嗜麦芽假单胞菌碱性蛋白酶的纯化及性质研究

A STUDY ON ISOLATION AND BIOCHEMISTRY CHARACTERS OF PROTEINASE FROM PSEUDOMONAS MALTOPHILIA 27

嗜麦芽假单胞菌(Pseudomonas maltophilia)27分泌的胞外碱性蛋白酶(经硫酸铵分级盐析、Sephadex G-200和 Sephadex G-75柱层析、聚丙烯酰胺凝胶制备电泳分离得到纯酶。此酶分子量为76000,由两个不同亚基构成(亚基分子量分别为31000和44000)。两条肽链 N 末端氨基酸是 Gly 和 Thr.酶作用最适 pH9.5,最适温度50℃,在40℃以内稳定。以酪蛋白为底物测得其 K_m 值为2.86 mg·ml^(-1)。此酶被 Hg^(2+)抑制,但在 Ca^(2+)、Mg^(2+)、Co^(2+)、Mn^(2+)、Cu^(2+)、Zn^(2+)中有相近活力,4mmol·L^(-1)的 EDTA 可抑制约36%的酶活。用 DFP 和 NBS 修饰对酶有强烈抑制作用,而 PMSF、pCMB、溴乙酸、丁二酮则对酶没有影响。

Alkaline proteinase secreted by Pseudomonas maltophilia 27 has been purified to homogeneity by using ammonium sulphate fraction,sephadex-G 200,sephadex G-75 gel filtration and polyaerylamide gel preparative electrophoresis.Molecular weight of the native enzyme was found to be 76000 by sephadex G 200 gel filtration.Electrophoresis in SDS polyacrylamide gel has shown two bands of molecular weight of 31040 and 44000 respectively in the gel,indicating that the alkaline proteinase was a dimer of different subunits.The N terminal amino acids of the proteinase were glycine and threoline.The optimal pH was 9.5,the optimal temperature was 50℃,the enzyme was stable below 40℃,The Km of the proteinase was 2.86 mg·ml_(-1) with casein as the substrate.The enzyme was inhibited by Hg^2,but its activities were changed a little by Ca^2,Mg^2,CO^2,Mn^2,Cu^2.The activity of the enzyme was inhibited 36% in the presence of EDTA(4mmol·L^(-1).The enzyme activiLy was greatly inhibited by chemical modification with DFP and NBS,but no effec can be detected when the enzyme was chemically modified with PMSF,PCMB,bromate acetate and 2,3 butanedione.