACTThe acidic α-amylase Gs4j-AmyA from deep-sea thermophile Geobacillus sp. is more acid-resistant than the commercial sources as it displays more than 50% of the optimum at a range of pH 4.5–7. This may allow the removal of the step of pH adjustment in starch processing and save costs. Unfortunately, this amylase is not very stable at 90–95°C. Therefore, to develop a new commercial acidic α-amylase targeted to the food industry, it is necessary to further improve the thermostability of the α-amylase Gs4j-AmyA. In this study, 11 different α-amylase mutants were obtained by site-mutagenesis. Among them, the mutant IG181–182* (IG181–182 deletion) showed significant improvement in thermostability, whose half-life at 70°C was 63.4 times longer than the wild type. Interestingly, single-site mutants C363G and N463T showed no enhancement on thermostability, while the half-lives of the combination mutants IG181–182*/C363G and IG181–182*/N463T at 70°C were further extended by 16.8 and 38.7%, respectively. Unfortunately, the catalytic constants (kcat) of the mutants C363G, N463T, IG181–182*/C363G and IG181–182*/N463T declined by 59, 49, 37 and 16%, respectively. The optimum temperature (65–70°C) and pH (5.5–5.6) of the mutants was unchanged. The thermostability improvement by IG181–182 deletion could be strengthened by synchronous C363G or N463T mutation.