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L68菌株中的邻苯二酚 2 ,3 双加氧酶经硫酸铵分级沉淀、DEAE SepharoseFastFlow柱层析、Hydroxyapatite柱层析、Sephadex G15 0凝胶过滤分离 ,并经SDS 聚丙烯酰胺凝胶电泳检测 ,达到了电泳纯 .提纯倍数为 2 34 .1倍 ,收率为 2 0 .2 % .酶相对分子质量为 (132± 10 )kDa ,由 4个相对分子质量相同的亚基组成 .最适反应温度 35℃ ,且酶活力在 15～ 65℃比较稳定 ;最适 pH为 8.0 ,在 pH 7.0～ 10 .5酶活力较高且比较稳定 .某些金属离子尤其是Fe3+ 对酶活力有强烈的抑制作用 .以邻苯二酚为底物 ,Km 为 2 0 .63μmol/L ,Vmax为 2 .82 μmol/ (min·mg)
The catechol 2,3 dioxygenase in L68 strain was precipitated by ammonium sulfate, separated by DEAE Sepharose Fast Flow column chromatography, followed by Hydroxyapatite column chromatography and Sephadex G15 0 gel filtration column. SDS-polyacrylamide gel electrophoresis , Reached electrophoretic purity.The purification fold was 2 34.1 times, and the yield was 20.2% .The relative molecular mass of the enzyme was (132 ± 10) kDa, which consisted of four subunits with the same relative molecular mass.The optimum The reaction temperature is 35 ℃, and the enzyme activity is relatively stable at 15-65 ℃; the optimum pH is 8.0, and the enzyme activity is relatively high and stable at pH 7.0-10.5 Some metal ions, especially Fe3 +, have strong enzyme activity Inhibition with catechol as substrate, Km was 2.063μmol / L, Vmax was 2.82 μmol / (min · mg)