Bacillus sp. C3细胞色素P450 CYP102A16酶活性研究

doi:10.3785/j.issn.1008-9209.2012.01.061

浙江大学学报(农业与生命科学版)

2012, Vol. 38

Issue (6): 662-668 DOI: 10.3785/j.issn.1008-9209.2012.01.061

生物科学与技术

Bacillus sp. C3细胞色素P450 CYP102A16酶活性研究

李泽莉¹，丁海涛²，杨玉义²，陈雪娇²，赵宇华²，周启发¹

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

Activities of cytochrome P450 enzyme CYP102A16 from Bacillus sp. C3

LI Ze-li¹, DING Hai-tao², YANG Yu-yi², CHEN Xue-jiao², ZHAO Yu-hua^2*, ZHOU Qi-fa¹

1. Institute of Plant Science, College of Life Sciences, Zhejiang University, Hangzhou 310058, China; 2. Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou 310058, China

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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P450 CYP102A16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 °C，最适反应pH为6.5~7.5，该酶在45 °C以下、pH 5.0~10.0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) (0.003~0.02 g/L) 和聚乙二醇辛基苯基醚 (Triton X-100) (0.1~0.2 g/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 (sodium dodecyl sulphate, SDS) (0.004~0.008 g/L) 对该酶活性无显著影响；1~20 mmol/L K⁺、20~50 mmol/L Na⁺、0.05 mmol/L Cd²⁺对CYP102A16酶活性表现出轻微的促进效应，NH₄⁺、Ca²⁺、Mg²⁺、Fe³⁺、Co²⁺、Mn²⁺、Zn²⁺、Cu²⁺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1~100 mmol/L时抑制效应表现为Ca²⁺> Mg²⁺> NH₄⁺ > EDTA，抑制作用的大小总体上与离子浓度呈正相关

Abstract: 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 of xenobiotic 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 coli. 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 CYP102A16 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 CYP102A16 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-HCl buffer (Fig. 1C). CYP102A16 was stable under a wide range of pH, 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) U at 35 ºC, and kept stability at the temperatures below 45 ºC with residual activity of above 95 % (Fig. 1A, B). CYP102A16 was completely stable in 20 % dimethylsulfoxide (DMSO), 30 % methanol, 10 % ethanol and 20 % acetone, and could retain above 45 % activity in 10 % acetonitrile, n-propanol and isopropanol, but was almost inactivated in 10 % n-butanol (Table 1). The enzyme activity increased about 40 % and 60 % when low levels of cetylpyridine chloride (CPC) (0.003-0.02 g/L) and Triton X-100 (0.1-0.2 g/L) were added respectively, while non-significant effect was found with the addition of low concentration of sodium dodecyl sulphate (SDS) (0.004-0.008 g/L). The activity decreased gradually with increasing the surfactant concentration, and it was almost inactivated at 0.09 g/L CPC, 5.0 g/L Triton X-100 or 0.08 g/L SDS (Fig. 2). Addition of 1-20 mmol/L K⁺, 20-50 mmol/L Na⁺ and 0.05 mmol/L Cd²⁺ increased CYP102A16 activity slightly, while NH₄⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Mn²⁺, Zn²⁺, Cu²⁺ and ethylene diamine tetraacetic acid (EDTA) exhibited negative effects on enzyme activity (Fig. 3). Inhibition at 1-100 mmol/L concentrations was observed by the order of Ca²⁺ > Mg²⁺ > NH₄⁺ > EDTA, and the effect was positively correlated with the concentration of metal and non-metal ions. Exploration of the in vitro catalytic conditions for CYP102A16 provides a reliable basis for constructing a better in vitro catalytic system for CYP102A16, which is of significant importance for its practical application in environmental remediation.

出版日期: 2012-11-20

基金资助:

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；2010C13G2010074)；浙江省科技计划国际合作资助项目(2008C14038).

通讯作者: 赵宇华，E-mail: yhzhao225@zju.edu.cn

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	李泽莉1
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引用本文:

李泽莉1，丁海涛2，杨玉义2，陈雪娇2，赵宇华2,周启发1. Bacillus sp. C3细胞色素P450 CYP102A16酶活性研究[J]. 浙江大学学报(农业与生命科学版), 2012, 38(6): 662-668.

LI Ze-li1, DING Hai-tao2, YANG Yu-yi2, CHEN Xue-jiao2, ZHAO Yu-hua2*, ZHOU Qi-fa1. Activities of cytochrome P450 enzyme CYP102A16 from Bacillus sp. C3. , 2012, 38(6): 662-668.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2012.01.061 或 http://www.zjujournals.com/agr/CN/Y2012/V38/I6/662

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