Bacillus sp．C3细胞色素P450CYP102A16酶活性研究  

Activities of cytochrome P450 enzyme CYP102A16 from Bacillus sp. C3

作　　者：李泽莉 丁海涛[2] 杨玉义[2] 陈雪娇[2] 赵宇华[2] 周启发[1] 

机构地区：[1]浙江大学生命科学学院植物科学研究所,浙江杭州310058 [2]浙江大学生命科学学院微生物研究所,浙江杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2012年第6期662-668,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(31070079);浙江省科技计划资助项目(2008C13014-3;74);浙江省科技计划国际合作资助项目(2008C14038).*

摘　　要：通过异源表达及Ni-NTA亲和层析纯化获得重组Bacillus sp．C3细胞色素P450CYP102A16蛋白；以CYP102A16纯酶为研究对象，系统研究温度、pH、有机溶剂、表面活性剂、金属和非金属离子等对该酶活性及其稳定性的影响；用还原型烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate，NADPH）消减法测定CYP102A16的酶活性．结果表明：CYP102A16的最适反应温度为35℃，最适反应pH为6．5-7．5，该酶在45℃以下、pH5．0～10．0的范围内稳定；CYP102A16能完全耐受20％二甲基亚砜（dimethylsulfoxide，DMSO）、30％甲醇、10％乙醇和20％丙酮，经10％乙腈、正丙醇和异丙醇处理后残余酶活性在45％以上，经10％正丁醇处理几乎失活；添加低质量浓度氯代十六烷基吡啶（cetylpyridine chloride，CPC）（0．003-0．02g/L）和聚乙二醇辛基苯基醚（Triton X-100）（0．1-0．2g／L）可使CYP102A16酶活性分别提高40％和60％左右，而添加低质量浓度十二烷基硫酸钠（sodiumdodecyl sulphate，SDS）（O．004-0．008g／L）对该酶活性无显著影响；1-20mmol／L K^＋、20-50mmol/L Na^＋、0．05mmol／L Cd^2＋对CYP102A16酶活性表现出轻微的促进效应，NH4^＋、Ca^2＋、Mg^2＋、Fe^3＋、CO^2＋、Mn^2＋、Zn^2＋、Cu^2＋和乙二胺四乙酸（ethylene diamine tetraacetic acid．EDTA）均能不同程度地抑制CYP102A16的活性，在离子浓度为1-100mmol／L时抑制效应表现为Ca^2＋〉Mg^2＋〉NH^4＋〉EDTA，抑制作用的大小总体上与离子浓度呈正相关．Cytochrome P450 monooxygenases （P450s or CYPs） belong to a large family of hemoproteins which are widely distributed among various organisms ranging from eukaryotes to prokaryotes. P450s play a pivotal role in the biosynthesis of bioregulators such as prostaglandins, leucotrienes and steroid hormones, and participate in the metabolism ofxenobiotic such as poisons, drugs, and environmental pollutants. In recent years, more and more researches reported that P450s were potentially useful in the environmental remediation. And bacterial CYPs have been found to be more promising than those from plants and animals because of more solubility and higher stability, not membrane-associated and higher reaction rates. However, the activity of wild type prokaryotic P450s towards recalcitrant contaminants has been less reported as compared to eukaryotic P450s. In our laboratory, a novel cytochrome P450 monooxygenase CYP102A16, which belonged to CYP102 family, was cloned from Bacillus sp. C3 and expressed in Escherichia coil CYP102 family represents a bacterial P450 one naturally fused with cytochrome P450 and cytochrome P450 reductase, making them potentially more useful in biotechnological applications due to their self-sufficiency, while the reaction catalyzed by cytochrome P450 requires cytochrome P450 reductase for transfer electron. The recombinant CYP 102A16 was then purified by Ni-NTA affinity chromatography. The effects of temperature, pH, organic solvents, surfactants, metal and non-metal ions on the activity and stability of purified CYP 102A 16 were investigated based on nicotinamide adenine dinucleotide phosphate （NADPH） oxidation assay. The results showed that the optimum pH for CYP102A16 was 6.5-7.5, with a highest NADPH consumption rate of （885.5± 11.7） U at pH 7.5 in Tris-HC1 buffer （Fig. 1C）. CYP102A16 was stable under a wide range ofpH, retaining more than 80 % of its activity after incubated at pH 5.0-10.0 for 30 min （Fig. 1D）. The enzyme reached a highest activity of（511.0 ± 20.5）

关 键 词：细胞色素P450 CYP102A16蛋白 酶活性 影响因素 

分 类 号：Q939.99[生物学—微生物学]