CAJ | 학술논문

用PCR随机诱变方法，研究氨基酸置换对耐热邻苯二酚2，3-双加养酶性质的影响。比较分析了突变体ro229Ser和Glu243Gly与野生型酶的酶学性质。结果显示点突变Pro229→Ser或Glu243→Gly并未改变酶的最适反应温度（均为60℃）；突变体Pro229Ser（Kcat／Km＝4.89±0.01×106mol－1s－1）以及突变体Glu243Gly(Kcat／Km＝5.88±0.01×106mol－1s－1)的催化能力与野生型酶（Kcat／Km＝6.97±0.01×106mol－1s－1）相比有所下降；点突变Pro229→Ser大幅度降低了酶的热稳定性（ΤTm≈10.2℃），而点突变Glu243→Gly对酶的热稳定性影响较小（Tm≈1.5℃）。推测Pro229对耐热邻苯二酚2，3双加养酶的热稳定性有重要贡献。
용PCR수궤유변방법，연구안기산치환대내열린분이분2，3-쌍가양매성질적영향。비교분석료돌변체ro229Ser화Glu243Gly여야생형매적매학성질。결과현시점돌변Pro229→Ser혹Glu243→Gly병미개변매적최괄반응온도（균위60℃）；돌변체Pro229Ser（Kcat／Km＝4.89±0.01×106mol－1s－1）이급돌변체Glu243Gly(Kcat／Km＝5.88±0.01×106mol－1s－1)적최화능력여야생형매（Kcat／Km＝6.97±0.01×106mol－1s－1）상비유소하강；점돌변Pro229→Ser대폭도강저료매적열은정성（ΤTm≈10.2℃），이점돌변Glu243→Gly대매적열은정성영향교소（Tm≈1.5℃）。추측Pro229대내열린분이분2，3쌍가양매적열은정성유중요공헌。
In order to investigate the effects of amino acid replacement onthe characters of thermostable catechol 2, 3-dioxygenase, two mutants (Pro229Ser and Glu243Gly) of this enzyme were obtained by using the method of PCR random mutagenesis. The wild type thermostable catechol 2, 3-dioxygenase and these two mutants (Pro229Ser, Glu243Gly) were over expressed in E. Coli TG1 and purified. The enzymatic characters and thermostability of the wild type enzyme and the two mutants (Pro229Ser, Glu243Gly) were analyzed. The results revealed that the optimum enzymatic temperature of the two mutants were the same as that of the wild type enzyme (60℃) and the Kcat／Km value of Pro229Ser and Glu243Gly (4.89±0.01×106mol－1s－1 and 5.88±0.01×106mol－1s－1, respectively) were reduced compared with the wild type enzyme (6.97±0.01×106mol－1s－1). However, the thermostability of Pro229Ser extremely decreased 10.2℃ and the thermostability of Glu243Gly slightly increased 1.5℃. It was proposed that Pro229 played an important role on the thermostability of thermostable catechol 2, 3-dioxygenase.