Abstract: This study aimed to analyze the effect of active site mutations on the specificity of bile salt hydrolase (BSH) g1294 from Ligilactobacillus cholophilus BD7642. Glycine scanning technology was used to investigate systematic mutations of BSH g1294’s active center, and the activity of mutants was assessed by the spot-plate method. High performance liquid chromatography was utilized to analyze the specificity of the mutants toward six bile salts. Additionally, saturation mutagenesis was conducted at the N81 site, followed by molecular docking simulations to analyze the differences between the mutants and the wild type. Results revealed that mutations at some sites led to the inactivation of the BSH activity. However, the N81G and N174G mutants exhibited enhanced substrate specificity, the former being superior to the latter. Among the mutants at the N81 site, significant variations in the enzymatic activity and hydrolytic capability were observed. Molecular docking simulations indicated that the N81G mutant acquired hydrolytic capability by reducing the steric hindrance between the catalytic site Cys2 and glycocholic acid. In conclusion, this mutant showed improved specificity than did the wild type. The molecular recognition of bile acids by BSH may not be simply based on amino acid recognition, which could provide new insights for rational design for BSH engineering.

Key words: Ligilactobacillus cholophilus; bile salt hydrolase; specificity; heterologous expression; saturation mutagenesis

摘要： 以Ligilactobacillus cholophilus BD7642来源的胆盐水解酶（bile salt hydrolase，BSH）g1294为研究对象，重点探讨其活性位点突变对酶特异性的影响。首先，通过甘氨酸扫描技术对BSH g1294活性中心进行系统性突变，并采用平板点样法评估突变体酶活性；利用高效液相色谱分析突变体对6 种胆盐的水解特异性。针对关键位点N81位点进行饱和突变，结合分子模拟对接分析突变体与野生酶的差异。结果表明，与野生酶相比，部分位点突变导致酶活性消失，N81G和N174G突变体特异性增加，其中N81G水解特异性更优。N81位点饱和突变中的多个突变体酶活性变化明显，且水解能力各异。分子模拟对接表明，N81G突变体可通过减少催化位点Cys2与甘氨胆酸之间的空间位阻获得水解能力。综上，N81G突变体具有优于野生酶的特异性，且BSH对胆酸的分子识别机制可能并非简单的氨基酸识别，这可为理性设计BSH g1294酶工程改造提供新思路。

关键词: Ligilactobacillus cholophilus；胆盐水解酶；特异性；异源表达；饱和突变