地中海诺卡氏菌丙氨酸脱氢酶的纯化和性质  

PURIFICATION AND PROPERTIES OF L-ALANINE DEHYDROGENASE FROM NOCARDIA MEDITERRANEI

作　　者：刁蓉 焦瑞身[1] 

机构地区：[1]中国科学院上海植物生理研究所

出　　处：《微生物学报》1991年第3期206-212,共7页Acta Microbiologica Sinica

基　　金：全国自然科学基金

摘　　要：采用硫酸铵分部沉淀、DEAE纤维素-52柱层析和亲和蓝柱层析的方法,分离纯化了地中海诺卡氏菌(Nocardia mediterranei)U-32丙氨酸脱氢酶(ADH),用聚丙烯酰胺凝胶电泳鉴定为单一组份。以聚丙烯酰胺凝胶梯度电泳测得丙氨酸脱氢酶的分子量为228000,SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为38000,表明地中海诺卡氏菌U-32丙氢脱氢酶由六个相同的亚基组成。ADH加氨反应最适pH为8.5,脱氨反应最适pH为11.5,ADH的pH稳定范围在pH7.5—11.5。脱氨反应的最适温度为50℃。ADH的米氏常数K_m为:丙酮酸4.88×10^(-4)mol/L;NH_4^+ 4.03×10^(-3)mol/L;NADH 6.02×10^(-3)mol/L;L-Ala 7.45×10^(-3)mol/L;NAD^+ 6.67×10^(-3)mol/L。Hill作图法求得ADH的Hill系数n为:ADH对丙酮酸、NADH和NAD^+的Hill系数都为1;对L-Ala和NH_4^+的Hill系数n值分别为0.52和0.51,ADH对L-Ala和NH_4^+有负协同效应,由此初步推测ADH是一个调节酶。L-Alanine Dehydrogenase (L-Alanine: NAD oxidoreductase, deaminating, EC 1.4.1.1) was purified to homogeneity from a cell-free extract of Nocardia mediterranei U-32 by precipitation with ammonium sulfate and column chromatography on DEAE-cellulose, Blue-Sepharose. By electrophoresis with polyacryamide and SDS-polyacrylamide gel, it was demonstrated that the molecular weight of ADH was 228000 and it was composed of six identical subunits with a molecular weight of 38000. The enzyme is stable in a wide pH range (pH 7.5—11.5), and the optimal pH is 11.5 for the deamination and 8.5 for the amination. The optimal temperature of deamination is 50℃. The Michaelis constants of ADH are as follows:Pyrurate 4.88×10^(-4)mol/L;NH_4^+ 4.03×10^(-3)mol/L;NADH 6.02×10^(-5)mol/L;NAD^+ 6.67×10^(-5)mol/L;L-Alanine 7.45×10^(-3)mol/L.The hill constants of ADH are as follows:Pyrurate (n, 1); NADH (n, 1); NAD (n, 1); L-Ala (n, 0.52); NH_4^+(n, 0.51).We suggest that ADH could be an allosteric enzyme.

关 键 词：地中海诺卡菌 丙氨酸脱氢酶 纯化 

分 类 号：Q939.106[生物学—微生物学]