地中海拟无枝酸杆菌甲基丙二酰CoA转羧基酶的纯化及酶学特性

经硫酸铵沉淀，ＤＥＡＥ－纤维素吸附，磷酸纤维素吸附和Ｓｅｐｈａｒｏｓｅ４Ｂ分子筛层析四步从地中海拟无枝酸杆菌纯化得到电泳纯ＭＣＴ酶，酶比活力为３．２１Ｕ／ｍｇ，纯化倍数１７８，酶活回收１４．９％。酶反应的最适ｐＨ和温度分别为７．０和３５℃。纯化ＭＣＴ酶对底物丙酰ＣｏＡ和草酰乙酸的米氏常数分别为０．０２７ｍｍｏｌ／Ｌ０．０３５０９ｍｍｏｌ／Ｌ．经ＳｅｐｈａｄｅｘＧ－１５０测定酶分子量为２０００００，ＳＤＳ－取丙烯酰胺电泳凝胶显示一条分子量６８０００的亚基蛋白带，说明该酶由三个等大小亚基组成．薄层等电聚焦测定酶等电点为ｐＩ６．０．二价金属离子Ｃｏ￣（２＋）和Ｆｅ￣（３＋）促进酶活力．采用原生质体裂解的方法发现ＭＣＴ酶是可能分布于胞浆和细胞膜上．

Purification and Characteristics of Methylmalonyl-CoA Transcarboxylase from Rifamycin SV Synthesizing Amycolatopsis mediterranei U32

Methylmalonyl-CoA transcarboxylase was purified 178-fold to homogeneity from rifamycin SV-producing Amycolatopsis mediterranei U32 using ammonium sulfate fractionation,DEAE-cellulose,cellulose phosphate and gel filtration with sepharose 4B. The enzyme was distributed both in the cytoplasma and membrane fractions. The specific activity of final enzyme preparation was 3. 21 U/mg protein. The M_r of the native enzyme was 200 000 as determined by gel filtration on sephadex G-150 and the subunit M_r were 68 000 as estimated by SDS-PAGE,suggesting that the enzyme consists of one type of subunit which was different from the early reports. The enzyme showed a PH and temperature optimum of 7. 0 and 35 ℃ . The enzyme showed typical Michaelis-Menten type substrate saturation patterns with Km of 0. 027 mmol/L for propionyl-CoA and 0. 03509 mmol/L for oxaloacetate. The isoelectric point of the purified enzyme was determined as 6. 0.