甜叶菊糖基转移酶UGT76G1的克隆表达及其性质研究
Cloning, expression and characteristic of glycosyl-transferase UGT76G1 from Stevia rebaudiana
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摘要: 目的:利用甜叶菊糖基转移酶UGT76G1特异性催化甜菊糖中含量高并且具有较强后苦味的甜菊甙生成高甜度的莱鲍迪甙A。方法:将合成的UGT76G1编码基因插入pYES2载体的EcoRⅠ和XhoⅠ酶切位点之间,成功构建了pYES2-UGT重组质粒。重组质粒导入表达宿主酿酒酵母YPH499中,利用2%半乳糖对重组菌进行诱导表达。结果:确定了最佳诱导时机为菌体培养后48h,诱导表达时间为12h。并对重组酶粗酶液性质进行了初步研究,确定其最适反应pH为8.0,最适反应温度为40℃,最佳反应时间为36h。结论:为建立经济高效的生物催化法对甜菊糖口味改质奠定了基础。Abstract: Objective:Stevioside which was high in content but has a strong bitter sweet in Steviosides is specifically catalyzed to generate Rebaudioside A high in sweetness by the use of glycosyl transferase UGT76G1.Methods:The synthetic glycosyl-transferase UGT76G1 coding gene after modified was inserted into the vector pYES2 with the restriction site of EcoR I and Xho I in order to construct the recombinant plasmid pYES2-UGT which was then imported to Saccharomyces cerevisiae YPH499.The recombinant strain was induced to express by 2% galactose.Results:Determined the best induction start time was 12h.The nature of this restructuring enzyme was investigated and the optimal conditions were investigated as following:the pH of phosphate salt solution was 8.0, the reaction temperature was 40℃, the reaction time was 36h.Condusion:This study laid a foundation for building an economic and efficient biological catalysis method to modify the taste of Stevioside.
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