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醛还原酶和单胺氧化酶的性质研究及分子改造
Alternative TitleCharacterization and Enzyme Enginering of Aldehyde reductase and Monoamine Oxidase
李广悦
Subtype博士
Thesis Advisor朱敦明
2013-01
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword醛还原酶 化学选择性 胺基氧化酶 定向进化 去消旋化反应
Abstract氧化还原酶中具有高度化学选择性的醛还原酶和高度立体选择性的单胺氧化酶在药物中间体和精细化学品的合成中具有广泛的应用前景,然而这两种酶的研究报道还相对较少,因此本文对这两类酶进行了研究探索,以期对这两类酶有更深入的认识并获得具有应用价值的生物催化剂。我们通过基因挖矿的方法从海洋螺杆菌Oceanospirillum sp. MED92筛选到一个功能注释为氧化还原酶的基因,并进行了功能性表达和纯化,该酶命名为OsAR。对该酶的酶学性质研究表明OsAR是一种NADPH依赖性的单体蛋白。该酶对苯甲醛的Km和 kcat分别为 0.89±0.08 mM、11.07±0.99 s?1,而对NADPH分别为 0.04±0.01 mM 、 6.05±1.56 s?1。底物特异性研究表明该酶具有广谱的醛基还原活性,但对酮底物没有活力。对醛酮复合物4-乙酰基苯甲醛和醛酮混合物正己醛和2-壬酮的转化反应表明,该酶是一个高度化学选择性的醛基还原酶,在醛基化学选择性还原中具有潜在的应用价值。在2012年加拿大教授Peter Lau实验室报道了一种细菌来源的单胺氧化酶环己胺氧化酶CHAO,该酶能实现对部分一级胺的去消旋化反应,是一种具有潜在应用价值的生物催化剂。为了进一步挖掘该酶的应用潜力,本实验室与Peter Lau教授实验室合作对CHAO进行了酶工程改造。首先基于CHAO与MAOB的空间结构分析比对,对CHAO进行了定点突变,获得了6个突变体(L199A、 M226A、 Y321A、 Y321F和L353M)。其中两个突变体M226A和L353M对底物的活力分别提高了5–400 %(M226A)和 7–445 %(L353M)。随后,应用突变体L353M结合硼铵去消旋化成功实现了对抗抑郁药物Norsertraline手性前体(R)-1,2,3,4-四氢-1-萘胺的合成,分离产率76%,ee值>99%。尽管该酶及其突变体表现出较高的立体选择性和底物广谱性,但是对大部分二级胺无活力或活力较低不能实现底物转化。二级胺底物喹啉的衍生物是手性胺类药物的重要组成模块,具有重要的应用价值。为了进一步挖掘该酶的应用潜力,我们选择了喹啉衍生物中最简单的2-甲基-1,2,3,4-四氢喹啉作为筛选底物对CHAO进行了定向进化。采用CASTing结合平板显色高通量筛选方法对CHAO进行了三轮进化筛选获得了6个活力提高的突变体(T198F,L199T,M226F,Y459T,T198FL199S,T198FL199SM226F),这些突变体对2-甲基-1,2,3,4-四氢喹啉及其衍生物活力明显提高。通过动力学测定发现,相对于野生型酶6个突变体对2-甲基-1,2,3,4-四氢喹啉的催化效率最高提高了406倍。同时这些突变体仍然保持着高度的立体选择性。随后,应用突变体T198FL199SM226F结合硼铵成功实现了2-甲基四氢喹啉的去消旋化合成,分离产率76%,ee值98%, 展示了这些突变体的应用潜力。对CHAO进行定向进化和高通量筛选获得了一系列有价值的突变体,但是也存在一个明显的缺陷就是只能针对一个或几个底物,无法提高筛选底物的通量。为进一步拓宽底物谱获得有价值突变体同时阐明关键催化位点与底物特异性之间的关系,我们选取了8个关键目标突变位点(198,199,226,321,351,368,422,459)将其突变成性质各不相同的氨基酸,从而构建多样性的突变体库(60个突变体组成)。然后构建多样性的底物库,将突变体库与底物库之间做乘法式的筛选。这些功能位点和不同结构底物催化活力之间的关系可以初步总结出功能位点和底物特异性的内在规律。根据底物特异性的研究结果可知321位点影响着CHAO对底物1-(4-甲氧苄基)-1,2,3,4,5,6,7,8-八氢异喹啉的催化活力,而S-1-(4-甲氧苄基)-1,2,3,4,5,6,7,8-八氢异喹啉是合成药物右美沙芬的重要前体,因此通过定点饱和突变和高通量筛选的方法获得了突变体321I。突变体321I成功实现了去消旋转化合成了S-1-(4-甲氧苄基)-1,2,3,4,5,6,7,8-八氢异喹啉,分离产率为76.5%,ee值为93.5%。这些研究结果表明通过对CHAO的进化和改造可以进一步开发该酶在手性胺合成中的应用。
Other AbstractAmong oxidoreductases, high chemoselective aldehyde reductases and stereoselective monoamine oxidases are very important enzyme catalysts for the synthesis of the intermediates of pharmaceuticals and fine chemicals. However, for these two kinds of enzymes, only few are reported and such study are still in demand. So we initiated the studies on these enzymes with the aim to obtain deep understanding of their catalytic properties and explore the application potentials in organic synthesis.A putative aldehyde reductase gene from Oceanospirillum sp. MED92 was discoveried by gene mining. The enzyme was successfully overexpressed in Escherichia coli and purified using affinity chromatography with a HisTrapTMFF crude column. The recombinant protein (OsAR) was characterized as a monomeric NADPH-dependent aldehyde reductase. The kinetic parameters Km and kcat of OsAR were 0.89±0.08 mM and 11.07±0.99 s?1 for benzaldehyde, 0.04±0.01 mM and 6.05±1.56 s?1 for NADPH, respectively. This enzyme exhibited high activity toward a variety of aromatic and aliphatic aldehydes, but no activity toward ketones. As such, it catalyzed the chemoselective reduction of aldehydes in the presence of ketones, as demonstrated by the reduction of 4-acetylbenzaldehyde or the mixture of hexanal and 2-nonanone, showing the application potential of this marine enzyme in such selective reduction of synthetic importanceIn 2012, Peter Lau et al reported a monoamine oxidase of bacterial origin, cyclohexylamine oxidase (CHAO), which was shown to be a potentially useful catalyst in the deracemization of racemic primary amines. To further explore the properties and application of this enzyme, the enzyme engineering of CHAO was carried out. First, five single-amino acid substitution mutants (L199A, M226A, Y321A, Y321F, and L353M) were created based on superimposition of the tertiary structure of CHAO and the monoamine oxidase (MAO) B homolog. Among the mutant enzymes, M226A displayed an enhanced activity (5–400 %) towards most substrates, and L353M showed 7–445 % higher activity towards primary aliphatic amines with cycloalkane or aromatic moieties. The variant L353M in combination with a borane–ammonia complex as reducing agent was applied to the deracemization of 1-aminotetraline to give the (R)-enantiomer, a precursor of an antidepressant drug Norsertraline, in good yield (76 %) and high ee value (>99%), demonstrating its application potential in chiral amine synthesis.CHAO and obtained mutants showed high stereoselectivity and wide substrate specificity. However, the application of CHAO was limited to the primary amine as it showed little or no activity toward secondary amines. Chiral secondary amines, such as optically active tetrahydroquinoline derivatives, are an important class of building blocks for asymmetric synthesis of pharmaceuticals, agrochemicals and natural products. To expand the application of the cyclohexylamine oxidase (CHAO) in the deracemization of secondary amines, directed evolution of wild-type CHAO was performed and the resulting focused libraries were screened with 2-methyl-1, 2, 3, 4-tetrahydroquinoline as the substrate. Six positive mutants T198F, L199T, M226F, Y459T, T198FL199S and T198FL199SM226F were obtained, which displayed obvious higher catalytic activity for 2-methyl-1, 2, 3, 4-tetrahydroquinoline and other 2-substitued-1, 2, 3, 4-tetrahydroquinolines than wt CHAO. The six mutants showed up to four hundred and six times higher catalytic efficiency than wt CHAO for 2-methyl-1, 2, 3, 4-tetrahydroquinoline. Meanwhile, the mutants also had high stereoselectivity on 2-methyl-1, 2, 3, 4-tetrahydroquinoline and other 2-substitued-1, 2, 3, 4-tetrahydroquinolines. The whole cells expressed T198FL199SM226F mutant efficiently catalyzed the deracemization of 2-methyl-1, 2, 3, 4-tetrahydroquinoline giving (R)-2-methyl-1, 2, 3, 4-tetrahydroquinoline with high isolated yield (76%) and ee value (98%), demonstrating the application potential of muta
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/15501
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
李广悦. 醛还原酶和单胺氧化酶的性质研究及分子改造[D]. 中国科学院研究生院,2013.
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