环糊精葡萄糖基转移酶182位点定点改造催化合成糖基化染料木素

染料木素及其单糖苷衍物在食品和医药领域具有重要作用,但难溶于水的特性极大地限制了其应用范围,研究表明糖基化反应可有效提高其水溶性。文中针对来源于软化芽孢杆菌的环糊精葡萄糖基转移酶,研究其对染料木素单糖苷衍生物槐角苷的糖基化反应。通过对D182位点的定点饱和突变,突变酶D182C较WT转化率提高了13.42%,主要糖基化产物（槐角苷单糖苷、二糖苷、三糖苷）分别提高了39.35%、56.05%和64.81%。酶学性质研究发现,D182C较WT的环化、水解、歧化活力均有所提高。且最适pH和温度分别为6和40℃。动力学研究发现,D182C针对底物（糖基供体和受体）的K_m值较WT均有所降低,且催化效率k_cat/K_m则明显提高,说明D182C对底物的亲和力相较于WT有大幅提升。同源建模及分子对接结果表明,突变酶D182C糖基化效率提高的原因可能与底物作用力增加有关。

Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields,whereas their applications are limited by the low water solubility.Glycosylation is regarded as one of the effective approaches to improve water solubility.In this paper,the glycosylation of sophoricoside(genistein monoglucoside)was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans(PmCGTase).Saturation mutagenesis of D182 from PmCGTase was carried out.Compared with the wild-type(WT),the variant D182C showed a 13.42%higher conversion ratio.Moreover,the main products sophoricoside monoglucoside,sophoricoside diglucoside,and sophoricoside triglucoside of the variant D182C increased by 39.35%,56.05%and 64.81%compared with that of the WT,respectively.Enzymatic characterization showed that the enzyme activities(cyclization,hydrolysis,disproportionation)of the variant D182C were higher than that of the WT,and the optimal pH and temperature of the variant D182C were 6 and 40℃,respectively.Kinetics analysis showed the variant D182C has a lower K_m value and a higher k_cat/K_m value than that of the WT,indicating the variant D182C has enhanced affinity to substrate.Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.