林肯链霉菌丙氨酸脱氢酶的纯化和性质

PURIFICATION AND PROPERTIES OF ALANINE DEHYDROGENASE FROM STREPTOMYCES LINCOLNENSIS

采用硫酸铵分级沉淀、DEAE-纤维素52柱层析、亲和蓝柱层析和琼脂糖凝胶Sepharose6B柱层析的方法，分离纯化了林肯链霉菌丙氨酸脱氢酶，用聚丙烯酰胺凝胶电泳鉴定为单一组分。以凝胶过滤和聚丙烯酰胺梯度凝胶电泳测得该酶的分子量为170000，SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为42500，表明林肯链霉菌丙氨酸脱氢酶由四个相同的亚基组成。该酶加氨反应最适pH为9．0，脱氨反应最适pH为9．5，加氨反应和脱氨反应的最适温度均为50℃。加氨反应丙氨酸脱氢酶的表现米氏常数km值为：丙酮酸2．08×10－4mol／L，NH4+2．00×10-2mol／L，NADH2．38×10-5mol／L；脱氨反应的Km为：L-Ala1．43×10-2mol／L；NAD+6．67×10-5mol／L。

Alanine Dehydrogenase (L-Alanine: NAD+ oxidoreductase, deaminating, EC1.4.1.1) was purified from Sireptomyces lincolnensis through four steps :(NH4), SO4precipitation, DEAE-cellulose 52, Affi-Gel Blue and Sepharose 6B. Molecular weightof the enzyme was determined as 170 000 by gel filtration and concentration gradientPAGE. SDS-PAGE showed only one band of 42 500, demonstrating that ADH fromStreptomyces lincolnensis was consisted of four identical subunits. The optimal pH foramination was 9.0, for deamination 9.5. The optimal temperature for both aminationand deamination was 50℃. The Km valuse for pyruvate, NH4+, NADH L-Ala andNAD+ were 2.08 × 10-4mol / L, 2.00 ×10-2mol / L, 2.38 ×10-5mol / L, 1.43 ×10-2mol / L and 6.67 ×10-5 mol/ L, respectively.