Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute named by B.P.Konstantinov of National Research Centre "Kurchatov Institute", Orlova Roscha 1, Postal Code 188300, Gatchina, Russia.
BioMedical Technology Department, Kurchatov Institute, Akademika Kurchatova square 1, Postal Code 123182, Moscow, Russia.
Protein Eng Des Sel. 2019 Dec 31;32(6):251-259. doi: 10.1093/protein/gzz048.
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.
基于易错 PCR 的随机诱变，利用定向进化方法，从丝状真菌 Aspergillus awamori X100 中构建了新型耐热性糖化酶 (GA) 变体。该基因位于毕赤酵母细胞中新的附加型表达载体 pPEHα 上的糖化酶基因的催化结构域区域。在筛选的 3000 个酵母转化体中，鉴定并研究了 6 种具有氨基酸取代的新型耐热性 GA 变体，即 Val301Asp、Thr390Ala、Thr390Ala/Ser436Pro、Leu7Met/His391Tyr、Asn9His/Ile82Phe 和 Ser8Arg/Gln338Leu。为了估计每个取代对双突变体的影响，我们通过定点诱变构建了相关的 GA 单突变体，并分析了它们的热性质。分析结果表明，只有 Ile82Phe 和 Ser8Arg 取代本身增加了酶的热稳定性。而 Leu7Met、Asn9His 和 Gln338Leu 的取代降低了 GA 的热稳定性，而 Leu7Met/His391Tyr、Asn9His/Ile82Phe 和 Ser8Arg/Gln338Leu 的双突变体的协同效应与野生型 GA 相比，导致显著的热稳定性提高。Thr390Ala 和 Thr390Ala/Ser436Pro 突变体变体在 80°C 时具有最高的热稳定性，自由活化能变化 ΔΔG 分别为 2.99 和 3.1 kJ/mol。
