酶促合成手性氨基酸的研究进展

doi:10.12211/2096-8280.2024-015

摘要/Abstract

摘要：

手性氨基酸是一类重要的高价值化学品，广泛应用于食品、医药、化工、农药等多个领域。手性氨基酸常用的制备方法可以分为四类，包括化学合成、蛋白质水解、发酵和酶促合成。其中，酶促合成手性氨基酸以其反应条件温和、立体选择性高、步骤简单、应用范围广等优势备受关注。近年来，得益于生物信息学和蛋白质工程等技术的快速发展，大量性能优异的酶制剂被开发，并成功应用于多种手性氨基酸的制备。本文重点综述了酶促不对称合成和去消旋化合成两种路径在手性氨基酸合成中的应用，包括关键酶制剂氨基酸脱氢酶、转氨酶、氨裂解酶、醛缩酶、氨基酸氧化酶、氨基酸脱氨酶等的开发与改造，及其在草铵膦、叔亮氨酸、西格列汀中间体等高价值手性氨基酸合成中的应用。同时，总结了酶促合成手性氨基酸领域面临的主要困境，如关键酶元件缺乏，以及野生酶非对映体选择性低、底物谱窄、催化活性低、稳定性差、反应条件局限等。最后，展望了自动化实验装置、机器学习和人工智能等前沿技术在酶改造领域的应用，以及通过反应器设计和反应过程控制，开发更为高效和环境友好的催化工艺，推动酶促合成手性氨基酸技术更广泛的工业应用。

关键词: 高价值手性化学品, 手性氨基酸, 不对称合成, 去消旋化合成, 蛋白质工程, 多酶级联

Abstract:

Chiral amino acids represent a crucial class of chiral building blocks with significant value in food, medicine, chemical industry, and agriculture. The market scale of pharmaceuticals, pesticides, food, and chemical industries relying on chiral amino acids is substantial and has been attracting increasing attention. The pursuit of efficient, environmentally friendly, and cost-effective synthesis of chiral amino acids has long been a goal for scientists. Commonly used preparation methods for chiral amino acids fall into four categories: protein hydrolysis, fermentation, chemical synthesis, and enzyme-catalyzed synthesis. Among these, enzyme-catalyzed synthesis has demonstrated great potential due to its mild reaction conditions, high stereo-selectivity, simplicity of steps, and wide application range. In recent years, with the rapid development of bioinformatics, protein engineering, and computational biology, there has been an increasing number of high-performance enzyme preparations developed, leading to a steady increase in the diversity of enzymes and the gradual diversification of catalyzed reactions, further promoting the wide application of enzyme-catalyzed synthesis of chiral amino acids. The enzyme-catalyzed synthesis of chiral amino acids can be categorized into three groups: asymmetric synthesis, deracemization synthesis, and kinetic resolution. Kinetic resolution, due to its theoretical yield of only 50% and low atom economy, is not suitable for industrial applications. In contrast, asymmetric synthesis and deracemization synthesis with theoretical yield of 100% find wider industrial application. This article reviews the application of enzymatic asymmetric synthesis and deracemization synthesis in the synthesis of chiral amino acids. It includes the development and modification of key enzyme such as amino acid dehydrogenase, transaminase, ammonia lyase, aldolase, amino acid oxidase, and amino acid deaminase, as well as their application in the synthesis of high-value chiral amino acids such as phosphinothricin, tert-leucine, and intermediate of sitagliptin. Additionally, it summarizes the main challenges faced in the field of enzymatic synthesis of chiral amino acids, such as the lack of key enzyme components, and low enantioselectivity, narrow substrate spectra, low catalytic activity, poor stability, limited reaction conditions of wild-type enzymes. Finally, it looks ahead to the application of cutting-edge technologies such as automated experimental devices, machine learning, and artificial intelligence in the field of enzyme modification, as well as the development of more efficient and environmentally friendly catalytic processes through reactor design and reaction process control. These endeavors collectively aim to facilitate the broader industrial application of enzymatic synthesis for chiral amino acids.

Key words: High-value chiral chemicals, Chiral amino acids, Asymmetric synthesis, Deracemization synthesis, Protein engineering, Multi-enzymatic cascade

中图分类号:

Q814

王子渊, 杨立荣, 吴坚平, 郑文隆. 酶促合成手性氨基酸的研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-015.

Ziyuan WANG, Lirong YANG, Jianping WU, Wenlong ZHENG. A review on enzyme-catalyzed synthesis of chiral amino acids.[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-015.

图/表 33

图1 手性氨基酸的应用

Fig. 1 The application of chiral amino acids.

表1 酶促合成手性氨基酸的三种常用方法比较

Table 1 Comparison of three common methods for enzyme-catalyzed synthesis of chiral amino acids

方法

Methods

酶制剂

Enzyme

底物

Substrate

立体选择性

Stereosele-ctivity

理论产率

Theoretical yield

原子经济性

Atomic economy

典型案例

Typical examples

Asymmetric synthesis

Amino acid dehydrogenase,

Transaminase,

Ammonia lyase,

Amino mutase,

Aldolase,

Hydroxymethyltransferase,

etc.

Keto acids,

α, β-unsaturated carboxylic acids,

Amino acids and Aldehydes

High

100%

High

L-tert-Leucine ^[11-12]，

(R)-3-Amino-4-(2,4,5-trifluorophenyl)butyric acid^[13]

Racemization synthesis

Amino acid dehydrogenase,

Transaminase,

Ammonia lyase,

Amino mutases, Aldolase,

Hydroxymethyltransferase,

Amino acid oxidase,

Amino acid deaminase,

Amino acid racemase,

etc.

L-Phosphinothricin^[14-15]，

L-Phenylglycine ^[16]

Dynamic kinetic resolution

Amino acid oxidase,

Amino acid deaminase,

Amino acid dehydrogenase,

Amino acid racemase,

etc.

图2 氨基酸脱氢酶（AADH）催化酮酸不对称胺化

Fig. 2 Asymmetric reductive amination of keto acids by amino acid dehydrogenases (AADH)

图3 谷氨酸脱氢酶结构（A）来源于枯草芽孢杆菌的谷氨酸脱氢酶晶体结构（PDB ID： 3K92）；（B）来源于枯草芽孢杆菌的谷氨酸脱氢酶的单亚基结构；（C）谷氨酸脱氢酶的“开放”与“封闭”结构

Fig. 3 The structure of glutamate dehydrogenase (A) The crystal structure of glutamate dehydrogenase from Bacillus subtilis (PDB ID: 3K92); (B) The single subunit structure of glutamate dehydrogenase from Bacillus subtilis; (C) The "open" and "close" structure of glutamate dehydrogenase

图4 氨基酸脱氢酶催化机理

Fig. 4 The catalytic mechanism of amino acid dehydrogenases

图5 利用L-苏氨酸合成L-2-氨基丁酸的多酶级联体系（TD-苏氨酸脱氨酶，LeuDH-亮氨酸脱氢酶）

Fig. 5 Multi-enzymatic cascade system for synthesizing L-2-aminobutyric acid from L-threonine (TD-Threonine deaminase, LeuDH-Leucine dehydrogenase)

图6 转氨酶（TA）催化氨基不对称转移反应

Fig. 6 Asymmetric transfer of amino groups to keto acids by transaminase (TA)

图7 转氨酶（TA）催化反应一般通式

Fig. 7 General catalytic reaction formula for transaminases (TA)

图8 转氨酶晶体结构（A）来源于Bacillus megaterium的（S）-选择性酶晶体结构（PDB ID：5G09）（B）来源于Aspergillus fumigatus的（R）-选择性酶晶体结构（PDB ID：4UUG）

Fig. 8 The crystal structure of transaminase (A) The crystal structure of (S)-transaminase from Bacillus megaterium (PDB ID: 5G09); (B) The crystal structure of (R)-transaminase from Aspergillus fumigatus (PDB ID: 4UUG)

图9 转氨酶催化机理

Fig. 9 The catalytic mechanism of transaminase

图10 氨基不对称转移反应合成手性α-氨基酸（A）级联支链氨基酸转氨酶（BCAT）和鸟氨酸转氨酶（OrnAT）合成手性α-氨基酸；（B）级联转氨酶（TA）、谷氨酸脱氢酶（GluDH）和醇脱氢酶（ADH）合成L-草铵膦（L-PPT）；（C）基于丙氨酸脱氢酶（ALD）-甲酸脱氢酶（FDH）胺循环系统，级联苏氨酸脱氨酶（TD）和TA合成L-2-氨基丁酸；（D）级联化学法与天冬氨酸转氨酶（AspAT）合成L-3，4-二甲氧基苯丙氨酸

Fig. 10 Synthesis of chiral α-amino acids by asymmetric transfer of amino groups to keto acids (A) Cascade synthesis of chiral α-amino acids by branched-chain amino acid aminotransferase (BCAT) and ornithine aminotransferase (OrnAT); (B) Cascade synthesis of L-phosphinothricin (L-PPT) by transaminase (TA), glutamate dehydrogenase (GluDH), and alcohol dehydrogenase (ADH); (C) Based on the alanine dehydrogenase (ALD) - formate dehydrogenase (FDH) amine cycle system, cascade synthesis of L-2-aminobutyrate by threonine deaminase (TD) and TA; (D) Chemical methods are coupled with aspartate transaminase (AspAT) to synthesize L-3,4-dimethoxyphenylalanine.

图11 氨基不对称转移反应合成西格列汀中间体（TA-转氨酶）

Fig. 11 Synthesis of intermediate of sitagliptin by asymmetric transfer of amino groups to keto acids (TA-transaminase)

图12 氨基不对称转移反应合成非α-手性氨基酸（A）偶联不同的转氨酶（TA）合成（S）-4-氨基戊酸；（B）级联腈水解酶和ω-TA合成（S）-β-苯丙氨酸

Fig. 12 Synthesis of chiral non-α-amino acids by asymmetric transfer of amino groups to keto acids (A) Coupling different transaminases (TA) to synthesize (S)-4-aminopentanoic acid; (B) Cascade synthesis of (S)-β-phenylalanine by nitrilase and ω-TA

图13 解氨酶（AL）或氨基变位酶（AM）催化α，β-不饱和羧酸的选择性胺化加成反应

Fig. 13 Enantioselective addition of ammonia to α,β-unsaturated acids by ammonia lyase (AL) or amino mutase (AM)

图14 用于手性氨基酸合成的解氨酶的催化反应一般通式（DAL-天冬氨酸解氨酶，MAL-甲基天冬氨酸解氨酶，PAL-苯丙氨酸解氨酶，HAL-组氨酸解氨酶，TAL-酪氨酸解氨酶）

Fig. 14 General catalytic reaction formula for ammonia lyases used for chiral amino acid synthesis (DAL-Aspartate ammonia-lyase, MAL-Methylaspartate ammonia-lyase, PAL-Phenylalanine ammonia-lyase, HAL-Histidine ammonia-lyase, TAL-Tyrosine ammonia-lyase.)

图15 解氨酶催化机制（A）烯醇机制（B）MIO电子受体机制

Fig. 15 The catalytic mechanism of transaminases of ammonia-lyase (A) enolate mechanism. (B) MIO electrophile mechanism

图16 苯丙氨酸解氨酶（PAL）催化α，β-不饱和羧酸选择性胺化（A）多种芳香族L-氨基酸；（B）EMA401中间体

Fig. 16 Enantioselective addition of ammonia to α,β-unsaturated acids by phenylalanine ammonia lyase (PAL) (A) Various aromatic L-amino acids; (B) EMA401 intermediate.

图17 计算重设计天冬氨酸解氨酶（DAL）用于合成多种非天然氨基酸

Fig. 17 Computational redesign of aspartate ammonia lyase (DAL) for the synthesis of several unnatural amino acids

图18 甲基天冬氨酸裂解酶（MAL）催化α，β-不饱和羧酸选择性胺化（A）维生素B5衍生物（B）L-多巴（ADC-天冬氨酸-α-脱羧酶，CrpG-β-甲基天冬氨酸-α-脱羧酶，GAD-谷氨酸脱羧酶，PS-泛酸盐合成酶）

Fig. 18 Enantioselective addition of ammonia to α,β-unsaturated acids by Methylaspartate ammonia lyases (MAL) (A) Vitamin B5 derivatives (B) L-dopa (ADC-aspartate-α-decarboxylase, CrpG-β-methylaspartate-α-decarboxylase, GAD-glutamate decarboxylase, PS-pantothenate synthetase)

图19 醛缩酶或羟甲基转移酶（HMT）催化氨基酸的醛缩反应

Fig. 19 Aldol condensation of an amino acid to aldehydes by aldolase or hydroxymethyltransferase (HMT)

图20 苏氨酸醛缩（TA）酶催化反应一般通式

Fig. 20 General catalytic reaction formula for threonine aldolase (TA)

图21 苏氨酸醛缩酶晶体结构（A）来源于Aeromonas jandaei的L-苏氨酸醛缩酶晶体结构（PDB ID： 3WGB）及活性中心；（B）来源于Achromobacter xylosoxidans的D-苏氨酸醛缩酶晶体结构（PDB ID： 4V15）及活性中心

Fig. 21 The crystal structure of threonine aldolase (A) The crystal structure of L-threonine aldolase from Aeromonas jandaei (PDB ID: 3WGB); (B) The crystal structure of D-threonine aldolase from Achromobacter xylosoxidans (PDB ID: 4V15)

图22 苏氨酸醛缩酶催化机理（A）L-苏氨酸醛缩酶催化机理；（B）D-苏氨酸醛缩酶催化机理

Fig. 22 The catalytic mechanism of threonine aldolase (A) The catalytic mechanism of L-threonine aldolase; (B) The catalytic mechanism of D-threonine aldolase

图23 “路径假说”的示意图。醛类（MTB）通过顺式路径或反式路径攻击醛胺PLP-Gly的Cα形成相应的产品构型［92］。

Fig. 23 Schematic diagram of the path hypothesis. Aldehydes (MTB) attack Cα of aldimine PLP-Gly through the syn path or anti path to form the corresponding configuration of products[92].

图24 丝氨酸羟甲基转移酶（SHMT）酶催化反应一般通式

Fig. 24 General catalytic reaction formula for Serine hydroxymethyltransferase (SHMT)

图25 去消旋化合成（A）多酶级联去消旋化合成；（B）化学-酶法去消旋化合成

Fig. 25 Deracemization synthesis (A) Multi-enzymatic deracemization synthesis; (B) Chemo-enzymatic deracemization synthesis

图26 氨基酸氧化酶（AAO）催化反应一般通式

Fig. 26 General catalytic reaction formula for Amino acid oxidase (AAO)

图27 氨基酸氧化酶参与去消旋化合成手性氨基酸（A）L-草铵膦；（B）L-2-氨基丁酸。（AAO-氨基酸氧化酶，AADH-氨基酸脱氢酶，CAT-过氧化氢酶，TA-转氨酶）

Fig. 27 Amino acid oxidase are involved in in the deracemization synthesis of chiral amino acids (A) L-phosphinothricin; (B) L-2-aminobutyric acid. (AAO-amino acid oxidase, AADH-amino acid dehydrogenase, CAT-catalase, TA-transaminase)

图28 L-氨基酸脱氨酶（L-AAD）催化反应一般通式

Fig. 28 General catalytic reaction formula for L-Amino acid deaminase (L-AAD)

图29 氨基酸脱氨酶参与去消旋化合成手性氨基酸（AAD-氨基酸脱氨酶，TA-转氨酶）

Fig. 29 Amino acid deaminase are involved in in the deracemization synthesis of chiral amino acids (AAD-amino acid deaminase, TA-transaminase)

图30 氨基酸脱氢酶参与去消旋化合成手性氨基酸（A）D-丙氨酸；（B）L-苯甘氨酸（ALADH-丙氨酸脱氢酶，TA-转氨酶，MR-桃酸消旋酶、DMDH-D-扁桃酸脱氢酶，LeuDH-亮氨酸脱氢酶）

Fig. 30 Amino acid dehydrogenase are involved in in the deracemization synthesis of chiral amino acids (A) D-alanine; (B) L-phenylglycine. (ALADH-alanine dehydrogenase, TA-transaminase, MR-mandelate racemase, DMDH- D-mandelate dehydrogenase, LeuDH-leucine dehydrogenase.)

图31 化学酶法去消旋化合成手性氨基酸（A）D-氨基酸；（B）D-苯丙氨酸（AAO-氨基酸氧化酶，PAL-苯丙氨酸解氨酶，AAD-氨基酸脱氨酶）

Fig. 31 Chemo-enzymatic deracemization synthesis of chiral amino acids (A) D-Amino acids; (B) D-Phenylalanine (AAO-amino acid oxidase, PAL-phenylalanine ammonia-lyase, AAD-amino acid deaminase.)

表2 部分应用于生产实践的酶促合成路线

Table 2 Partially applied enzymatic synthetic routes in production

产品 Products	应用 Applications	合成路线 Synthetic routes	酶制剂 Enzyme	参考文献 References
L-phosphinothricin	Broad-spectrum herbicides	Asymmetric reductive amination of keto acids	Glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[22,28,35]
L-phosphinothricin	Broad-spectrum herbicides	Deracemization synthesis	D-amino acid oxidase, catalase, glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[14-15]
L-tert-leucine	Intermediate of azanavir, animal feed additive, nutritional fortifier	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[11-12, 30,32]
L-tert-leucine		Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
L-2-aminobutyric acid	Intermediate of antituberculosis ethambutol and antiepileptic drug levetiracetam	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，threonine deaminase，Glucose dehydrogenase	[33-34]
		Asymmetric transfer of amino groups to keto acids	Transaminase，Glutamate dehydrogenase，Alcohol dehydrogenase	[63]
		Deracemization synthesis	D-amino acid oxidase, ω- Transaminase	[118]
L- phenylglycine	Intermediate of β-lactam antibiotics	Asymmetric reductive amination of keto acids	Amino acid dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[29, 36-37]
L- phenylglycine	Intermediate of β-lactam antibiotics	Deracemization synthesis	Mandelate racemase, D-mandelate dehydrogenase, Leucine dehydrogenase	[16]
(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid	Intermediate of siagliptin	Asymmetric transfer of amino groups to keto acids	Transaminase	[13]
L-norvaline	Intermediate of perindopril	Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
(2R, 4S) -ethyl-5-([1,1′- biphenyl]-4-yl) -4- ((tert butoxycarbonyl) amino)-2-methylvaleric acid	Intermediate of sacubitril	Asymmetric transfer of amino groups to keto acids	Transaminase	[53]
L-3,4-dimethoxyphenylalanine	Drug intermediates, chemical sensors, chiral catalysts, etc	Asymmetric transfer of amino groups to keto acids	Transaminase	[64]
(3S)-5-(Benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid	Intermediate of olodanrigan (EMA401)	Enantioselective addition of ammonia to α,β-unsaturated acids	Phenylalanine ammonia-lyase	[80]
(R)-pantothenic acid	Intermediate of antimicrobials against plasmodium falciparum and multidrug-resistant staphylococcus aureus	Enantioselective addition of ammonia to α,β-unsaturated acids	3-methylaspartate ammonia lyase, Aspartate-α-decarboxylase, β-methylaspartate-α-decarboxylase/glutamate decarboxylase, Pantothenate synthetase	[84]
L-syn-p-methylsulfonylphenylserine	Intermediate of flufenicol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[114-116]
β-(2-furyl)serine	Intermediate of furan antibiotic and 2-amino-1-(2-furanyl)ethanol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[117]

表2 部分应用于生产实践的酶促合成路线

Table 2 Partially applied enzymatic synthetic routes in production

产品 Products	应用 Applications	合成路线 Synthetic routes	酶制剂 Enzyme	参考文献 References
L-phosphinothricin	Broad-spectrum herbicides	Asymmetric reductive amination of keto acids	Glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[22,28,35]
L-phosphinothricin	Broad-spectrum herbicides	Deracemization synthesis	D-amino acid oxidase, catalase, glutamate dehydrogenase, Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[14-15]
L-tert-leucine	Intermediate of azanavir, animal feed additive, nutritional fortifier	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[11-12, 30,32]
L-tert-leucine		Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
L-2-aminobutyric acid	Intermediate of antituberculosis ethambutol and antiepileptic drug levetiracetam	Asymmetric reductive amination of keto acids	Leucine dehydrogenase，threonine deaminase，Glucose dehydrogenase	[33-34]
		Asymmetric transfer of amino groups to keto acids	Transaminase，Glutamate dehydrogenase，Alcohol dehydrogenase	[63]
		Deracemization synthesis	D-amino acid oxidase, ω- Transaminase	[118]
L- phenylglycine	Intermediate of β-lactam antibiotics	Asymmetric reductive amination of keto acids	Amino acid dehydrogenase，Alcohol dehydrogenase/Glucose dehydrogenase/Formate dehydrogenase	[29, 36-37]
L- phenylglycine	Intermediate of β-lactam antibiotics	Deracemization synthesis	Mandelate racemase, D-mandelate dehydrogenase, Leucine dehydrogenase	[16]
(R)-3-amino-4-(2,4,5-trifluorophenyl)butyric acid	Intermediate of siagliptin	Asymmetric transfer of amino groups to keto acids	Transaminase	[13]
L-norvaline	Intermediate of perindopril	Asymmetric transfer of amino groups to keto acids	Transaminase	[62]
(2R, 4S) -ethyl-5-([1,1′- biphenyl]-4-yl) -4- ((tert butoxycarbonyl) amino)-2-methylvaleric acid	Intermediate of sacubitril	Asymmetric transfer of amino groups to keto acids	Transaminase	[53]
L-3,4-dimethoxyphenylalanine	Drug intermediates, chemical sensors, chiral catalysts, etc	Asymmetric transfer of amino groups to keto acids	Transaminase	[64]
(3S)-5-(Benzyloxy)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic Acid	Intermediate of olodanrigan (EMA401)	Enantioselective addition of ammonia to α,β-unsaturated acids	Phenylalanine ammonia-lyase	[80]
(R)-pantothenic acid	Intermediate of antimicrobials against plasmodium falciparum and multidrug-resistant staphylococcus aureus	Enantioselective addition of ammonia to α,β-unsaturated acids	3-methylaspartate ammonia lyase, Aspartate-α-decarboxylase, β-methylaspartate-α-decarboxylase/glutamate decarboxylase, Pantothenate synthetase	[84]
L-syn-p-methylsulfonylphenylserine	Intermediate of flufenicol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[114-116]
β-(2-furyl)serine	Intermediate of furan antibiotic and 2-amino-1-(2-furanyl)ethanol	Aldol condensation of an amino acid to aldehydes	L-threonine aldolase	[117]

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