Novel thermostable variants of glucoamylase (GA) from filamentous fungus Aspergillus awamori X100 were constructed using the directed evolution approach based on random mutagenesis by error-prone PCR of the catalytic domain region of glucoamylase gene located on a new episomal expression vector pPEHα in Pichia pastoris cells. Out of 3000 yeast transformants screened, six new thermostable GA variants with amino acid substitutions Val301Asp, Thr390Ala, Thr390Ala/Ser436Pro, Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu were identified and studied. To estimate the effect of each substitution in the double mutants, we have constructed the relevant single mutants of GA by site-directed mutagenesis and analyzed their thermal properties. Results of the analysis showed that only Ile82Phe and Ser8Arg substitutions by themselves increased enzyme thermostability. While the substitutions Leu7Met, Asn9His and Gln338Leu decreased the thermal stability of GA, the synergistic effect of double mutant variants Leu7Met/His391Tyr, Asn9His/Ile82Phe and Ser8Arg/Gln338Leu resulted in significant thermostability improvement as compared to the wild type GA. Thr390Ala and Thr390Ala/Ser436Pro mutant variants revealed the highest thermostability with free activation energy changes ΔΔG of 2.99 and 3.1 kJ/mol at 80°C, respectively.

中文翻译：

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泡盛曲霉葡糖淀粉酶的热稳定性通过其位于毕赤酵母细胞中游离表达载体上的基因的定向进化而得到改善。

基于定向诱变方法，基于随机诱变，通过易错PCR技术对位于巴斯德毕赤酵母细胞中新游离表达载体pPEHα上的葡糖淀粉酶基因催化结构域区域进行定向诱变，构建了来自丝状真菌泡盛曲霉X100的葡糖淀粉酶（GA）的新型热稳定变异体。 。在筛选的3000个酵母转化子中，鉴定并研究了6个新的热稳定的GA变体，这些变体具有氨基酸取代Val301Asp，Thr390Ala，Thr390Ala / Ser436Pro，Leu7Met / His391Tyr，Asn9His / Ile82Phe和Ser8Arg / Gln338Leu。为了评估双突变体中每个取代的作用，我们通过定点诱变构建了GA的相关单突变体，并分析了它们的热特性。分析结果表明，只有Ile82Phe和Ser8Arg取代本身提高了酶的热稳定性。尽管取代Leu7Met，Asn9His和Gln338Leu降低了GA的热稳定性，但与野生型GA相比，双重突变变体Leu7Met / His391Tyr，Asn9His / Ile82Phe和Ser8Arg / Gln338Leu的协同作用导致了热稳定性的显着提高。Thr390Ala和Thr390Ala / Ser436Pro突变体变体显示出最高的热稳定性，在80°C下的自由活化能变化ΔΔG分别为2.99和3.1 kJ / mol。与野生型GA相比，Asn9His / Ile82Phe和Ser8Arg / Gln338Leu显着提高了热稳定性。Thr390Ala和Thr390Ala / Ser436Pro突变体变体显示了最高的热稳定性，在80°C下的自由活化能变化ΔΔG分别为2.99和3.1 kJ / mol。与野生型GA相比，Asn9His / Ile82Phe和Ser8Arg / Gln338Leu显着提高了热稳定性。Thr390Ala和Thr390Ala / Ser436Pro突变体变体显示出最高的热稳定性，在80°C下的自由活化能变化ΔΔG分别为2.99和3.1 kJ / mol。