嗜糖黄杆菌β-葡萄糖苷酶的功能及在稀有人参皂苷制备中的应用

Glucosidases from Flavobacterium saccharophilum: characterization and application in the preparation of minor ginsenosides

摘要:

【背景】有些稀有皂苷具有较好的药理活性，寻找活性高和专一性好的糖苷酶可能实现稀有皂苷的定向制备。嗜糖黄杆菌中含有丰富且未被表征的糖苷酶基因是寻找新酶的潜在来源。【目的】从嗜糖黄杆菌中发现活性高和专一性好的糖苷酶，用于制备稀有人参皂苷。【方法】重组表达嗜糖黄杆菌中15个假定的葡萄糖苷酶基因，系统研究重组酶的性质和功能，筛选可用于制备稀有皂苷的酶，利用薄层层析法和高效液相色谱法鉴定转化产物。【结果】从嗜糖黄杆菌中获得3种活性较好的β-葡萄糖苷酶，即SA2629、SA0236和SA2851。其中，SA2629具有最高的比酶活(78.7 U/mg)和催化效率[k_cat=(27.38±1.40) s⁻¹]，且SA2629能同时水解人参皂苷C-20位上的β-1,6-葡萄糖苷键和C-3位直接与苷元相连的葡萄糖苷键。SA2851和SA0236只对C-20位上的β-1,6-葡萄糖苷键具有水解活性，其中SA0236活力高。将SA2629和SA0236与课题组前期获得的一种β-1,2-葡萄糖苷酶分别组合，可以将高含量人参皂苷Rb1完全转化成稀有皂苷CK和F2。【结论】获得了可用于制备稀有人参皂苷的糖苷酶，填补了嗜糖黄杆菌β-葡萄糖苷酶研究的空白。

Abstract:

[Background] Some minor ginsenosides possess valuable pharmacological activities. Mining glycosidases with high activity and specificity can realize the directional preparation of minor ginsenosides.Flavobacterium saccharophilum carries abundant and uncharacterized glycosidase genes, which are potential sources for excavating new glycosidases.[Objective] To obtain highly active and specific glycosidases from F. saccharophilum for the preparation of minor ginsenosides. [Methods] Fifteen putative glucosidase genes were cloned from F. saccharophilum and expressed. To screen out the enzymes for the preparation of minor ginsenosides, we fully characterized the recombinants and identified the biotransformation products by employing thin layer chromatography and high performance liquid chromatography. [Results] Three β-glucosidases (SA2629, SA0236, and SA2851) with high activities were obtained from F. saccharophilum. SA2629 showed the highest specific activity (78.7 U/mg) and catalytic efficiency [k_cat=(27.38±1.40) s⁻¹]. Furthermore, it could simultaneously hydrolyze the β-1,6-glucosidic bond at the C−20 position and the glucosidic bond directly connected to the aglycone at the C-3 position. SA2851 and SA0236 only had hydrolysis activity on the β-1,6 glucosidic bond at the C-20 position, and SA0236 had higher activity. Furthermore, we completely transformed ginsenoside Rb1 to the minor ginsenosides CK and F2 by using SA2629 and SA0236, respectively, with a β-1,2-glucosidase obtained in our lab. [Conclusion] The glycosidases that can be employed to prepare minor ginsenosides were obtained, which filled the gap in the study of β-glucosidases from F. saccharophilum.