地中海拟无枝菌酸菌(Amycolatopsis mediteranei)U-32硝酸还原酶的定位、纯化及性质  

Localization,Purification and Characterization of Nitrate Reductase from Amycolatopsis mediterranei U 32

作　　者：李果龙 杨蕴刘[1] 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：《中国生物化学与分子生物学报》1998年第6期710-714,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金

摘　　要：在力复霉素ＳＶ研究中，发现硝酸盐对抗生素合成呈现多效性作用，不仅大幅度提高产量，还对产生菌——地中海拟无枝菌酸菌生理产生多方面的影响，从而提出整体性调节的结论．这一多效性作用是由硝酸盐所引起的，为此对硝酸还原酶进行了研究．首先，通过原生质体渗透裂解发现地中海拟无枝菌酸菌Ｕ－３２的硝酸还原酶是一个胞质酶．该酶极不稳定，缓冲液中加入硝酸钾、甘油等保护剂能极大地提高其稳定性．通过硫酸鱼精蛋白沉淀，硫酸铵分级分离，Ｐｈｅｎｙｌ－ＳｅｐｈａｒｏｓｅＣＬ４Ｂ、Ｂｉｏ－ＧｅｌＡ１．５ｍＤＥＡＥ－Ｓｅｐｈａｃｅｌ和ＳｅｐｈａｄｅｘＧ－７５柱层析等多步纯化得到了电泳纯的硝酸还原酶．该酶为一７９ｋＤ的单亚基酶，每分子酶含有约２．２９原子的钼，但并不含有非血红素铁、酸不稳定硫、ＦＭＮ及ＦＡＤ，其等电点为６．２，反应最适ｐＨ为７．２，最适温度为４０℃．对硝酸根的Ｋｍ值为１３．３μｍｏｌ／Ｌ．同时分析了该酶的吸收光谱．The localization of nitrate reductase was ascertained.Through osmotic lysis of protoplasts,nitrate reductase from Amediterranei U 32 was located in the cytoplasm.The enzyme was rather unstable,and inclusion of protective reagents in the buffer system greatly improved its stability.Electrophoretically purified enzyme was obtained through six steps of purification:Protamine sulfate precipitation,ammonium sulfate fractionation,Phenyl Sepharose CL 4B,Bio Gel A 1 5m,DEAE Sephacel and Sephadex G 75 column chromatography.The electrophoretically pure enzyme was a monomer of 79 kD,each mol of enzyme contained 2 29 mol Mo,but no non heme iron,acid labil sulfur,FMN and FAD.Through iso electric focusing,the isoelectric point of nitrate reductase was found to be 6 2.Its optimum pH was 7 2,and its optimum temperature 40℃.The K m value for nitrate was 13 3 μmol/L,and the absorption spectrum of this enzyme was also analyzed.

关 键 词：硝酸还原酶 定位 纯化 A.mediterranei 

分 类 号：Q936[生物学—微生物学] Q554.03