ecombinant expression of nitrile hydratase and its application in the synthesis of intermediates irbesartan 摘要厄贝沙坦主要针对原发性高血压以及合并高血压的2型糖尿病肾病的治疗。目前，厄贝沙坦的关键中间体2-丁基- 1，3二氮杂螺［4，4］壬-1-烯-4-酮用化学方法合成，使用大量的强酸强碱，而且伴有大量盐类生成，给分离纯化带来困难，也会造成一定的环境污染。腈水合酶可以转化1-戊酰胺基环戊腈为相应的酰胺，进而在碱性条件下水解、环化合成2-丁基- 1，3二氮杂螺［4，4］壬-1-烯-4-酮。该路线反应条件较温和，基于腈水合酶的催化专一性可以避免1-戊酰胺基环戊腈水解为相应的羧酸。同时，该工艺减少酸碱溶液的使用量，对环境较友好性，符合原子经济和绿色化学的发展方向。目前，腈水合酶作为酶工程领域产业化的成功范例，主要用于丙烯酰胺和烟酰胺的工业生产。因此，开发腈水合酶在合成厄贝沙坦等精细化学品中间体中的应用，具有重要的理论意义及应用前景。本论文中，主要筛选具有催化1-戊酰胺基环戊腈制备相应酰胺的腈水合酶，为后期厄贝沙坦关键中间体的酶催化工艺开发奠定基础。我们针对本实验室已构建的重组腈水合酶库进行筛选，实验发现来源于Pseudonocardia thermophila JCM3095和Silicibacter pomeroyi DSS-3的腈水合酶表现出1-戊酰胺基环戊腈的水合活力，然而其催化活力较低。进一步，采用同源建模策略构建S。 pomeroyi DSS-3腈水合酶的蛋白质模型，经过分子对接考察1-戊酰胺基戊腈与腈水合酶底物结合口袋的结合情况，为后期针对腈水合酶的分子改造奠定理论依据。76658

Abstract Irbesartan is mainly for essential hypertension in patients with hypertension and type 2 diabetic nephropathy。 Currently, irbesartan key intermediate 2-butyl - 1,3-diazaspiro [4,4] non-1-en-4-one synthesized by chemical methods, a large amount of strong acid and alkali, and with there are a lot of salt production, difficult to separation and purification, can also cause certain environmental pollution。 Can be converted to the nitrile hydratase valerolactam 1- cyclopentyl nitrile to the corresponding amide and then hydrolyzed under basic conditions, cyclization Synthesis of 2-butyl - 1,3-diazaspiro [4,4] non-1 en-4-one。 The route milder reaction conditions, specificity avoid pentene amide cyclopentyl based nitrilase catalyzed nitrile hydratase the corresponding carboxylic acid。 Meanwhile, the process reduces the amount of acid solution for more environmentally friendly, in line with the development direction of atom economy and green chemistry。 Currently, as the enzyme nitrile hydratase engineering industrial success stories, mainly for acrylamide and nicotinamide industrial production。 Therefore, the development of nitrile hydratase in fine chemicals intermediates in the synthesis of irbesartan and other applications, has important theoretical significance and application prospects。 In this thesis, the main filter having a catalytic pentene nitrile cyclopentyl amide group to prepare the corresponding amide nitrile hydratase, lay the foundation for the development of post-enzymatic process irbesartan key intermediates。 We screened our laboratory for the nitrile hydratase recombinant library, was found from Pseudonocardia thermophila JCM3095 Silicibacter pomeroyi DSS-3 and the nitrile hydratase exhibit dynamic hydration valerolactam cyclopentyl 1- carbonitrile, but its catalytic lower vitality。 Further, the use of protein homology modeling strategy to build the model S。 pomeroyi DSS-3 nitrile hydratase, after molecular docking study pentene amide groups valeronitrile with a nitrile hydratase binding of substrate binding pocket, for the latter for nitrile hydration enzyme molecule transformation lay a theoretical basis。

毕业论文关键词：腈水合酶； 厄贝沙坦； 筛选； 重组蛋白；分子对接