为了提高α-L-鼠李糖苷酶的热稳定性,对实验室前期构建的K406R、K440R、K573V、E631F四个不同氨基酸位点的单点突变体进行联合突变,得到了K406R-K573V、K406R-E631F、K440R-K573V、K440R-E631F四个联合突变体。结果表明,相比于野生型(wild type, WT),联合突变体K440R-K573V和K440R-E631F的热稳定性有所提高,在60 ℃时半衰期分别提高了1.13倍,1.44倍;在65 ℃的半衰期分别提高了1.64倍,1.64倍;在70 ℃ 的半衰期分别提高了1.88倍,1.68倍。对突变体K440R-E631F进行圆二色谱分析、分子动力学以及分析微观结构变化分析可知,K440R-E631F突变体与WT相比,内部疏水性有所提高,二级结构中α-螺旋、β-转角、无规则卷曲含量增多,可能与热稳定性的提高相关。
In order to improve the thermostability of α-L-rhamnosidase, four single site mutants: K406R, K440R, K573V, and E631F, were combined and four new mutants: K406R-K573V, K406R-E631F, K440R-K573V, and K440R-E631F, were obtained. In comparison to the wild type (WT), the thermostabilities of K440R-K573V and K440R-E631F improved, as their half-lives at 60 ℃ increased by 1.13-fold and 1.44-fold, respectively. Moreover, their half-lives were both 1.64 times higher than that of WT at 65 ℃, and 1.88-fold (K440R-K573V) and 1.68-fold (K440R-E631F) higher at 70 ℃. Based on circular dichroism, molecular dynamics and micro-structural analysis, it was found that mutant K440R-E631F in comparison to WT had increased internal hydrophobicity. Additionally, its contents of α-helix, 123456-turn and random coil also increased, which may be related to the increased thermostability of α-L-rhamnosidase.
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