CAJ | 학술논문

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。
위제고중조β-포도당철산감매적저물특이성，통과역착 PCR방법대기진행돌변병구건돌변체문고，이용박층층석화고효액상색보대돌변문고진행사선，획득료돌변주PGUS（M51）-E ，기저물특이성제고료41％。돌변매적매학특성연구발현，해돌변매적최괄pH치화온도교PGUS-E무현저변화，매활력하강료16．86％，Tm 치제고료5℃；서렬분석결과표명：PGUS（M51）-E공발생5처돌변，기중3처발생재당기결합역。인차，이용정향진화책략능유효제고β-포도당철산감매적저물식별특이성。
To improve the substrate specificity of recombinant β-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 select-ed 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 β-glucuronidase ,three of them located in the carbohydrate binding domain . Taken together ,directed evolution could improve the substrate specificity of β-glucuronidase effectively .