圈卷产色链霉菌硝基烷类氧化酶的分离纯化及酶学性质  

Purification and Properties of Nitroalkane-oxidizing Enzyme of Streptomyces ansochromogenes

作　　者：张集慧[1] 马文勃[1] 谭华荣[1] 

机构地区：[1]中国科学院微生物研究所,北京100080

出　　处：《微生物学报》2002年第5期587-593,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金重点项目 ( 39830 0 1 0 )资助~~

摘　　要：圈卷产色链霉菌硝基烷类氧化酶基因naoA在大肠杆菌中获得了成功表达 ,从含有重组质粒pNA1 0 1 (pET2 3b∷naoA)的工程菌株BL2 1 (DE3 )中分离纯化了硝基烷类氧化酶 ,SDS PAGE检测为均一。对纯酶进行了酶学性质及动力学研究。底物为 1 硝基丙烷、2 硝基丙烷和硝基乙烷时 ,在 0 4mol L的磷酸缓冲液中 ,酶的最适反应pH值为 7～ 8,最适反应温度为48℃～ 5 6℃。室温保存 6d后 ,酶的活性保持了 43 3 % ,但对 6 0℃以上的高温敏感。硫醇化合物如巯基乙醇、还原型谷胱甘肽不同程度地抑制酶活性 ,特别是NADH ,其浓度为 1mmol L时 ,酶活性几乎全部丧失。以 1 硝基丙烷为底物时 ,NaoA的Km 为 3 5 7mmol L ,Vmax 为0 1 99μmol (μg .min) 。The nitroalkane-oxidizing enzyme gene(naoA) of Streptomyces ansochromogenes 7100 was expressed in E.coli and the product(NaoA) was purified from the strain of BL21(DE3)/pNA101(pET23b∷naoA).The enzyme was identified to be homogeneity on SDS-PAGE after purification by chromatography.The results showed that the enzyme functioned optimally at pH 7～8 and 48℃～56℃ when using 1-nitropropane,2-nitropropane and nitroethane as substrates.The enzyme activity remained 43.3% after stored for 6 d at room temperature and it is sensitive to high temperature(over 60℃).The activity is partially inhibited by thiol-compounds such as β-mercaptoethanol and reduced glutathione and almost completely lost by 1mmol/L NADH,whereas 1mmol/L EDTA indicated slightly inhibitory effect.The K m coefficient of enzyme for 1-nitropropane is 35.7mmol/L and V max is 0.199 μmol/(μg.min).

关 键 词：圈卷产色链霉菌 硝基烷类氧化酶 分离纯化 酶学性质 

分 类 号：Q936[生物学—微生物学]