Objective To improve enzyme activity of Glucosamine-6-phosphate synthase (Glms) of Bacillus subtilis by site saturation mutagenesis at Leu 593 , Ala 594 , Lys 595 , Ser 596 and Val 597 based on computer-aided semi-rational design. Results The results indicated that L 593 S had the greatest effect on the activity of BsGlms and the enzyme activity increased from 5 to 48 U/mL. The mutation of L 593 S increased the yield of glucosamine by 1.6 times that of the original strain. The binding energy of the mutant with substrate was reduced from − 743.864 to − 768.246 kcal/mol. Molecular dynamics simulation results showed that Ser 593 enhanced the flexibility of the protein, which ultimately led to increased enzyme activity . Conclusion We successfully improved BsGlms activity through computer simulation and site saturation mutagenesis. This combination of methodologies may fit into an efficient workflow for improving Glms and other proteins activity.

中文翻译：

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通过半合理设计策略和计算机分析提高氨基葡萄糖-6-磷酸合酶的酶活性

目的基于计算机辅助半合理设计，通过位点饱和诱变Leu 593、Ala 594、Lys 595、Ser 596和Val 597，提高枯草芽孢杆菌6-磷酸氨基葡萄糖合酶(Glms)的酶活性。结果 结果表明，L 593 S 对BsGlms 的活性影响最大，酶活性从5 U/mL 增加到48 U/mL。L 593 S 的突变使氨基葡萄糖的产量是原始菌株的 1.6 倍。突变体与底物的结合能从 - 743.864 降低到 - 768.246 kcal/mol。分子动力学模拟结果表明，Ser 593 增强了蛋白质的柔韧性，最终导致酶活性增加。结论我们通过计算机模拟和位点饱和诱变成功地提高了 BsGlms 活性。