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题名	细胞色素P450体外催化除草剂代谢及其活性代谢化合物的研究
作者	向文胜
学位类别	博士
答辩日期	2003
授予单位	中国科学院过程工程研究所
授予地点	中国科学院过程工程研究所
导师	许志宏
关键词	小麦 细胞色素 异源表达 绿磺隆 醚苯磺隆 活性代谢 除草剂
其他题名	Herbicide metabolism by cytochrome P450 in vitro and bioactivity of metabolites
学位专业	应用化学
中文摘要	除草剂草甘磷能够抑制小麦细胞色素P450的活性.在酵母细胞中表达的小麦细胞色素P450微粒体CO差异光谱在502nm有吸收峰,草甘磷与细胞色素P450结合为典型的Ⅱ光谱.这表明草甘磷中的N原子与细胞色素P450中的血红素产生了结合,其分离常数Ks为70μmol/L;草甘磷抑制细胞色素P450酶活性的IC<,50>为11μmol/L.这一抑制机理与先前人们研究的除草剂的增效剂即细胞色素P450的抑制剂作用机理不同,因而,这为开发磺酰脲类除草剂的增效剂提供了新的母体化合物.为研究小麦细胞色素P450特性,异源表达的小麦细胞色素P450微粒体经DE-52纤维素柱色谱法和Mono-Q快速蛋白纯化系统(FPLC)分离纯化后,以除草剂绿磺隆为催化底物时比活性达到512.3nmolmin<'-1>mg<'-1>puotein,该酶被纯化1366.1倍,纯化后的细胞色素P450经SDS-PSGE检测为单一谱带,分子量为52.5kD.为了在体外快速研究除草剂的代谢,利用蚕丝素固定化了细胞色素P450.固定化细胞色素P450在连续使用10次以后,仍能维持原酶活性的80%;在4℃下贮存2个月,酶的相对活性保持在73.8%以上;此外,固定化细胞色素P450增强了酶对温度的稳定性和对pH的稳定性.固定化细胞色素P450与游离细胞色素P450对绿磺隆、醚苯磺隆具有相近的亲和常数.固定化细胞色素P450可方便、快速、稳定、持续地在微型反应器中进行除草剂的体外代谢研究.利用分子生物学,固定化技术构建方便、快捷的农药靶酶活性化合物体外筛选模型、代谢酶体外代谢模型.这一研究填补了中国进行高效筛选农药活性化合物的空白,满足了自动化进行农药体外代谢研究的需要;利用生物间农药差异代谢产物用于活性化合物筛选,发展农药体外代谢、活性化合物高效筛选与农药分子设计的偶合技术应用于新农药的创制中.可能成为创制新农药的一条捷径和获得活性化合物的源泉,有着广阔的研究前景.
英文摘要	Study on pesticide metabolism is very important in novel pesticide creation. The metabolites of bioactivation reaction could be model structures for discovery of novel active compounds. To deeply understand the molecular mechanisms by monocots rapidly metabolizing herbicides, a wheat cytochrome P450 cDNA (CYP7lC6vl) cloning by RT-PCR was expressed in yeast, and cytochrome P450 microsomal fractions generated from this strain were investigated for their potential to catalyze the metabolism of two sulfonylurea herbicides chlorsulfuron and triasulfuron. In vitro enzyme assays showed that the gene product of one heterologously expressed cytochrome P450 cDNA specifically catalyzed the metabolism of these two sulfonylurea herbicides. In vitro kinetic parameters Km for chlorsulfuron and'triasulfuron were 57 (±15) ujtnol/L and 38 (±16) [imol/L, respectively. Analysis of the metabolites demonstrated that the CYP71C6vl encoded enzyme functioned primarily as a 5-phenyl ring hydroxylase when chlorsulfuron and triasulfuron were the substrates. Metabolites 5-hydroxy-chlorsulfuron and 5-hydroxy-triasulfuron inhibit acetolactate synthase (ALS) activity. The difference in inhibition of acetolactate synthase activity ICsoby parent compound and 5-hydroxy-metabolites was very small in vitro. However, their metabolites injured neither wheat nor bean, when applied as postemergence treatment. In the case of chlorsulfuron and its metabolites IC50 for inhibition of wheat ALS activity were 7.1xlO"9 mol/L and 7.9^10"9 mol/L, respectively; IC50 for inhibition of bean ALS activity were 3.6^10"9 mol/L and 4.1xlO'9 mol/L, respectively. In the case of triasulfuron and its metabolites IC50 for inhibition of wheat ALS activity were 4.6x10"9 mol/L and 5.3xlO"9 mol/L, respectively; IC50 for inhibition of bean ALS activity were 4.7xlO"9 mol/L and 4.9x10" mol/L, respectively. These results showed proper groups on 5-position on phenyl ring of sulfonylurea molecule could also possess high herbicidal activity. Glyphosate is an inhibitor of a wheat cytochrome P450. The cytochrome P450 microsomal fractions generated from heterologously expressed in yeast retained normal spectral characteristics having a Soret peak at 502 nm in the reduced carbon monoxide difference spectrum. Addition of the herbicide glyphosate resulted in a Type II spectrum indicative of binding via the nitrogen group to haem of cytochrome P450 as a sixth ligand. A spectral dissociation constant Ks of 70 |imol/L was observed and an IC50 of 11 umol/L was observed for glyphosate inhibition of CYP71C6vl P450 activity. To investigate characteristics of wheat cytochrome P450, recombinant wheat cytochrome P450 monooxygenase heterologous expressed in yeat was purified by DE-52 cellulose chromatography and Fast Protein Liquid Chromatography (FPLC) with Mono-Q column. The degree of purification was 1366.1 fold. The specific activity of purified cytochrome P450 reached to 512.3 nmol min"1 mg"1 protein from the substrate of herbicide chlorsulfuron. The purified cytochrome P450 showed one band in SDS-polyacrylamide gel electrophoresis, molecular weight was 52.5 kD. To investigate herbicide metabolism further in vitro by cytochrome P450 with stable enzymatic activity, cytochrome P450 were immobilized in silk fibroin. The activity of immobilized cytochrome P450 was maintained above 80% after repeated batch experiments for 10 times. Moreover, the activity of immobilized cytochrome P450 was kept as relatively high as 73.8% after storage for two months at 4°C. In addition, immobilization could improve the temperature stability and pH stability of cytochrome P450. Immobilized cytochrome P450 had the similar affinity Km values for chlorsulfuron and triasulfuron as the free cytochrome P450o In the case of chlorsulfuron affinity Km value was 53 \xmo\JL for free cytochrome P450, and 63 ujnol/L for immobilized cytochrome P450. In the case of triasulfuron affinity Km value was 36 u,mol/L for free cytochrome P450, and 44 (imol/L for immobilized cytochrome P450. Immobilized cytochrome P450 could be applied to micro-bioreactor, and would be convenient, rapid, stable and continuous for study on herbicide metabolism in vitro.
语种	中文
公开日期	2013-09-16
页码	62
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1384]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	向文胜. 细胞色素P450体外催化除草剂代谢及其活性代谢化合物的研究[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2003.

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