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

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.