嗜麦芽假单胞菌碱性蛋白酶的纯化及性质研究

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 曾勇 Zheng Yong Huang Ningwu Zhu Rufan Peng Zhenrong 1 first-author 黄宁悟 1 朱汝璠 1 彭珍荣 1 武汉大学生物系 武汉大学生物系 Department of Biology,Wuhan University Department of Biology,Wuhan University 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. 嗜麦芽假单胞菌 碱性蛋白酶 纯化 生化特性 pseudomonas maltophilia alkaline proteinase purify Characters 1990-03-01 2021-04-01 3