点突变Pro229Ser和Glu243Gly对耐热邻苯二酚2，3—双加氧酶性质的影响

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±0.01×10

Effects of Pro229

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.