摘要
采用原生质体裂解方法确定甲基丙二酰CoA变位酶(MCM)和消旋酶(MCR)均是胞浆内酶。各经过四步纯化得到电泳纯酶。纯化MCM酶的比活力为12.84u/mg,纯化倍数为528,酶活回收为60％,纯化的MCM酶服从典型的米氏底物饱和曲线,对琥珀酰CoA和辅酶B_(12)的K_m值分别为9.723#mol/L和0.1277#mol/L。经SephadexG-150测定MCM分子量约为134.000±2000,SDS-聚丙烯酰胺凝胶电泳显示两条分子量分别为67000和65000的蛋白带,说明该酶由两个大小不等亚基组成。吸收光谱测定每摩尔纯化全酶含两摩尔辅酶B_(12)。纯化MCR酶比活力为2.305u/mg,纯化倍数96,酶活回收46.7％。MCR酶由两个分子量均为17500的亚基组成。MCR酶活性能被二价金属离子Cu^(2+)、Co^(2+)、Mg^(2+)、Mn^(2+)和Fe^(2+)所促进。两酶的酶学性质和其他生物来源的MCM、MCR酶明显相似。
Early studies have shown that the polyketide chain of rifamycin is formed by condensation of eight propionate and two acetate units via malonyl-CoA and methylmalonyl-Co A, which could be generated by the isomerization of succiny-CoA. Thus methylmalonyl-CoA mutase (MCM) and methylmalonyl-CoA racemase (MCR), the two key enzymes in the succinyl-CoA isomerization pathway, are important in rifamycin synthesis. By osmotic lysis of protoplast of A. mediterranei U32, MCM and MCR were determined as cytosolic enzymes. The MCM and MCR were purified as electrophoreticaly homogenous enzymes from the crude extract with four steps of purification. The purified MCM showed a typical Michaelis-Menten type substrate saturation pattern, with Km of 9.723μmol / L and 0.1277μmiol/ L for succinyl-CoA and B12 coenzyme, respectively. The molecular weight of MCM was determined as 134 000 ± 2000 by sephadex G-l 50 chromatography. SDS-PAGE showed the purified MCM was a heterodimer with molecular weight of 67 000 and 65 000. Coenzyme B12 concentration was estimated as about 2 moles of coenzyme B12 per mole purified holoenzyme from absorption spectrum. The molecular weight of MCR was 33 500 ± 1 '500 by sephadex G-75 chromatography measurement. SDS-PAGE showed that the enzyme was a homodimer of 17 500. The activity of MCR was increased by Cu2+, Co2+, Mg2+, Mn2+ and Fe2+. The properties of the purified MCM and MCR enzymes appear to be fairlysimilar to those of previously obtained from other sources.
出处
《微生物学报》
CAS
CSCD
北大核心
1996年第3期199-207,共9页
Acta Microbiologica Sinica
关键词
拟无枝酸杆菌
地中海菌
甲基丙二酰CoA
变位酶
A. mediterranei U32, Methylmalonyl-CoA mutase, Methylmalonyl-CoA racemase, Purification, Characterization.
