烟草节杆菌02181肌酸酶的纯化及理化特性  

Purification and Characteristics of Creatinase from Arthrobacter Nicotianae 02181

作　　者：罗侃 崔有宏 曾志南 徐东 贺燕鸿 

机构地区：[1]解放军兰州医学高等专科学校生化教研室,甘肃兰州730020

出　　处：《甘肃科学学报》2006年第1期60-63,共4页Journal of Gansu Sciences

基　　金：兰州军区医药卫生科研项目(LXH99-11)

摘　　要：采用DEAE-纤维素和羟基磷灰石层析联合使用的纯化程序,从烟草节杆菌02181株提取纯化了肌酸酶.比活性由0.92 U/m g上升至124.44 U/m g,酶被提纯了135.26倍,在非变性聚丙烯酰胺凝胶电泳上呈单一区带,最终收率达52.95%.特性研究显示此酶的相对分子量为84500,等电点为4.80,在磷酸盐缓冲体系中,最适pH为6.50,pH 7.00环境下耐受性最好,最适温度为35℃,米氏常数(Km)为46.50 mm ol/L,最大反应速度(Vm ax)为8.20μm ol/m in.Cu2+、SDS可使酶完全失活,Hg2+、Cd2+、Zn2+和Fe3+明显抑制酶活性,M g2+、M n2+、B rij 35、T riton X-100和邻菲饶啉对酶活性有一定的抑制作用,EDTA对酶活性无明显影响,而Ca2+和N aN3对酶有一定的激活作用.络合剂邻菲饶啉和EDTA对酶活性的抑制试验表明此酶属于金属酶类.The creatinase from Arthrobacter Nicotianae 02181 was purified by a procedure involving the precipitation of nucleic acid with streptomycin and chromatographies on DEAE-cellulose and hydroxylapatite. This process resulted in a 135. 26-fold increase in specific activity with a recovery of 52.95%. The preparation of creatinase was homogeneous on acrylamide gel electrophoresis （PAGE）. The optimal pH was 6. 50 and the most stable pH was 7.00 at 37℃. The Km was 46.50 mmol/L. The relative molecular weight as estimated by PAGE was approximately 84500. The enzyme was markedly inactivated by incubation with 1 mmol/L of Cu^2＋, Hg^2＋, Fe^2＋, Zn^2＋, Cd^2＋, and slightly activated by Ca^2＋ respectively. Inhibitory effects of EDTA and phananthroline on the activity of the enzyme suggested that this enzyme was a metalloenzyme.

关 键 词：烟草节杆菌 肌酸酶 纯化 特性 

分 类 号：Q554[生物学—生物化学] Q503