氨基酸脱氢酶的基因挖掘、定向改造及合成手性氨基酸的研究
发布时间：2019-03-04 21:39
【摘要】：手性氨基酸、氨基醇、氨基腈等手性胺是多种化合物合成的关键中间体,广泛应用于活性药物成分、精细化工品、农药以及染料等领域。手性胺合成的方法包括化学和酶法立体选择性酰化、氧化拆分与不对称氢转移。其中,氨基酸脱氢酶(Amino acid dehydrogenase,AADH)不对称还原潜手性酮酸或酮制备手性胺具有高立体选择性、高效率、反应温和以及环境友好等优势,是合成手性胺的重要途径之一。本研究通过基因挖掘策略,获得了来自克劳氏芽孢杆菌(Bacilllus clausii)和解淀粉芽孢杆菌(Bacilllus amyloliquefaciens)的两个有较高催化活性的氨基酸脱氢酶BcAADH和BaAADH。(1)对BcAADH的酶学性质研究发现:其还原胺化的最适pH为9.5,氧化脱氨的最适pH为10.5;该酶的最适反应温度为30℃,在30、40和50℃条件下的半衰期分别为315、86.6和4.49 h。BcAADH可高效还原芳基酮酸类底物,对苯甲酰甲酸的比活力为17.7 U?mg?1,kcat/Km为5.02 s 1 mmol·L 1。将其与来自巨大芽孢杆菌(Bacillus megaterium)的葡萄糖脱氢酶(Glucose dehydrogenase,GDH)共表达,对共表达重组菌合成L-苯甘氨酸的反应条件进行了优化。在6小时内可将400 mmol·L 1苯甲酰甲酸完全转化为L-苯甘氨酸,e.e.99.9%,底物与酶量的比值高达60 g·g 1,环境因子和酶消耗数分别为4.7和0.018。(2)对BaAADH的酶学性质研究发现:其还原胺化和氧化脱氨的最适pH分别为8.5和10.5;最适反应温度为45℃,在30、40和50℃的半衰期分别为462、117.6和6.17 h。BaAADH对苯甲酰甲酸的比活力为44.5 U·mg 1,kcat/Km为9.8 s 1·mmol·L 1。对五种AADH进行双突变显示,仅BaAADHK68S/N261L具有不对称还原烷基酮类底物的活力。进一步通过同源建模、分子对接和定向进化,使BaAADHK68S/N261L具有还原胺化苯乙酮的催化活性。构建其底物结合口袋的定点饱和突变文库,基于2,4-二硝基苯肼法对构建的突变文库进行高通量筛选。最终获得突变体BaAADHK68S/N261L/L74N和BaAADHK68S/N261L/L76N,对苯乙酮的比活力分别为75.6和46.8 mU·mg 1。综上所述,利用BcAADH可以绿色和高效地制备L-苯甘氨酸;通过定向进化研究鉴定获得BaAADH不对称还原胺化苯乙酮的关键位点L74和L76。本研究为手性非天然氨基酸的合成提供了高效的生物催化剂,为指导氨基酸脱氢酶的分子改造和合成手性芳基胺提供了突变热点并奠定了理论基础。
[Abstract]:Chiral amino acids, amino alcohols, amino nitriles and other chiral amines are key intermediates in the synthesis of various compounds. They are widely used in the fields of active drug components, fine chemicals, pesticides and dyes. The methods of chiral amine synthesis include chemical and enzymatic stereoselective acylation, oxidative resolution and asymmetric hydrogen transfer. Among them, the asymmetric reduction of amino acid dehydrogenase (Amino acid dehydrogenase,AADH) to chiral ketonic acids or ketones has the advantages of high stereoselectivity, high efficiency, mild reaction and environmental friendliness. It is one of the important ways to synthesize chiral amines. In this study, through the strategy of gene mining, Two amino acid dehydrogenase (BcAADH) and two amino acid dehydrogenase (BaAADH.) with high catalytic activity from Bacillus Claus (Bacilllus clausii) and Bacillus amylase (Bacilllus amyloliquefaciens) were obtained and their enzymatic properties on BcAADH were studied. It was found that the reductive amidation of the two amino acid dehydrogenase (BcAADH) and BaAADH. (1) to BcAADH. The optimal pH is 9.5. The optimum pH for oxidative deammoniation was 10.5; The optimum reaction temperature was 30 鈩，
本文编号：2434659
[Abstract]:Chiral amino acids, amino alcohols, amino nitriles and other chiral amines are key intermediates in the synthesis of various compounds. They are widely used in the fields of active drug components, fine chemicals, pesticides and dyes. The methods of chiral amine synthesis include chemical and enzymatic stereoselective acylation, oxidative resolution and asymmetric hydrogen transfer. Among them, the asymmetric reduction of amino acid dehydrogenase (Amino acid dehydrogenase,AADH) to chiral ketonic acids or ketones has the advantages of high stereoselectivity, high efficiency, mild reaction and environmental friendliness. It is one of the important ways to synthesize chiral amines. In this study, through the strategy of gene mining, Two amino acid dehydrogenase (BcAADH) and two amino acid dehydrogenase (BaAADH.) with high catalytic activity from Bacillus Claus (Bacilllus clausii) and Bacillus amylase (Bacilllus amyloliquefaciens) were obtained and their enzymatic properties on BcAADH were studied. It was found that the reductive amidation of the two amino acid dehydrogenase (BcAADH) and BaAADH. (1) to BcAADH. The optimal pH is 9.5. The optimum pH for oxidative deammoniation was 10.5; The optimum reaction temperature was 30 鈩，
本文编号：2434659
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