羧酸、醛和醇广泛应用于食品、医药和精细化学品等众多领域。卤化物等可以方便地制备腈化合物，腈化合物经水解、还原反应可以获得增加一个碳原子的羧酸、醛和醇。因此，腈水解和羧酸还原是由卤化物等制备增加一个碳原子的羧酸、醛和醇的关键步骤，也是有机合成中两类非常重要的化学反应。本文研究了腈水解酶催化潜手性二腈的去对称水解反应和羧酸还原酶催化羧酸的还原反应，为实现从腈化合物到羧酸、醛和醇的生物合成奠定了基础。γ-氨基丁酸类药物如加巴喷丁、普瑞巴林、巴氯芬等在临床上广泛用于治疗癫痫和骨骼肌松弛等多种神经疾病。以潜手性3-取代戊二腈为原料，经去对称水解、库尔提斯重排和酸水解可以有效合成这类物质。然而，腈化合物的传统化学水解需要强酸或强碱，无法去对称水解潜手性3-取代戊二腈生成相应的、光学纯的单腈单酸。本文以潜手性3-异丁基戊二腈（2.1a）和3-(4-氯苯基)戊二腈（2.1b）为底物对9个已报道的和5个尚未报道的腈水解酶进行筛选。发现腈水解酶HsNIT、BjNIT6402和SsNIT能够去对称水解2.1a和2.1b生成(S)-3-(氰甲基)-5-甲基己酸[(S)-2.2a]和(S)-3-(4′-氯苯基)-4-氰基丁酸[(S)-2.2b]，具有高的转化率和ee值（≥90%）。在这些酶中，仅AtNIT3能够催化2.1b生成(R)-2.2b（ee值为78%）。系统研究了AtNIT3、BjNIT6402和HsNIT对一些3-取代戊二腈的催化活力和立体选择性，发现酶的催化活力和立体选择性受底物3-取代基的影响。选择BjNIT6402和HsNIT分别用于2.1a和2.1b的制备级水解，生成产物(S)-2.2a（收率为93%）和(S)-2.2b（收率为80%）；(S)-2.2a和(S)-2.2b分别经过库尔提斯重排和酸水解合成(S)-普瑞巴林（ee值为89%）和(R)-巴氯芬（ee值为99%），从而建立了一种生物催化与化学反应融合合成普瑞巴林和巴氯芬等3-取代γ-氨基丁酸衍生物类药物的新方法。羧酸的化学还原需要强还原剂，操作危险，反应难以停留至中间产物醛（生成产物醇）；羧酸的酶法还原目前研究很少，是当前生物催化技术的难题之一，已确证的羧酸还原酶屈指可数，其底物谱信息更是缺乏，这极大地限制了它的应用。本研究筛选、确证两个新的羧酸还原酶（Segniliparus CAR和Mycobacterium CAR）。它们与已报道的Nocardia CAR有相似的反应机理，即翻译后的磷酸泛酰巯基乙胺基化修饰和辅因子Mg2+、ATP、NADPH是酶促反应所必须的。这两个酶均能够高效地还原一系列脂肪族和芳香族羧酸生成相应的醛。尤其是，Segniliparus CAR能有效地还原4-羟基苯甲酸（3.8a）和4-硝基苯甲酸（3.19a），而Mycobacterium CAR能更高效地还原布洛芬（3.33a），尚没有报道羧酸还原酶能有效地还原这三个底物。它们对含有C=C或C=O双键的羧酸有良好的化学选择性，而传统的化学还原反应难以实现这样的化学选择性。共表达Segniliparus CAR和已报道的Nocardia PPTase的E. coli全细胞[E. coli BL21(DE3)/pETDuet-1-PPTase-CAR]分别催化香草酸（3.20a）和3,4-二羟基苯乙酸（3.25a）得到香草醇（3.20c，产率为69%，时空产率为18.6 mg·L-1·h-1）和3,4-二羟基苯乙醇（羟基酪醇，3.25c，产率为42%，时空产率为11.3 mg·L-1·h-1），明显优于当前全细胞催化还原3.25a制备3.25c的文献报道（产率为19%，时空产率为2 mg·L-1·h-1）。推测宿主细胞内在的醛还原酶进一步还原羧酸还原酶催化生成的产物醛使全细胞催化的终产物为相应的醇。本部分的研究工作丰富了羧酸还原酶种类和底物谱等信息，为它的应用奠定了基础。另外，将Mycobacterium CAR催化的羧酸还原反应与Wittig反应耦合，得到了碳链延长两个碳原子的α,β-不饱和酸酯，从而建立了一种新的合成这类化合物的酶-化学方法。本研究工作中实现了腈水解酶和羧酸还原酶偶联催化苯甲腈、苯乙腈和苯基丙腈来制备苯甲醇、苯乙醇和苯丙醇，为进一步实现高附加值的腈化合物至（醛）醇的生物催化（比如潜手性的二腈经过酶促串联反应合成手性醇）奠定了基础。在该生物催化中，羧酸的还原（羧酸还原酶催化）非常缓慢，是主要障碍。针对羧酸还原酶的活力和稳定性的定向进化和其辅因子的供给是当前面临的主要问题，仍需要进一步研究。 

Carboxylic acids, aldehydes and alcohols are used widely in many fields, such as food, fine chemical and pharmaceutical industry. Alkyl halides are easily converted into nitriles and these can be hydrolyzed to carboxylic acids with extension of one carbon atom (compared with alkyl halides). These carboxylic acids can get further reduced to aldehydes and alcohols. Therefore, nitrile hydrolysis and carboxylic acid reduction are key steps in the conversion of alkyl halides to carboxylic acids, aldehydes and alcohols with extension of one carbon atom. They are also two kinds of important chemical reactions in organic synthesis. This paper gives a general study on desymmetrization of prochiral dinitriles catalyzed by nitrilases and the reduction of carboxylic acids catalyzed by carboxylic acid reductases (CAR), which provides guidance for the conversion of nitriles to carboxylic acids, aldehydes and alcohols. γ-Aminobutyric acid (GABA) analogues, such as Gabapentin, Pregabalin and Baclofen, have been used widely for treatment of a variety of neurological diseases, such as epilepsy and skeletal muscle relaxation. Prochiral 3-substituted glutaronitriles as raw materials are desymmetrized by enantioselective hydrolysis and a straightforward Curtius rearrangement of hydrolysis products, followed by the acidic hydrolysis, afford these analogues effectively. However, these corresponding optically active cyanocarboxylic acid derivatives are obtained by enantioselective hydrolysis and this cannot be achieved using traditional chemical hydrolysis, which is caused by strong acids or bases. In this study, nine reported and five new nitrilases were screened with prochiral 3-isobutylglutaronitriles (2.1a) or 3-(4′-chlorophenyl)glutaronitriles (2.1b) as the substrate. Nitrilases HsNIT, BjNIT6402 and SsNIT were found to catalyze the desymmetric hydrolysis of 2.1a and 2.1b to form optically active (S)-3-(cyanomethyl)-5- methylhexanoic acid [(S)-2.2a] and (S)-3-(4′-chlorophenyl)-4-cyanobutanoic acid [(S)-2.2b] with high conversions and enantiomeric excesse (ee, >90%), respectively. Among them, only AtNIT3 catalyzed the desymmetric hydrolysis of 2.1b to form (R)-2.2b (78% ee). We studied systematically the activities and stereoselectivities of these three nitrilases (AtNIT3, BjNIT6402 and HsNIT) toward some 3-substituted glutaronitriles. It was found that the 3-substituent of the substrates exerted great effect on the enzyme activity and stereoselectivity. BjNIT6402 and HsNIT were selected for further enzymatic desymmetrization of 2.1a and 2.1b at a preparative scale, respectively. (S)-2.2a and (S)-2.2b were isolated in 93% and 80% yields, respectively. They were converted to (S)-Pregabalin and (R)-Baclofen with 89% and 99% ee, respectively, via the Curtius rearrangement followed by acidic hydrolysis, which established a novel methodology using biological catalysis coupled with chemical reaction for the synthesis of optically active GABA analogues, such as Pregabalin and Baclofen. The chemical reduction of carboxylic acids requires strong reducing agent, which made for hazardous operations. And it is difficult to obtain aldehydes formed as intermediate products (alcohols easily obtained). Currently, biocatalytic reduction of carboxylic acids is studied rarely, and it is one of the difficult problems in the biocatalytic technology. Only a few CARs have been biochemically characterized, and the substrate profiles of the known CARs are also limited. This greatly limits its application. Two new CARs (Segniliparus CAR and Mycobacterium CAR) were identified in this study. Their reaction mechanisms were similar to that of the known Nocardia CAR. The post-translational phosphopantetheinylation and Mg2+, ATP, and NADPH as cofactors were necessary for their enzymatic reductions. They exhibited high activity toward a variety of aromatic and aliphatic carboxylic acids. Especially, Segniliparus CAR could effectively reduced 4-hydroxybenzoic acid (3.8a) and 4-nitrobenzoic acid (3.19a), and Mycobacterium CAR could effectively reduced ibuprofen (3.33a). None of the known CARs was reported to reduce these three substrates effectively. They showed good chemoselectivity for the reduction of some carboxylic acids containing C=C or C=O double bonds. It's hard to achieve the chemoselectivity using traditional chemical reduction. The recombinant E. coli cells co-expressing the Segniliparus CAR and the known Nocardia PPTase genes catalyzed the reductions of vanillic acid (3.20a) and 3,4-dihydroxyphenylacetic acid (3.25a) to give vanillyl alcohol (3.20c, 69% yield, 18.6 mg·L-1·h-1 productivity) and 3-hydroxytyrosol (3.25c, 42% yield, 11.3 mg·L-1·h-1 productivity), respectively. This was obviously higher than the currently reported synthesis of 3.25c (19% yield, 2 mg·L-1·h-1 productivity) through the reduction of 3.25a using whole-cell biotransformation. Aldehyde reductases in host cells were proposed to be responsible for the further reduction of the aldehydes, which were obtained from the reduction of carboxylic acids catalyzed by Segniliparus CAR. So the corresponding alcohols was obtained in whole-cell biotransformation. This study increases the number of CARs and the information of their substrate profiles, which provides guidance for their applications. In addition, Mycobacterium CAR catalyzed carboxylic acid reduction to give aldehydes, followed by a Wittig reaction to afford the products α,β-unsaturated esters with extension of two carbon atoms, demonstrating a new chemo-enzymatic method for the synthesis of these important compounds.In this study, nitrilases coupled with CARs catalyzed benzonitrile, phenylacetonitrile and 3-phenylpropanenitrile to give benzyl alcohol, phenethyl alcohol and 1-phenyl-1-propanol, respectively. It might provide a foundation for further study on some valuable examples about biotransformation of nitriles to aldehydes/alcohols, such as the conversion of prochiral dinitriles to chiral alcohols. The reduction of carboxylic acid catalyzed by CARs was very slow, which was a major obstacle. Directed evolution for improving enzyme (CAR) stability and stabilization and the supply of cofactors (ATP/NADPH) still need further study.