贵州传统发酵豆制品中水解银杏黄酮苷的微生物β-葡萄糖苷酶筛选

Screening of ginkgo-flavonoids-hydrolyzing microbial β-glucosidase from traditional fermented soybean in Guizhou

摘要:

[目的]微生物β-葡萄糖苷酶法水解银杏黄酮苷具有重要意义，不过目前这方面的研究极少。因此，本文目的是筛选到水解银杏黄酮苷的酶活高的微生物β-葡萄糖苷酶，并分析其底物选择性机制。[方法]以银杏叶提取物作为唯一碳源富集培养，从贵州传统发酵豆豉中筛选产对银杏黄酮苷水解酶活高的β-葡萄糖苷酶的菌株，并对该菌株进行鉴定。然后比较此β-葡萄糖苷酶对不同底物的选择性，同时测定此酶水解银杏黄酮苷反应的米氏常数K_m及最大反应速率V_max。最后，对不同的底物进行分子对接，分析其底物特异性机制。[结果]结果表明，筛选到的菌株GUXN01所产β-葡萄糖苷酶水解银杏黄酮苷的酶活最高，被鉴定为枯草芽孢杆菌。此β-葡糖糖苷酶对β构型的糖类以及苷类等具有广泛的底物特异性和不同的选择性，尤其对银杏黄酮苷具有很好的亲和性。分子对接研究表明枯草芽孢杆菌β-葡萄糖苷酶对银杏黄酮苷和其他糖苷类具有不同亲和性和选择性的原因主要是酶结构和底物分子结构的相互作用力的差异导致的。[结论]这些发现为GUXN01所产的β-葡萄糖苷酶应用于水解银杏黄酮苷类生产相应苷元奠定了良好的基础。

Abstract:

[Objective] Hydrolysis of ginkgo flavonoid glycosides by microbial β-glucosidase is less known. Therefore, we screened a microbial β-glucosidase with high enzymatic activity to hydrolyze ginkgo flavonoids and analyze its substrate-specificity. [Methods] β-Glucosidases with high enzymatic activity for the hydrolysis of ginkgo flavone glycoside were screened from traditional fermented soybean meal in Guizhou using Ginkgo biloba extract as the sole carbon source. The strain producing glucosidase was then identified, and the selectivity of the enzyme to different substrates was compared. The Michaelis constant K_m and maximum velocity V_max of the enzymatic reaction to hydrolyze ginkgo flavonoids were determined. Finally, the substrate-specific mechanism of the enzyme was analyzed via molecular docking studies. [Results] Among the strains observed, strain GUXN01 showed the highest β-glucosidase activity and was subsequently identified as Bacillus subtilis. β-glucosidase produced by GUXN01 had broad substrate specificity to sugars and glycosides with the β configuration. The glucosidase showed especially good affinity to ginkgo flavonoid glycosides. Molecular docking studies indicated that B. subtilis β-glucosidase had different affinities and selectivities toward ginkgo flavonoid glycosides and other glycosides due to differences in the interactions between the enzyme and substrate structures. [Conclusion] These findings may serve as a good foundation for the production of the corresponding aglycones via the hydrolysis of ginkgo flavonoid glycosides through the β-glucosidase produced by GUXN01.