重组β-葡萄糖醛酸苷酶转化甘草酸的定向性改造  

Directly Modified Recombinantβ-glucuronidase for Glycyrrhizin Transformation

作　　者：矫丽媛[1] 吕波[2] 王金娜[2] 李春[1,2] 

机构地区：[1]石河子大学化学化工学院/新疆兵团化工绿色过程重点实验室/省部共建国家重点实验室培育基地,石河子832003 [2]北京理工大学生命学院,北京100081

出　　处：《石河子大学学报（自然科学版）》2013年第5期640-644,共5页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(21176028)

摘　　要：为提高重组β-葡萄糖醛酸苷酶的底物特异性,通过易错PCR方法对其进行突变并构建突变体文库,利用薄层层析和高效液相色谱对突变文库进行筛选,获得了突变株PGUS(M51)-E,其底物特异性提高了41%。突变酶的酶学特性研究发现,该突变酶的最适pH值和温度较PGUS-E无显著变化,酶活力下降了16.86%,Tm值提高了5℃;序列分析结果表明:PGUS(M51)-E共发生5处突变,其中3处发生在糖基结合域。因此,利用定向进化策略能有效提高β-葡萄糖醛酸苷酶的底物识别特异性。To improve the substrate specificity of recombinant ~ glucuronidase, error prone PCR was performed to construct a mutant library. One mutant was selected by thin-layer chromatography and high performance liquid chromatography. The selected mutant was named PGUS（M51）-E, substrate specificity of which was improved by 41%. Enzymatic properties analysis showed that the optimum pH and temperature of the catalytic reaction were not changed obviously compared with wild type. The activity of PGUS（M51）-E was decreased by 16.86%, while the Tm value was increased by 5 ℃. Sequence analysis revealed that there were five mutations in the sequence of 13 glucuronidase, three of them located in the carbohydrate binding domain. Taken together, directed evolution could improve the substrate specificity of β-glucuronidase effectively.

关 键 词：Β-葡萄糖醛酸苷酶 易错PCR 甘草酸 底物特异性 

分 类 号：S852.65[农业科学—基础兽医学] S858.26[农业科学—兽医学]