This study reported simultaneously improved thermostability and hydrolytic pattern of α-amylase from Bacillus subtilis CN7 by rationally engineering the mostly conserved central beta strands in TIM barrel fold. Nine single point mutations and a double mutation were introduced at the 2nd site of the β7 strand and 3rd site of the β5 strand to rationalize the weak interactions in the beta strands of the TIM barrel of α-amylase. All the five active mutants changed the compositions and percentages of maltooligosaccharides in final hydrolytic products compared to the product spectrum of the wild-type. A mutant Y204V produced only maltose, maltotriose, and maltopentaose without any glucose and maltotetraose, indicating a conversion from typical endo-amylase to novel maltooligosaccharide-producing amylase. A mutant V260I enhanced the thermal stability by 7.1 °C. To our best knowledge, this is the first report on the simultaneous improvement of thermostability and hydrolytic pattern of α-amylase by engineering central beta strands of TIM barrel and the novel “beta strands” strategy proposed here may be useful for the protein engineering of other TIM barrel proteins.

该研究报道了枯草芽孢杆菌同时提高了热稳定性和α-淀粉酶的水解模式CN7通过合理地改造TIM桶形折叠中最保守的中央β链。在β7链的第二个位点和β5链的第三个位点引入了九个单点突变和双突变，以合理化TIM桶中α-淀粉酶的β链中的弱相互作用。与野生型产物谱相比，所有五个活性突变体均改变了最终水解产物中麦芽低聚糖的组成和百分数。突变体Y204V仅产生麦芽糖，麦芽三糖和麦芽五糖，而没有任何葡萄糖和麦芽四糖，表明从典型的内切淀粉酶转变为产生麦芽低聚糖的新型淀粉酶。突变型V260I将热稳定性提高了7.1°C。据我们所知，