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烯酮/烯酯还原酶和D-氨基酸脱氢酶的筛选、性质及定向进化
Alternative TitleDiscovery, Characterization and Directed Evolution of Enoate Reductase and D-amino acid dehydrogenase
高秀珍
Subtype博士
Thesis Advisor朱敦明
2013-04-01
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline生物化工
Keyword烯酮/烯酯还原酶   不对称氢化   d-氨基酸脱氢酶   内消旋-二氨基庚二酸脱氢酶   不对称胺化
Abstract在有机合成领域中,采用酶催化代替化学催化进行化合物的制备不仅可以满足绿色发展的需求,而且酶催化所具有的选择性可以用于化学催化不能实现或难以实现的手性化合物的合成。在过去几年中,利用氧化还原酶类制备的光学活性化合物的数量日益增多。在本文的工作中,我们对氧化还原酶类中的烯酮/烯酯还原酶和D-氨基酸脱氢酶进行了相关的研究。 烯酮/烯酯还原酶(Enoate reductase)可以不对称催化还原α,β-不饱和羰基化合物中的碳碳双键,从而形成两个手性中心,所得到的手性化合物在药物、精细化工品以及高级材料的合成中具有广泛的用途。尽管目前开发烯酮/烯酯还原酶用于手性模块的合成上已经取得了较大的进展,但是这一家族酶的应用潜能尚未被完全挖掘,难点仍在于针对于某一目标化合物尚不能很快找到合适的烯酮/烯酯还原酶进行催化反应,因此需要进一步扩展这一工具箱。为了扩展该类酶的酶种,在本文的工作中,我们对来源于Lactobacillus casei str. Zhang的古老黄色酶(LacER)基因克隆到pET21a(+)表达载体中,并在Escherichia coli BL21(DE3)中表达,表达后的蛋白经硫酸铵沉淀和酸性缓冲液处理的方法进行了纯化。对纯化的蛋白进行了生化性质的分析。鉴定发现,LacER为NADH依赖型的烯酮/烯酯还原酶。当以2-环己烯酮为底物时,LacER对NADH的Km和kcat分别是0.034 ± 0.006 mM和3.2 ± 0.2 s-1;而其对环己烯酮的Km和kcat分别是1.90 ± 0.04 mM和8.4 ± 0.2 s-1。酶学性质分析确定,LacER的最适pH为8.0-9.0,最适反应温度50-55 °C,在60 °C以下时较为稳定。底物谱分析发现LacER对脂肪族的烯醛/酮及α,β-不饱和环酮表现出较高的活性。LacER对(R)-香芹酮的转化率大于99%,对产物的非对映立体选择性(de值)高达98%,这一结果表明了LacER在不对称合成方面所具有的应用潜能。 内消旋-二氨基庚二酸脱氢酶(meso-diaminopimelate dehydrogenase,meso-DAPDH)是一类特殊的D-氨基酸脱氢酶,催化可逆的内消旋-二氨基庚二酸的D-手性中心氧化脱氨生成L-2-氨基-6-氧代庚酸。在我们的工作中,我们对来源于Symbiobacterium thermophilum IAM14863的meso-DAPDH的编码基因在E. coli BL21(DE3)中进行了表达,并对目的蛋白进行了纯化。除了对天然底物内消旋-二氨基庚二酸表现出活性外,该酶对D-丙氨酸、D-缬氨酸和D-赖氨酸也具有氧化脱氨的活性。更令人兴奋的是,该酶可以还原胺化一系列2-酮酸底物,例如以丙酮酸为底物生成D-丙氨酸,转化率和ee值均高达99%。目前报道的meso-DAPDH专一催化内消旋-二氨基庚二酸,仅对某些2-酮酸或羊毛硫氨酸表现出微弱的活性,因此,该酶是目前发现的第一个具有D-氨基酸生成能力的天然的内消旋-二氨基庚二酸脱氢酶。酶学性质分析表明,该酶是目前报道的同家族中耐热性最好的酶。鉴于该酶较好的耐热性及宽松的底物谱,我们通过氨基酸序列比对确定了该酶底物L-手性中心周围的氨基酸残基,Phe146、Thr171、Arg181和His227,并对四个位点分别进行定点饱和突变,以期提高该酶对大位阻底物,如苯基丙酮酸的还原胺化活性。以苯基丙酮酸为底物对突变文库进行筛选后,得到了T171P、T171S、R181F、H227C、H227V五个活性明显提高的突变子,其中H227V对苯基丙酮酸的比活为2.39 ± 0.06 U?mg-1,相对野生型提高了35.1倍,且所有突变体对D-苯丙氨酸的ee值均大于99%。对阳性突变子进行组合突变后,活性并没有提高。对所有突变体及野生型进行了动力学参数的分析发现,突变的引入并没有引起酶对底物亲和力(Km)的改变,突变体活性的提高则是由酶转化数(kcat)提高导致的。对H227V和WT进行分子动力学模拟表明,突变后底物Cα与NADP+的烟酰胺环上的C4'之间的原子距离由4.8 ?缩短到3.6 ?,与活性分析结果一致。这一结果表明,底物L-手性中心周围的氨基酸残基的类型影响了底物的范围及酶活性,但是不影响反应的立体选择性。这些结果表明,在保证优异的立体选择性的前提下,我们有希望通过对StDAPDH的改造获得对不同底物有较好活性的D-氨基酸脱氢酶。
Other AbstractEnzymatic catalysis is increasingly recognized by organic chemists, because of its mild conditions and excellent specificity. Many successful reduction and oxidation processes have been realized by using oxidoreductases (E.C.1.x.x.x) . In our study, enoate reductase and D-amino acid dehydrogenase were targeted for exploitation. Carbon-carbon double bond of α, β-unsaturated carbonyl compounds can be reduced by enoate reductase (ER) (E.C.1.3.1.x) under mild reaction conditions with high chemo-, regio- and stereoselectivity, which is an important reaction in fine chemical synthesis. A putative enoate reductase gene from Lactobacillis casei str. Zhang was cloned into pET-21a(+) and expressed in Escherichia coli BL21 (DE3) host cells. The encoded enzyme (LacER) was purified by ammonium sulfate precipitation and treatment in an acidic buffer. This enzyme was identified as a NADH-dependent enoate reductase, which had a Km of 0.034 ± 0.006 mM and kcat of 3.2 ± 0.2 s-1 toward NADH using 2-cyclohexen-1-one as the substrate. Its Km and kcat toward substrate 2-cyclohexen-1-one were 1.94 ± 0.04 mM and 8.4 ± 0.2 s-1, respectively. The enzyme showed a maximum activity at pH 8.0-9.0. The optimum temperature of the enzyme was 50-55?C, and LacER was relatively stable below 60?C. The enzyme was active toward aliphatic alkenyl aldehyde, ketones and some cyclic anhydrides. Substituted groups of cyclic ?,?-unsaturated ketones and its ring size have positive or negative effects on activity. (R)-(-)-Carvone was reduced to (2R, 5R)-dihydrocarvone with 99% conversion and 98% (de) stereoselectivity, indicating a high synthetic potential of LacER in asymmetric synthesis. meso-Diaminopimelate dehydrogenase (meso-DAPDH, EC1.4.1.16) is a NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on the D-configuration of meso-2, 6-diaminopimelate to produce L-2-amino-6-oxopimelate. In this study, a meso-DAPDH from Symbiobacterium therophilum was cloned and expressed in E. coli. In addition to the native substrate meso-2, 6-diaminopimelate, the purified enzyme also showed activity toward D-alanine, D-valine, D-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generate D-amino acids in up to 99% conversion and 99% enantiomeric excess. Since meso-DAPDHs are known to be specific to meso-2, 6-diaminopimelate, this is the first wild-type meso-DAPDH with relaxed substrate specificity and potential for D-amino acid synthesis. The enzyme was the most stable meso-diaminopimelate dehydrogenase reported until now. Because of the high thermostability and relaxed substrate profile of S. therophilum mes-DAPDH, amino acid residues interacting with the L-center of meso-DAPDH, Phe146, Thr171, Arg181 and His227, were selected for site-saturation mutagenesis in an effort to enlarge the substrate binding site for accommodating larger 2-keto acids. By enzymatic activity screening with phenylpyruvic acid as the substrate, several mutants were obtained with improved activities. Among them, the single mutation H227V had a specific activity of 2.39 ± 0.06 U?mg-1, which was 35.1-fold enhancement over wild type enzyme. The positive mutants were combined to investigate the synergetic effects of different sites, but the resulting double and triple variants did not show higher activity. All the mutant enzymes did not show significant difference in Km comparing with wild type, but the kcat values ranged from about 1.10 to 3.98 s-1. Molecular dynamics simulations revealed that the distance between the Cα of imine intermediate and the C4' of NADPH nicotinamide ring in the wild type enzyme (4.8 ?) was longer than the mutant H227V (3.6 ?), consistent with the higher activity of H227V. These results suggested that efficient D-amino acid dehydrogenases for different substrates could be tailor-made by engineering of the meso-DAPDH from S. thermophilum while keeping the excellent stereospecificity.
Pages104
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/8302
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
高秀珍. 烯酮/烯酯还原酶和D-氨基酸脱氢酶的筛选、性质及定向进化[D]. 北京. 中国科学院研究生院,2013.
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