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

Abstract: In the human body, intestinal microbial bile salt hydrolase (BSH) enhances the host's bile acid pool and supports gut health by deconjugating primary bile acids (BAs). BSH shows hydrolytic specificity for bile acids, which is closely linked to the adaptability of strains and their ability to colonize the intestine, drawing considerable interest from researchers. This study further investigates the bile salt hydrolase g1294 found in Ligilactobacillus cholophilus BD7642 to better understand its characteristics. Methods and conclusions: We first conducted a glycine scan on the predicted active site. Using a sodium taurodeoxycholate plate assay, we discovered that mutations at specific sites led to a loss of enzyme activity for sodium taurodeoxycholate. Notably, the g1294-N81G mutant demonstrated a larger precipitation zone. High-performance liquid chromatography (HPLC) was employed to analyze the hydrolytic specificity of the mutants for six bile salts. The g1294-N81G and g1294-N174G mutants altered the specificity of the wild-type enzyme, with g1294-N81G showing superior performance. Saturation mutagenesis was conducted on N81 using random primers, revealing that several mutants at this site exhibited significant changes in enzyme activity and altered hydrolytic capacity for various bile acids. Molecular docking simulations compared the g1294-N81G mutant with the wild-type enzyme in their interaction with glycocholic acid. The analysis showed that the mutant reduced steric hindrance between Cys2 at the catalytic site and glycocholic acid, which may explain its enhanced hydrolysis of this bile acid. Our findings suggest that the specificity of bile salt hydrolase for bile acids involves more than just the recognition of amino acids and bile acid structures. Future studies on the structure of the g1294-N81G mutant protein could deepen our understanding of bile salt hydrolases and inform strategies for rationally modifying their activity in synthetic biology.

Key words: Ligilactobacillus, bile salt hydrolase, Specificity, heterogeneously expressed, Saturation mutagenesis