枯草杆菌6-磷酸葡萄糖酸脱氢酶的分离纯化及部分性质研究  

Isolation and Studing Some Properties of 6-Phosphogluconate Dehydrogenase From Bacilllus Subtilis

作　　者：秦岭[1] 刘克武[1] 莫宏春[1] 江琰[1] 国锦林[1] 刘祝君[1] 喻东[1] 

机构地区：[1]四川大学生命科学学院,成都610064

出　　处：《四川大学学报（自然科学版）》2003年第5期939-944,共6页Journal of Sichuan University(Natural Science Edition)

摘　　要：采用DEAE Sepharose离子交换层析,Blue SepharoseCL 6B特异结合层析和SephadexG 200凝胶过滤技术,对枯草芽孢杆菌中的6 磷酸葡萄糖酸脱氢酶进行了分离纯化.纯化倍数为112.3倍,比活为1.46U/mg,回收率为8.2%.纯化酶液经聚丙烯酰胺凝胶电泳检验为单一带.测得全酶相对分子质量为107kD,具有两个分子量相同的亚基,亚基相对分子质量为51kD.最适pH值为8.0,最适温度为30℃,对热不稳定.以6 磷酸葡萄糖酸和NADP+为底物,其Km值分别为Km1(NADP+)=19.8μmol/L和Km2(6 GPA)=22.6μmol/L.在5mmol/L～50mmol/L浓度范围内,Mg2+,Ca2+和Mn2+对酶有激活作用,Fe3+和Cu2+对酶有抑制作用,K+,Na+,Cl-,NO-3,SO-4对酶几乎没有影响.用PMSF,TNBS,NBS,DTT和BrAc对酶进行修饰,实验结果表明,丝氨酸、苏氨酸、赖氨酸和组氨酸残基可能是该酶活性中心的功能基团.6Phosphogluconate dehydrogenase(6PGADase,EC 1.1.1.44) was isolated by homogenate, ammunium sulfate fractionation, DEAESepharose chromatography, BlueSepharose Affinity chromatography and gel filtration with Sephadex G 200 from bacillus subtilis, and some properties of the enzyme had been studied. A 113.8fold purification was obtained with a 8.2% yield. The purified enzyme moved as a single electrophoretic band in PAGE and SDSPAGE. The molecule weight of the 6PGADase is 107 kD,which is measured by AKTA FPLC. The 6PGADase consists of subunits of a molecule weight of 51kD, therefore one 6PGADase has two same subunits. The optimum of the activity was found at about pH8.0 and at 30℃ ,with a Km value of 19.8 μmol/L for NADP and 22.6μmol/L for 6phosphogluconic acid. The 6PGADase is activated by Mg2+, Mn2+ and Zn2+,while the enzyme is inhibited by cations of Cu2+ and Fe3+. Na+,K+ and anions such as Cl-, NO-3, SO2-4 have no effect on the 6PGADase in concentration ranging from 5 mmol/L to 50 mmol/L.PMSF,TNBS,NBS,DTT and BrAc are used to modify the 6PGADase, and the results illustrate that Ser,Thr,Lys and His are critical for the activity of the enzyme.

关 键 词：枯草芽孢杆菌 6-磷酸葡萄糖酸脱氢酶 分离纯化 性质 

分 类 号：Q841.1[生物学]