WO2020107783A1

WO2020107783A1 - Method for preparing (s)-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid by means of multiple enzyme coupling

Info

Publication number: WO2020107783A1
Application number: PCT/CN2019/083916
Authority: WO
Inventors: 吴坚平; 居述云; 施俊巍; 杨立荣; 钱明心
Original assignee: 苏州同力生物医药有限公司; 浙江大学
Priority date: 2018-11-30
Filing date: 2019-04-23
Publication date: 2020-06-04
Also published as: CN111254170B; CN111254170A

Abstract

Disclosed is a method for preparing (S)-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid by means of multiple enzyme coupling, comprising using a racemate of 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid or a racemate of 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylate as a substrate, reacting the (R)-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid in the substrate under the catalytic action of an oxidative dehydrogenase to produce an imidic acid represented by formula (II); and transforming the imidic acid represented by formula (II) into the (S)-1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid in the presence of a piperidine acid reductase and a coenzyme capable of donating a hydride ion. The invention has characteristics such as mild reaction conditions, a strong stereoselectivity, a high reaction efficiency, and a high conversion rate.

Description

一种多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸的方法Method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by multiple enzyme coupling

技术领域Technical field

本发明属于生物催化技术领域，具体涉及一种多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸的方法。The invention belongs to the technical field of biocatalysis, and in particular relates to a method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by multi-enzyme coupling.

背景技术Background technique

(S)-1，2，3，4-四氢异喹啉-3-甲酸(1，2，3，4-tetrahydroisoquinoline-3-carboxylic acid)是一种重要的药物中间体，被广泛应用于多种有机小分子药物以及肽基药物的合成。例如，(S)-1，2，3，4-四氢异喹啉-3-甲酸是降压药喹那普利的重要组成部分(Diversity-oriented synthesis of medicinally important 1，2，3，4-tetrahydroisoquinoline-3-carboxylic acid(Tic)derivatives and higher analogs[J].Org Biomol Chem，2014，12(45)：9054-91.)。此外，(S)-1，2，3，4-四氢异喹啉-3-甲酸可用于合成含有四氢异喹啉母核的小分子拮抗剂，作用于趋化因子受体CXCR4，从而有望用于治疗HIV等疾病(Discovery of tetrahydroisoquinoline-based CXCR4 antagonists[J].ACS Med Chem Lett，2013，4(11)：1025-30.)。(S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) is an important pharmaceutical intermediate and is widely used in Synthesis of various organic small molecule drugs and peptide-based drugs. For example, (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is an important component of the antihypertensive drug quinapril (Diversity-oriented synthesis of medically imported 1, 2, 3, 4 -tetrahydroisoquinoline-3-carboxylic(acic(Tic)derivatives and higher analogs[J].Org Biomol Chem, 2014, 12(45):9054-91.). In addition, (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid can be used to synthesize small molecule antagonists containing tetrahydroisoquinoline nucleus, acting on the chemokine receptor CXCR4, thus It is expected to be used to treat HIV and other diseases (Discovery of tetrahydroisoquinoline-based CXCR4 antagonists [J]. ACS Med Chem Lett, 2013, 4(11): 1025-30.).

现有技术中，制备光学纯(S)-1，2，3，4-四氢异喹啉-3-甲酸的方法有化学手性合成和生物催化动力学拆分两种。研究人员最初利用Pictet-Spengler反应制备光学纯(S)-1，2，3，4-四氢异喹啉-3-甲酸，以L-苯丙氨酸为原料，在浓酸与高温条件下，与甲醛缩合生成目标产物，该法工艺相对简单，但生成的产物会发生部分消旋化。随后，Bischler-Nepieralski反应，[2+2+2]环加成方法等也被用于制备(S)-1，2，3，4-四氢异喹啉-3-甲酸及其衍生物，该类方法路线较复杂，成本高(Diversity-oriented synthesis of medicinally important 1，2，3，4-tetrahydroisoquinoline-3-carboxylic acid(Tic)derivatives and higher analogs[J].Org Biomol Chem，2014，12(45)：9054-91.)。近年来，Kurata等通过臭氧分解、氧化及去保护作用三步不对成合成(S)-1，2，3，4-四氢异喹啉-3-甲酸(Synthesis of Optically Pure(R)-and(S)-Tetrahydroisoquinoline-1-and-3-Carboxylic Acids[J].Synthesis，2015，47(09)：1238-44.)。该法产率低，步骤较多，不易于工业化应用。龚等利用化学酶法制备(S)-1，2，3，4-四氢异喹啉-3-甲酸即以外消旋苯丙氨酸为原料，经Pictet-Spengler反应合成外消旋1，2，3，4-四氢异喹啉-3-甲酸，然后经酯化作用、脂肪酶动力学拆分制备(S)-构型产物。23.8g外消旋酯盐酸盐(0.1mol)，脂肪酶和底物质量比为0.2，反应48h，产物ee＞99％，收率为49.1％。该法所得产物立体选择性高，工艺相对简单，但仍存在最大理论产率只有50％的问题(化学酶法合成光学纯(S)-1，2，3，4-四氢喹啉-3-羧酸的研究[J].现代化工，2003，23(12)：23-5.)。In the prior art, there are two methods for preparing optically pure (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid: chemical chiral synthesis and biocatalytic kinetic resolution. The researchers initially used the Pictet-Spengler reaction to prepare optically pure (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, using L-phenylalanine as the raw material under concentrated acid and high temperature conditions. , Condensation with formaldehyde to form the target product, the process of this method is relatively simple, but the resulting product will undergo partial racemization. Subsequently, the Bischler-Nepieralski reaction, [2+2+2] cycloaddition method, etc. were also used to prepare (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and its derivatives, This kind of method has a complicated route and high cost. 45): 9054-91.). In recent years, Kurata et al. synthesized (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Synthesis of Optically Pure(R)-and (S)-Tetrahydroisoquinoline-1-and-3-Carboxylic Acids [J]. Synthesis, 2015, 47(09): 1238-44.). The method has low yield and many steps, which is not easy for industrial application. Gong et al. prepared (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (racemic phenylalanine) as a raw material by chemical enzymatic method, and synthesized the racemic 1 by Pictet-Spengler reaction. 2,3,4-Tetrahydroisoquinoline-3-carboxylic acid, followed by esterification and lipase kinetic resolution to prepare (S)-configuration product. 23.8g of racemic ester hydrochloride (0.1mol), the mass ratio of lipase to substrate is 0.2, the reaction is 48h, the product ee>99%, the yield is 49.1%. The product obtained by this method has high stereoselectivity and the process is relatively simple, but there is still the problem that the maximum theoretical yield is only 50% (chemical enzymatic synthesis of optically pure (S)-1,2,3,4-tetrahydroquinoline-3 -Research on carboxylic acid [J]. Modern Engineering, 2003, 23(12): 23-5.).

发明内容Summary of the invention

本发明的目的在于克服现有技术的不足，提供一种新的制备(S)-1，2，3，4-四氢异喹啉-3-甲酸的方法。The purpose of the present invention is to overcome the shortcomings of the prior art and provide a new method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid.

为实现上述目的，本发明采取的技术方案如下：To achieve the above objectives, the technical solutions adopted by the present invention are as follows:

一种多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸(I)的方法，A method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (I) by multiple enzyme coupling,

所述方法包括如下步骤：The method includes the following steps:

以1，2，3，4-四氢异喹啉-3-甲酸外消旋体或1，2，3，4-四氢异喹啉-3-甲酸盐的外消旋体为底物，使该底物中(R)-1，2，3，4-四氢异喹啉-3-甲酸在氧化脱氢酶的催化作用下反应生成式(II)所示的亚胺酸；1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid racemate or 1,2,3,4-Tetrahydroisoquinoline-3-carboxylate racemate as substrate , The (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the substrate is reacted under the catalytic action of oxidative dehydrogenase to form the imidic acid represented by formula (II);

使所述式(II)所示的亚胺酸在哌啶酸还原酶与能够供给氢负离子的辅酶的存在下转化为所述(S)-1，2，3，4-四氢异喹啉-3-甲酸。The imine acid represented by the formula (II) is converted into the (S)-1,2,3,4-tetrahydroisoquinoline in the presence of pipecolic acid reductase and a coenzyme capable of supplying hydride ions -3-carboxylic acid.

进一步地，所述1，2，3，4-四氢异喹啉-3-甲酸盐可以为1，2，3，4-四氢异喹啉-3-甲酸的碱金属盐或铵盐等，具体例如1，2，3，4-四氢异喹啉-3-甲酸钠、1，2，3，4-四氢异喹啉-3-甲酸钾、1，2，3，4-四氢异喹啉-3-甲酸铵。Further, the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid salt may be an alkali metal salt or ammonium salt of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Etc., for example, sodium 1,2,3,4-tetrahydroisoquinoline-3-carboxylate, potassium 1,2,3,4-tetrahydroisoquinoline-3-carboxylate, 1,2,3,4-tetrakis Hydrogen isoquinoline-3-carboxylic acid ammonium.

根据本发明，所述氧化脱氢酶是能够选择性催化(R)-1，2，3，4-四氢异喹啉-3-甲酸的酶，且选择性大于等于80％，优选大于等于90％。According to the present invention, the oxidative dehydrogenase is an enzyme capable of selectively catalyzing (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and the selectivity is greater than or equal to 80%, preferably greater than or equal to 90%.

根据本发明的一些优选方面，所述氧化脱氢酶为D-氨基酸氧化酶。According to some preferred aspects of the invention, the oxidative dehydrogenase is a D-amino acid oxidase.

根据本发明，所述D-氨基酸氧化酶为选自如下D-氨基酸氧化酶中的一种或多种的组合：来源于三角酵母(Trigonopsis variabilis)CBS 4095的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自禾谷镰刀菌(Fusarium graminearum)CS3005的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自梨孢镰刀菌(Fusarium poae)2516的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶，来自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶。According to the present invention, the D-amino acid oxidase is a combination of one or more selected from the following D-amino acid oxidases: D-amino acid oxidase derived from Trigonopsis variabilis CBS 4095 or a mutant thereof Or other D-amino acid oxidases with amino acid sequence homology greater than 80%, D-amino acid oxidase from Fusarium Graminearum CS3005 or its mutants or other homology with amino acid sequence homology greater than 80% D-amino acid oxidase, D-amino acid oxidase from Fusarium poae 2516 or its mutants or other D-amino acid oxidases with amino acid sequence homology greater than 80%, from Fusarium solanacearum ( Fusarium (solani) M-0718 D-amino acid oxidase or its mutants or other D-amino acid oxidases with amino acid sequence homology greater than 80%.

优选地，所述D-氨基酸氧化酶具有如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的氨基酸序列。Preferably, the D-amino acid oxidase has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

根据本发明的一些具体且优选的方面，所述D-氨基酸氧化酶的添加量以8000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应体系重量的1～5％。According to some specific and preferred aspects of the present invention, the added amount of the D-amino acid oxidase is based on the wet weight of the cells after centrifugation at 8000 rpm for 10 minutes, and the added amount of the cells is 1 to 5% of the weight of the reaction system.

根据本发明的一些具体且优选的方面，所述D-氨基酸氧化酶的使用形式为离体的D-氨基酸氧化酶，或者离体的D-氨基酸氧化酶的粗酶液或者纯酶或者固定化酶，或胞内表达D-氨基酸氧化酶的细胞。According to some specific and preferred aspects of the present invention, the use form of the D-amino acid oxidase is isolated D-amino acid oxidase, or a crude enzyme solution or pure enzyme of the isolated D-amino acid oxidase or immobilization Enzymes, or cells that express D-amino acid oxidase intracellularly.

进一步地，所述细胞为表达D-氨基酸氧化酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述D-氨基酸氧化酶基因连接在所述表达载体pET-28a(+)上。Further, the cell is an engineered bacterium that expresses D-amino acid oxidase and contains an expression vector pET-28a(+), and the host cell of the engineered bacterium is E. coli BL21(DE3); wherein, the D-amino acid The oxidase gene is linked to the expression vector pET-28a(+).

根据本发明，所述哌啶酸还原酶为选自如下哌啶酸还原酶中的一中或多种的组合：来源于恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于绿脓杆菌(Pseudomonas aeruginosa)PAO1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于荧光假单胞菌(Pseudomonas fluorescens)Pf0-1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶。According to the present invention, the pipecolic acid reductase is a combination of one or more selected from the following pipecolic acid reductases: pipecolic acid reductase derived from Pseudomonas putida KT2440 or A mutant or a pipecolic acid reductase with an amino acid sequence homology greater than 80%, a pipecolic acid reductase derived from Pseudomonas aeruginosa PAO1 or a mutant thereof or a homology with an amino acid sequence greater than 80% Pipecolic acid reductase, pipecolic acid reductase derived from Pseudomonas fluorescens Pf0-1 or a mutant thereof, or pipecolic acid reductase with amino acid sequence homology greater than 80%, derived from insects Piperonic acid reductase of Pseudomonas entomophila str. L48 or its mutants or pipecolic acid reductase with amino acid sequence homology greater than 80%.

优选地，所述哌啶酸还原酶具有如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7或SEQ ID NO.8所示的氨基酸序列。Preferably, the pipecolic acid reductase has an amino acid sequence as shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8.

根据本发明的一些具体且优选的方面，所述哌啶酸还原酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应体系重量的0.1～5％。According to some specific and preferred aspects of the present invention, the added amount of pipecolic acid reductase is based on the wet weight of the cells after centrifugation at 4000 rpm for 10 minutes, and the added amount of the cells is 0.1 to 5% of the weight of the reaction system.

根据本发明的一些具体且优选的方面，所述哌啶酸还原酶的使用形式为离体的哌啶酸还原酶，含有离体的哌啶酸还原酶的粗酶液或者纯酶或者固定化酶，或胞内表达哌啶酸还原酶的细胞。According to some specific and preferred aspects of the present invention, the use form of the pipecolic acid reductase is isolated pipecolic acid reductase, a crude enzyme liquid or pure enzyme containing the isolated pipecolic acid reductase or immobilized Enzymes, or cells that express pipecolic acid reductase intracellularly.

进一步地，所述细胞为表达哌啶酸还原酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述哌啶酸还原酶基因连接在所述表达载体pET-28a(+)上。Further, the cell is an engineered bacterium expressing pipecolic acid reductase and contains an expression vector pET-28a(+), and the host cell of the engineered bacterium is E. coli BL21(DE3); wherein, the pipecolic acid The reductase gene is linked to the expression vector pET-28a(+).

根据本发明的一些优选方面，所述能够供给氢负离子的辅酶为NADH和/或NADPH。According to some preferred aspects of the present invention, the coenzyme capable of supplying hydride ions is NADH and/or NADPH.

根据本发明的一些优选方面，使所述生成亚胺酸的反应还在黄素腺嘌呤二核苷酸(FAD)的存在下进行。使反应在FAD存在下进行，有助于进一步提高转化率。进一步地，FAD与所述的底物等当量或者是过量的。一般情况下，所制备的D-氨基酸氧化酶的粗酶液中已经含有足够量的FAD，在直接采用粗酶液的情况下，无需再另外添加FAD。在使用D-氨基酸氧化酶纯酶的情况下，可以根据需要再外加适量的FAD。According to some preferred aspects of the present invention, the reaction for producing imidic acid is also carried out in the presence of flavin adenine dinucleotide (FAD). Performing the reaction in the presence of FAD helps to further increase the conversion rate. Further, FAD is equivalent to or excessive to the substrate. In general, the prepared crude enzyme solution of D-amino acid oxidase already contains a sufficient amount of FAD. In the case of directly using the crude enzyme solution, it is not necessary to add FAD. In the case of using pure D-amino acid oxidase enzyme, an appropriate amount of FAD may be added as needed.

根据本发明的一些优选方面，使所述生成亚胺酸的反应还在过氧化氢酶的存在下进行。According to some preferred aspects of the present invention, the reaction to produce imidic acid is also carried out in the presence of catalase.

根据本发明的一些具体且优选的方面，使生成所述亚胺酸的反应在设定温度和有氧环境中进行。According to some specific and preferred aspects of the present invention, the reaction to form the imidic acid is performed in a set temperature and an aerobic environment.

根据本发明的优选方面，所述设定温度为20～70℃。更优选地，所述设定温度为20～50℃。进一步优选地，所述设定温度为30～40℃。According to a preferred aspect of the present invention, the set temperature is 20 to 70°C. More preferably, the set temperature is 20-50°C. Further preferably, the set temperature is 30-40°C.

根据本发明的一些具体且优选的方面，所述方法的实施过程包括：首先构建反应体系，然后控制所述反应体系处于设定温度和有氧环境中进行反应，所述反应体系包括所述底物、所述氧化脱氢酶、所述哌啶酸还原酶、辅酶、辅酶再生***、溶剂，所述反应体系还选择性地包括pH缓冲剂和/或pH调节剂，所述辅酶包括NAD+(氧化型烟酰胺腺嘌呤二核苷酸)和/或NADH(还原型烟酰胺腺嘌呤二核苷酸)，或者，所述辅酶包括NADP+(氧化型烟酰胺腺嘌呤二核苷酸磷酸)和/或NADPH(还原型烟酰胺腺嘌呤二核苷酸磷酸)。According to some specific and preferred aspects of the present invention, the implementation process of the method includes: first constructing a reaction system, and then controlling the reaction system to perform the reaction in a set temperature and an aerobic environment, the reaction system includes the bottom Substances, the oxidative dehydrogenase, the pipecolic acid reductase, the coenzyme, the coenzyme regeneration system, the solvent, the reaction system also optionally includes a pH buffer and/or pH adjuster, and the coenzyme includes NAD+( Oxidized nicotinamide adenine dinucleotide) and/or NADH (reduced nicotinamide adenine dinucleotide), or, the coenzyme includes NADP+ (oxidized nicotinamide adenine dinucleotide phosphate) and// Or NADPH (reduced nicotinamide adenine dinucleotide phosphate).

根据本发明的一些优选方面，控制所述反应体系的pH值为6～9。更优选地，控制所述反应体系的pH值为7～8.5。According to some preferred aspects of the present invention, the pH value of the reaction system is controlled to 6-9. More preferably, the pH of the reaction system is controlled to 7-8.5.

根据本发明的一些优选方面，控制所述反应体系中起始底物的浓度为1～20g/L。According to some preferred aspects of the present invention, the concentration of the starting substrate in the reaction system is controlled to be 1-20 g/L.

根据本发明的一个具体且优选的方面，所述pH缓冲剂为磷酸盐，将其溶于水可以配制成磷酸盐缓冲溶液。According to a specific and preferred aspect of the present invention, the pH buffering agent is phosphate, which can be formulated into a phosphate buffer solution by dissolving it in water.

根据本发明的一些优选方面，所述的pH调节剂为氨水、碱金属氢氧化物或其水溶液。According to some preferred aspects of the present invention, the pH adjusting agent is ammonia water, alkali metal hydroxide or its aqueous solution.

根据本发明的一个具体且优选方面，所述的pH调节剂为20wt％～35wt％氨水。According to a specific and preferred aspect of the present invention, the pH adjusting agent is 20 wt% to 35 wt% ammonia.

根据本发明的又一具体方面，所述的pH调节剂为氢氧化钠或氢氧化钾的水溶液。According to still another specific aspect of the present invention, the pH adjusting agent is an aqueous solution of sodium hydroxide or potassium hydroxide.

根据本发明的一些具体且优选的方面，所述辅酶的添加量为底物浓度的1‰～1％。According to some specific and preferred aspects of the present invention, the amount of the coenzyme added is 1‰-1% of the substrate concentration.

根据本发明，所述辅酶再生***包括辅酶再生酶和辅酶再生底物。According to the present invention, the coenzyme regeneration system includes a coenzyme regeneration enzyme and a coenzyme regeneration substrate.

根据本发明的一些优选方面，所述辅酶再生酶为葡萄糖脱氢酶，所述辅酶再生底物为葡萄糖；或者，所述辅酶再生酶为醇脱氢酶，所述辅酶再生底物为异丙醇。根据本发明的一个具体方面，所述葡萄糖具体使用D-葡萄糖。According to some preferred aspects of the present invention, the coenzyme regenerating enzyme is glucose dehydrogenase and the coenzyme regenerating substrate is glucose; or, the coenzyme regenerating enzyme is alcohol dehydrogenase and the coenzyme regenerating substrate is isopropyl alcohol. According to a specific aspect of the present invention, the glucose specifically uses D-glucose.

根据本发明的一个具体方面，所述葡萄糖脱氢酶来源于枯草芽胞杆菌(Bacillus subtilis)168；和/或，所述醇脱氢酶来源于乳酸杆菌(Lactobscillus kefir)DSM20587。According to a specific aspect of the present invention, the glucose dehydrogenase is derived from Bacillus subtilis (Bacillus subtilis) 168; and/or the alcohol dehydrogenase is derived from Lactobscillus kefir DSM20587.

优选地，所述葡萄糖脱氢酶具有如SEQ ID NO.9所示的氨基酸序列。Preferably, the glucose dehydrogenase has the amino acid sequence shown in SEQ ID NO.9.

优选地，所述醇脱氢酶具有如SEQ ID NO.10所示的氨基酸序列。Preferably, the alcohol dehydrogenase has the amino acid sequence shown in SEQ ID NO. 10.

根据本发明的一些优选方面，所述反应体系还包括过氧化氢酶。According to some preferred aspects of the present invention, the reaction system further includes catalase.

根据本发明的一些优选方面，所述过氧化氢酶为牛肝过氧化氢酶冻干粉。根据本发明的一个具体方面，所述牛肝过氧化氢酶冻干粉的酶活为4000U/mg。According to some preferred aspects of the present invention, the catalase is bovine liver catalase lyophilized powder. According to a specific aspect of the present invention, the enzyme activity of the lyophilized powder of bovine liver catalase is 4000 U/mg.

根据本发明的一些优选方面，所述过氧化氢酶与所述氧化脱氢酶的酶活比为1000～2000∶1。According to some preferred aspects of the present invention, the enzyme activity ratio of the catalase to the oxidative dehydrogenase is 1000-2000:1.

根据本发明的一些优选方面，所述反应体系还包括黄素腺嘌呤二核苷酸。According to some preferred aspects of the present invention, the reaction system further includes flavin adenine dinucleotide.

根据本发明的一些具体且优选的方面，所述方法还包括分离步骤。According to some specific and preferred aspects of the invention, the method further includes a separation step.

根据本发明，所述分离步骤包括：将反应后的反应体系的pH值调至5.0-6.0，加热使蛋白变性析出，抽滤，滤液浓缩后，冷却析晶，干燥，即得所述式(I)所示的化合物的S型异构体。According to the present invention, the separation step includes: adjusting the pH value of the reaction system after the reaction to 5.0-6.0, heating to denature and precipitate the protein, suction filtration, after the filtrate is concentrated, cooling and crystallization, and drying to obtain the formula ( I) S-isomer of the compound shown.

由于以上技术方案的实施，本发明与现有技术相比具有如下有益效果：Due to the implementation of the above technical solutions, the present invention has the following beneficial effects compared with the prior art:

本发明发现在哌啶酸还原酶与能够供给氢负离子的辅酶的共同存在下可高效地使亚胺酸转化得到(S)-1，2，3，4-四氢异喹啉-3-甲酸，其选择性好，收率高，反应条件温和，制备得到的产物中S型异构体相对R型异构体的ee值＞99％，且工艺相对简单。The present invention finds that in the presence of pipecolic acid reductase and a coenzyme capable of supplying hydride ions, it can efficiently convert imidic acid to obtain (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid It has good selectivity, high yield and mild reaction conditions. The ee value of the S-isomer relative to the R-isomer in the prepared product is >99%, and the process is relatively simple.

附图说明BRIEF DESCRIPTION

图1为实施例3中反应体系中0小时取样的底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的两个光学异构体高效液相检测图谱；1 is a high performance liquid phase detection spectrum of two optical isomers of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid sampled at 0 hours in the reaction system in Example 3;

其中，保留时间8.877min为(R)-1，2，3，4-四氢异喹啉-3-甲酸；保留时间11.308min为(S)-1，2，3，4-四氢异喹啉-3-甲酸；Among them, the retention time 8.877min is (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; the retention time 11.308min is (S)-1,2,3,4-tetrahydroisoquinoline Porphyrin-3-carboxylic acid;

图2为实施例3中反应12小时取样的高效液相色谱检测图谱。FIG. 2 is a detection spectrum of high performance liquid chromatography for sampling of the reaction in Example 3 for 12 hours.

具体实施方式detailed description

本发明提供一种制备(S)-1，2，3，4-四氢异喹啉-3-甲酸的新方法，本发明方法具有反应条件温和、立体选择性强、反应效率高、产率高等特点，具有工业化应用前景。The invention provides a new method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. The method of the invention has mild reaction conditions, strong stereoselectivity, high reaction efficiency and yield High characteristics, with industrial application prospects.

根据本发明的一个具体方面，该方法以外消旋1，2，3，4-四氢异喹啉-3-甲酸为底物，经多酶体系催化获得(S)-1，2，3，4-四氢异喹啉-3-甲酸，所述多酶体系可由氧化脱氢酶(优选为D-氨基酸氧化酶)、过氧化氢酶、哌啶酸还原酶和辅酶(优选为NADP ⁺和/或NADPH)、辅酶再生***等组成。 According to a specific aspect of the present invention, the method uses racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid as a substrate, which is catalyzed by a multi-enzyme system to obtain (S)-1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, the multi-enzyme system can be composed of oxidative dehydrogenase (preferably D-amino acid oxidase), catalase, pipecolic acid reductase and coenzyme (preferably NADP ⁺ and And/or NADPH), coenzyme regeneration system, etc.

具体原理为：以外消旋1，2，3，4-四氢异喹啉-3-甲酸为底物，利用D-氨基酸氧化酶立体选择性催化(R)-1，2，3，4-四氢异喹啉-3-甲酸进行氧化脱氢生成亚胺酸，(S)-1，2，3，4-四氢异喹啉-3-甲酸基本未被催化而保留在反应体系中。反应过程中产生的过氧化氢经过氧化氢酶催化分解成水和氧气。亚胺酸被哌啶酸还原酶不对称还原为(S)-1，2，3，4-四氢异喹啉-3-甲酸。在此过程中，还原型辅酶II即还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)被氧化为NADP ⁺(氧化型烟酰胺腺嘌呤二核苷酸磷酸)，NADP ⁺经辅酶再生***还原为NADPH。 The specific principle is as follows: racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid as a substrate, stereoselective catalysis using D-amino acid oxidase (R)-1,2,3,4- Oxidative dehydrogenation of tetrahydroisoquinoline-3-carboxylic acid to form imidic acid, (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is basically not catalyzed but remains in the reaction system. The hydrogen peroxide produced during the reaction is decomposed into water and oxygen by catalase. Imine acid is asymmetrically reduced by pipecolic acid reductase to (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. In this process, reduced coenzyme II, reduced nicotinamide adenine dinucleotide phosphate (NADPH), is oxidized to NADP ⁺ (oxidized nicotinamide adenine dinucleotide phosphate), and NADP ⁺ is reduced by the coenzyme regeneration system For NADPH.

反应过程示意如下：The reaction process is as follows:

进一步地，优选使生成亚胺酸的反应还在黄素腺嘌呤二核苷酸(FAD)存在下进行，在反应过程中，黄素腺嘌呤二核苷酸(FAD)被还原为FADH ₂，随后，一分子氧被还原为过氧化氢(H ₂O ₂)，而FADH ₂则被氧化为FAD。过氧化氢在过氧化氢酶的催化下分解成水和氧气。反应过程示意如下： Further, it is preferred that the reaction to generate imidic acid is also carried out in the presence of flavin adenine dinucleotide (FAD). During the reaction, flavin adenine dinucleotide (FAD) is reduced to FADH ₂ , Subsequently, a molecule of oxygen is reduced to hydrogen peroxide (H ₂ O ₂ ), and FADH ₂ is oxidized to FAD. Hydrogen peroxide is decomposed into water and oxygen under the catalysis of catalase. The reaction process is as follows:

作为优选，所述D-氨基酸氧化酶来源于三角酵母、禾谷镰刀菌、梨孢镰刀菌、茄病镰刀菌。具体地，所述D-氨基酸氧化酶来源于三角酵母(Trigonopsis variabilis)CBS 4095、禾谷镰刀菌(Fusarium graminearum)CS3005、梨孢镰刀菌(Fusarium poae)2516或茄病镰刀菌(Fusarium solani)M-0718。Preferably, the D-amino acid oxidase is derived from Triangle yeast, Fusarium graminearum, Fusarium oxysporum, and Fusarium solani. Specifically, the D-amino acid oxidase is derived from Trigonopsis variabilis CBS 4095, Fusarium graminearum CS3005, Fusarium poae 2516, or Fusarium solani M -0718.

作为优选，所述哌啶酸还原酶来源于恶臭假单胞菌，绿脓杆菌，荧光假单胞菌，虫媒假单胞菌。具体的，所述哌啶酸还原酶源于恶臭假单胞菌(Pseudomonas putida)KT2440、绿脓杆菌(Pseudomonas aeruginosa)PAO1、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1或虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶。Preferably, the pipecolic acid reductase is derived from Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas arborea. Specifically, the pipecolic acid reductase is derived from Pseudomonas putida KT2440, Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens Pf0-1, or Pseudomonas arborea Piperonic acid reductase of Pseudomonas entomophila str. L48 or a mutant thereof or a pipecolic acid reductase with a homology greater than 80% in amino acid sequence.

作为优选，所述辅酶再生***包括辅酶再生酶和辅酶再生底物，所述辅酶再生酶来源于枯草芽胞杆菌，乳酸杆菌。具体地，所述辅酶再生酶来源于枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶，来源于乳酸杆菌(Lactobscillus kefir)DSM20587的醇脱氢酶。Preferably, the coenzyme regeneration system includes a coenzyme regeneration enzyme and a coenzyme regeneration substrate. The coenzyme regeneration enzyme is derived from Bacillus subtilis and Lactobacillus. Specifically, the coenzyme regenerating enzyme is derived from the glucose dehydrogenase of Bacillus subtilis (Bacillus subtilis) 168 and the alcohol dehydrogenase of LSM (Lactobscillus kefir) DSM20587.

具体地，反应体系中，多酶体系中的酶的使用形式可以为离体酶、粗酶液，或者是纯酶，或者固定化酶，或者表达重组酶的工程菌静息细胞。Specifically, in the reaction system, the use form of the enzyme in the multi-enzyme system may be an ex vivo enzyme, a crude enzyme solution, or a pure enzyme, or an immobilized enzyme, or a resting cell of an engineered bacteria expressing a recombinant enzyme.

作为优选，反应体系中起始底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的浓度为1～20g/L。Preferably, the concentration of the starting substrate racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction system is 1-20 g/L.

作为优选，反应体系中，D-氨基酸氧化酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应液重量的1～5％。Preferably, in the reaction system, the added amount of D-amino acid oxidase is calculated as the wet weight of the cells after centrifugation at 4000 rpm for 10 min. The added amount of the cells is 1 to 5% of the weight of the reaction solution.

作为优选，反应体系中，过氧化氢酶为牛肝过氧化氢酶冻干粉末，酶活为4000U/mg，过氧化氢酶与D-氨基酸氧化酶的酶活比为1000～2000∶1。Preferably, in the reaction system, the catalase is bovine liver catalase lyophilized powder, the enzyme activity is 4000 U/mg, and the enzyme activity ratio of catalase to D-amino acid oxidase is 1000-2000:1.

作为优选，反应体系中，哌啶酸还原酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应液重量的0.1～5％。Preferably, in the reaction system, the added amount of pipecolic acid reductase is calculated as the wet weight of the cells after centrifugation at 4000 rpm for 10 minutes, and the added amount of the cells is 0.1 to 5% of the weight of the reaction solution.

作为优选，反应体系中，辅酶再生酶的添加量以4000rpm离心10min后的细胞湿重计，所述细胞的添加量为反应液重量的0.1～5％。Preferably, in the reaction system, the added amount of coenzyme regenerating enzyme is based on the wet weight of the cells after centrifugation at 4000 rpm for 10 minutes, and the added amount of the cells is 0.1 to 5% of the weight of the reaction solution.

作为优选，反应体系中，起始的辅酶可以添加氧化型烟酰胺腺嘌呤二核苷酸磷酸(NADP ⁺)，其添加量为1‰～1％。 Preferably, in the reaction system, oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) may be added to the initial coenzyme in an amount of 1‰ to 1%.

作为优选，反应体系中，反应的温度为20～70℃，时间为6～72小时，反应液的pH值为6～9；更优选，温度为30～40℃，时间为12～48小时。磷酸缓冲溶液控制反应的pH值为7～8.5。Preferably, in the reaction system, the reaction temperature is 20 to 70°C, the time is 6 to 72 hours, and the pH of the reaction solution is 6 to 9; more preferably, the temperature is 30 to 40°C and the time is 12 to 48 hours. Phosphate buffer solution controls the pH of the reaction from 7 to 8.5.

以下结合具体实施例，对本发明做进一步说明。应理解，以下实施例仅用于说明本发明而非用于限制本发明的范围。The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention rather than to limit the scope of the present invention.

本发明实施例中的实验方法如无特别说明均为常规方法。Unless otherwise specified, the experimental methods in the embodiments of the present invention are conventional methods.

本发明实施例中所用基因由生工生物工程(上海)股份有限公司合成。E.coli BL21(DE3)菌种购自Novagen公司；DNA marker、PrimeStar DNA聚合酶、低分子量标准蛋白等分子生物学实验试剂购自TaKaRa。基因克隆及表达具体操作可参见J.萨姆布鲁克等编的《分子克隆实验指南》。The genes used in the examples of the present invention are synthesized by Biotechnology (Shanghai) Co., Ltd. E.coli BL21 (DE3) strains were purchased from Novagen; DNA marker, PrimeStar DNA polymerase, low molecular weight standard protein and other molecular biological reagents were purchased from TaKaRa. For specific operations of gene cloning and expression, please refer to "Molecular Cloning Experiment Guide" edited by J. Sambrook et al.

本发明通过高效液相色谱(HPLC)分析催化反应的各个产物和底物。外消旋1，2，3，4-四氢异喹啉-3-甲酸的HPLC分析方法为：色谱柱/

ZWIX(-)；柱温/25℃；流速/0.5mL/min；检测波长/UV210nm；流动相：HPLC级甲醇/乙腈(50/50，v/v)(加入50mM甲酸和25mM二已胺)。具体各相关物质出峰情况见图1。 The present invention analyzes each product and substrate of the catalytic reaction by high-performance liquid chromatography (HPLC). The HPLC analysis method of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is: chromatography column/

ZWIX(-); column temperature/25℃; flow rate/0.5mL/min; detection wavelength/UV210nm; mobile phase: HPLC grade methanol/acetonitrile (50/50, v/v) (add 50mM formic acid and 25mM dihexylamine) . The specific peaks of related substances are shown in Figure 1.

实施例1基因工程菌菌种构建Example 1 Construction of genetically engineered strains

1.1 D-氨基酸氧化酶的筛选及表达D-氨基酸氧化酶的基因工程菌的构建1.1 Screening of D-amino acid oxidase and construction of genetically engineered bacteria expressing D-amino acid oxidase

根据底物特异性的不同，微生物来源的D-氨基酸氧化酶可分为两大类：1)偏好底物侧链基团较小的氨基酸(如D-丙氨酸)，如尖孢镰刀菌(Fusarium oxysporum)来源的D-氨基酸氧化酶；2)偏好底物侧链基团较大的氨基酸(如D-苯丙氨酸)，如三角酵母(Trigonopsis variabilis)来源的D-氨基酸氧化酶(POLLEGIONI L，MOLLA G，SACCHI S，et al.Properties and applications of microbial D-amino acid oxidases：current state and perspectives[J].Appl Microbiol Biotechnol，2008，78(1)：1-16.)。分别用这两种D-氨基酸氧化酶的氨基酸序列在美国国立生物技术信息中心(NCBI)数据库(https：//www.ncbi.nlm.nih.gov/)中进行BLASTp分析，选取序列一致性不同的4种D-氨基酸氧化酶作进一步研究(如表1所示)。According to different substrate specificities, microbial-derived D-amino acid oxidases can be divided into two categories: 1) Preference for amino acids with smaller substrate side chain groups (such as D-alanine), such as Fusarium oxysporum (Fusarium oxysporum)-derived D-amino acid oxidase; 2) Preference for amino acids with larger substrate side chain groups (such as D-phenylalanine), such as D-amino acid oxidase from Trigonopsis variabilis ( POLLEGIONI L, MOLLA G, SACCHI S, et al. Properties, and applications, of microbial, D-amino acid, oxidationases: current state, and perspectives [J]. Appl, Microbiol, Biotechnol, 2008, 78 (1): 1-16. Use the amino acid sequences of these two D-amino acid oxidases to carry out BLASTp analysis in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/), and select different sequence identity The 4 kinds of D-amino acid oxidase for further study (as shown in Table 1).

表1四种不同来源的D-氨基酸氧化酶Table 1 Four different sources of D-amino acid oxidase

将上述D-氨基酸氧化酶基因序列经密码子优化后送生工生物工程(上海)股份有限公司进行全基因合成，并克隆到重组表达质粒pET-28a(+)上。重组质粒转入表达宿主E.coli BL21(DE3)中，经测序验证无误后，向所得工程菌菌液中加入起始浓度为25％的甘油并置于-80℃保藏备用。The above D-amino acid oxidase gene sequence was codon optimized and sent to Biotech (Shanghai) Co., Ltd. for full gene synthesis, and cloned into the recombinant expression plasmid pET-28a(+). The recombinant plasmid was transferred into the expression host E. coli BL21 (DE3). After verification by sequencing, glycerol with an initial concentration of 25% was added to the obtained engineering bacterial solution and stored at -80℃ for later use.

1.2表达哌啶酸还原酶的基因工程菌的构建1.2 Construction of genetically engineered bacteria expressing pipecolic acid reductase

分别从恶臭假单胞菌(Pseudomonas putida)KT2440、绿脓杆菌(Pseudomonas aeruginosa)PAO1、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1、虫媒假单胞菌(Pseudomonas entomophila str.)L48基因组中克隆哌啶酸还原酶基因(如表2所示)。From Pseudomonas (Pseudomonas putida) KT2440, Pseudomonas (Pseudomonas aeruginosa) PAO1, Pseudomonas (Pseudomonas fluorescens) Pf0-1, Pseudomonas (Pseudomonas entomophila str.) L48 genome The pipecolic acid reductase gene was cloned (as shown in Table 2).

表2四种不同来源的哌啶酸还原酶Table 2 Four different sources of pipecolic acid reductase

根据相应基因DNA序列设计PCR上游引物和下游引物。Design PCR upstream and downstream primers according to the corresponding gene DNA sequence.

来源于Pseudomonas putida KT2440的哌啶酸还原酶的引物：Primers of pipecolic acid reductase from Pseudomonas putida KT2440:

KT2440-F：5’-CG GGATCCATGTCCGCACCTTCCACCAGCAC-3’(BamH I) KT2440-F: 5'-CG GGATCC ATGTCCGCACCTTCCACCAGCAC-3 '(BamH I)

KT2440-R：5’-CCC AAGCTTTCAGCCAAGCAGCTCTTTCAGG-3’(Hind III) KT2440-R: 5'-CCC AAGCTT TCAGCCAAGCAGCTCTTTCAGG-3' (Hind III)

来源于Pseudomonas aeruginosa PAO1的哌啶酸还原酶的引物：Primers of pipecolic acid reductase from Pseudomonas aeruginosa PAO1:

PAO1-F：5’-CG GGATCCGTGATCCGAATGACGCTGGAC-3’(BamH I) PAO1-F: 5'-CG GGATCC GTGATCCGAATGACGCTGGAC-3 '(BamH I)

PAO1-R：5’-CCC AAGCTTTCACTCCAGCAACGCCAGC-3’(Hind III) PAO1-R: 5'-CCC AAGCTT TCACTCCAGCAACGCCAGC-3'(Hind III)

来源于Pseudomonas fluorescens Pf0-1的哌啶酸还原酶的引物：Primers of pipecolic acid reductase derived from Pseudomonas fluorescens Pf0-1:

Pf0-1-F：5’-CG GGATCCATGTCTGCGCCACACGATC-3’(BamH I) Pf0-1-F: 5'-CG GGATCC ATGTCTGCGCCACACGATC-3 '(BamH I)

Pf0-1-R：5’-CCG CTCGAGTTACTCGCCGGCCAGTTCAC-3’(Xho I) Pf0-1-R: 5'-CCG CTCGAG TTACTCGCCGGCCAGTTCAC-3' (Xho I)

来源于Pseudomonas entomophila str.L48的哌啶酸还原酶的引物：Primers of pipecolic acid reductase from Pseudomonas enteromophila str.L48:

L48-F：5’-CG GGATCCGTGCGCGTAGCCTTCAAC-3’(BamH I) L48-F: 5'-CG GGATCC GTGCGCGTAGCCTTCAAC-3 '(BamH I)

L48-R：5’-CCC AAGCTTTCACCTCGCCAGCGCCTTC-3’(Hind III) L48-R: 5'-CCC AAGCTT TCACCTCGCCAGCGCCTTC-3' (Hind III)

在上、下游引物中分别加入限制性内切酶切位点，如下划线所示，具体限制性内切酶见引物序列括号内。分别以恶臭假单胞菌(Pseudomonas putida)KT2440、绿脓杆菌(Pseudomonas aeruginosa)PAO1、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1、虫媒假单胞菌(Pseudomonas entomophila str.)L48基因组DNA为模板，利用相应的上下游引物分别进行PCR扩增反应，PCR反应体系和反应条件如下：Add restriction enzyme sites to the upstream and downstream primers respectively, as shown by the underline. For specific restriction enzymes, see the primer sequence brackets. Pseudomonas (Pseudomonas putida) KT2440, Pseudomonas (Pseudomonas aeruginosa) PAO1, Pseudomonas (Pseudomonas fluorescens) Pf0-1, Pseudomonas (Pseudomonas entomophila str.) L48 genomic DNA As a template, use corresponding upstream and downstream primers to perform PCR amplification reactions respectively. The PCR reaction system and reaction conditions are as follows:

PCR扩增体系：PCR amplification system:

PCR扩增条件：PCR amplification conditions:

1)预变性：95℃5min；1) Pre-denaturation: 95℃5min;

2)变性：95℃10s；退火：58℃15s；延伸：72℃10s；共循环30次；2) Denaturation: 95℃10s; Annealing: 58℃15s; Extension: 72℃10s; 30 cycles in total;

3)延伸：72℃10min；3) Extension: 72℃10min;

4)4℃保温。4) Insulation at 4℃.

PCR扩增反应结束后，利用1.0％琼脂糖凝胶电泳检测扩增结果，结果显示扩增产物为单一条带，大小约为1000bp。用DNA回收纯化试剂盒回收目的条带，具体步骤参照纯化试剂盒说明书。After the PCR amplification reaction was completed, the amplification result was detected by 1.0% agarose gel electrophoresis. The result showed that the amplification product was a single band with a size of about 1000 bp. Use DNA recovery and purification kit to recover the target band. For specific steps, please refer to the instructions of the purification kit.

表达载体pET-28a(+)和PCR扩增产物分别用相应的限制性内切酶进行双酶切。酶切完成后用DNA回收纯化试剂盒回收目的条带。随后，利用T4DNA连接酶将双酶切后的PCR扩增产物连接到具有相对应黏性末端的表达载体pET-28a(+)上，连接体系如下表3所示：The expression vector pET-28a(+) and PCR amplification products were double-digested with the corresponding restriction enzymes. After the digestion is completed, use DNA recovery purification kit to recover the target band. Afterwards, the double-digested PCR amplification product was ligated to the expression vector pET-28a(+) with corresponding sticky ends using T4DNA ligase. The ligation system is shown in Table 3 below:

表3重组表达质粒构建体系Table 3 Recombinant expression plasmid construction system

将酶连产物转化至E.coli DH5a感受态细胞中，涂平板、挑单菌落到LB液体基中培养，菌液PCR鉴定阳性转化子，并送测序公司来验证***序列的正确性。从验证无误的阳性转化子中提取质粒，相关方法参照质粒提取试剂盒。再将重组表达载体转入表达宿主E.coli BL21(DE3)中，经菌液PCR和测序验证无误后，向所得工程菌菌液中加入起始浓度为25％的甘油并置于-80℃保藏备用。The enzyme-linked product was transformed into E. coli DH5a competent cells, plated, single colonies were cultured in LB liquid base, bacterial solution PCR identified positive transformants, and sent to the sequencing company to verify the correctness of the inserted sequence. Extract the plasmids from the positive transformants that have been verified, and refer to the plasmid extraction kit for related methods. Then transfer the recombinant expression vector into the expression host E. coli BL21 (DE3), after verification by bacterial solution PCR and sequencing, add the initial concentration of 25% glycerol to the obtained engineering bacterial solution and place at -80℃ Save for future use.

1.3表达辅酶再生酶的基因工程菌的构建1.3 Construction of genetically engineered bacteria expressing coenzyme regeneration enzyme

分别从枯草芽胞杆菌(Bacillus subtilis)168基因组中克隆葡萄糖脱氢酶(NCBI登录号：NP_388275.1，SEQ ID NO.9)基因；从乳酸杆菌(Lactobacillus kefiri)DSM20587基因组中克隆醇脱氢酶(NCBI登录号：AAP94029.1，SEQ ID NO.10)基因。具体方法步骤参考1.2中哌啶酸还原酶表达菌种的构建方法。相关PCR上游引物和下游引物如下：The glucose dehydrogenase (NCBI accession number: NP_388275.1, SEQ ID NO. 9) gene was cloned from the Bacillus subtilis 168 genome; the alcohol dehydrogenase was cloned from the genome of Lactobacillus kefiri DSM20587 ( NCBI accession number: AAP94029.1, SEQ ID NO.10) gene. For the specific method steps, please refer to the construction method of the strain expressing pipecolic acid reductase in 1.2. The relevant PCR upstream and downstream primers are as follows:

来源于Bacillus subtilis 168的葡萄糖脱氢酶的引物：Primers derived from Bacillus subtilis 168 glucose dehydrogenase:

BGdh-F：5’-GA AGATCTGATGTATCCGGATTTAAAAGGAAAAGTC-3’(Bgl II) BGdh-F: 5'-GA AGATCT GATGTATCCGGATTTAAAAGGAAAAGTC-3'(Bgl II)

BGdh-R：5’-CATG CCATGGTTAACCGCGGC-3’(Nco I) BGdh-R: 5'-CATG CCATGG TTAACCGCGGC-3' (Nco I)

来源于Lactobacillus kefiri DSM20587的醇脱氢酶的引物：Primer of alcohol dehydrogenase from Lactobacillus Kefiri DSM20587:

LAdh-F：5’-CC GAATTCATGACCGATCGTCTGAAGGGC-3’(EcoR I) LAdh-F: 5'-CC GAATTC ATGACCGATCGTCTGAAGGGC-3'(EcoR I)

LAdh-R：5’-CCC AAGCTTTCACTGTGCGGTATACCCGCC-3’(Hind III)。 LAdh-R: 5'-CCC AAGCTT TCACTGTGCGGTATACCCGCC-3' (Hind III).

实施例2Example 2

2.1微生物的培养2.1 Cultivation of microorganisms

液体LB培养基组成：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，用去离子水溶解后定容，121℃灭菌20min，待用。若为固体LB培养基，则另加15g/L琼脂。The composition of liquid LB medium: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in deionized water, set the volume, sterilized at 121℃ for 20min, and ready for use. If it is solid LB medium, add 15g/L agar.

将含有D-氨基酸氧化酶基因的工程菌接种于5mL液体LB(含50μg/ml卡那霉素)培养基中，37℃，200rpm振荡培养8小时左右。按1％(V/V)的接种量接种于100mL液体LB(含50μg/ml卡那霉素)培养基中培养，OD600达到0.6-0.8后，加入诱导剂异丙基硫代半乳糖苷(起始浓度为0.1mM)，18℃诱导15h。培养结束后，将培养液倒入100mL离心管中4000rpm离心10min，弃上清，收集菌体细胞，用50mM磷酸缓冲液(pH 8.0)洗涤细胞两次，放于-80℃超低温冰箱中保存，备用。The engineered bacteria containing the D-amino acid oxidase gene were inoculated in 5 mL liquid LB (containing 50 μg/ml kanamycin) medium, and cultured at 37° C. and shaking at 200 rpm for about 8 hours. Inoculated at 1% (V/V) inoculated in 100mL liquid LB (containing 50μg/ml kanamycin) culture medium, OD600 reached 0.6-0.8, add the inducer isopropyl thiogalactoside ( The initial concentration was 0.1 mM) and induced at 18°C for 15h. After the cultivation, the culture solution was poured into a 100 mL centrifuge tube and centrifuged at 4000 rpm for 10 min. The supernatant was discarded, the bacterial cells were collected, the cells were washed twice with 50 mM phosphate buffer (pH 8.0), and stored in an ultra-low temperature refrigerator at -80℃ spare.

2.2粗酶液的制备2.2 Preparation of crude enzyme solution

将菌体重悬于25mL磷酸缓冲液(50mM，pH值＝8.0)中，超声破碎菌悬液，离心后得到的上清为含D-氨基酸氧化酶或哌啶酸还原酶或辅酶再生酶的粗酶液。The bacteria were resuspended in 25mL phosphate buffer (50mM, pH=8.0), the bacteria suspension was sonicated, and the supernatant obtained after centrifugation was the crude containing D-amino acid oxidase or pipecolic acid reductase or coenzyme regenerating enzyme Enzyme liquid.

实施例3 FsDAAO-PpdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸Example 3 FsDAAO-PpdpkA multi-enzyme coupling preparation (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

按照实施例2的方法，分别制备源自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶粗酶液、恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。According to the method of Example 2, crude D-amino acid oxidase enzyme solution derived from Fusarium solani M-0718 and crude pipecolic acid reductase enzyme from Pseudomonas putida KT2440 were prepared respectively And Bacillus subtilis (Bacillus subtilis) 168 glucose dehydrogenase crude enzyme liquid.

称取0.16g外消旋1，2，3，4-四氢异喹啉-3-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FsDAAO粗酶液(粗酶液中已含有足量辅酶FAD，因此，粗酶液反应体系中不需额外添加FAD)，5ml PpdpkA粗酶液，5ml葡萄糖脱氢酶粗酶液，20mg过氧化氢酶，NADP ⁺以及D-葡萄糖，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的浓度为4g/L，NADP ⁺的浓度为0.025mM，D-葡萄糖浓度为15mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，氨水调节反应pH在8～8.5范围内，反应12小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-3-甲酸两种构型的浓度(g/L)。 Weigh 0.16g of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid into a 100ml reaction flask, add 10ml of phosphate buffer (50mM, pH=8.0) and mix well, then use 30% ammonia water Adjust the pH of the solution to 8.0. Add 20ml FsDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, so no additional FAD is needed in the crude enzyme solution reaction system), 5ml PpdpkA crude enzyme solution, 5ml glucose dehydrogenase crude enzyme solution, 20mg Catalase, NADP ⁺ and D-glucose make the concentration of the substrate 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the initial reaction system 4g/L, the concentration of NADP ⁺ It is 0.025 mM and the D-glucose concentration is 15 mM. After mixing, immediately take a sample as "0 hours". Using a water bath to control the reaction temperature was 30 ℃, magnetic stirring, ammonia water to adjust the reaction pH in the range of 8 ~ 8.5, the reaction was sampled for 12 hours. The content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution The concentration of isoquinoline-3-carboxylic acid in two configurations (g/L).

检测结果如图1和图2所示，FsDAAO表现出严格的R-构型立体选择性，反应液中(S)-1，2，3，4-四氢异喹啉-3-甲酸的反应收率为85.7％(反应收率＝实际产物浓度(g/L)/理论产物浓度(g/L)×100％)，ee值为99.6％。The detection results are shown in Figure 1 and Figure 2. FsDAAO exhibits strict R-configuration stereoselectivity, and the reaction of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction solution The yield was 85.7% (reaction yield = actual product concentration (g/L)/theoretical product concentration (g/L)×100%), and the ee value was 99.6%.

实施例4 FpDAAO-PadpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸Example 4 FpDAAO-PadpkA multi-enzyme coupling preparation (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

按照实施例2的方法，分别制备源自梨孢镰刀菌(Fusarium poae)2516的D-氨基酸氧化酶粗酶液、绿脓杆菌(Pseudomonas aeruginosa)PAO1的哌啶酸还原酶粗酶液以及乳酸杆菌(Lactobacillus kefiri)DSM20587的醇脱氢酶粗酶液。According to the method of Example 2, a crude D-amino acid oxidase enzyme solution derived from Fusarium poae 2516, a crude pipecolic acid reductase enzyme solution of Pseudomonas aeruginosa PAO1, and lactobacilli were prepared respectively (Lactobacillus Kefiri) DSM20587 alcohol dehydrogenase crude enzyme solution.

称取0.2g外消旋1，2，3，4-四氢异喹啉-3-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FpDAAO粗酶液，2ml PadpkA粗酶液，8ml醇脱氢酶粗酶液，50mg过氧化氢酶，NADP ⁺以及异丙醇，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的浓度为5g/L，NADP ⁺的浓度为0.03mM，异丙醇浓度为20mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，反应16小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-3-甲酸两种构型的浓度(g/L)。反应液中(S)-1，2，3，4-四氢异喹啉-3-甲酸的收率为82.1％(计算方式同实施例3)，ee值为99.4％。 Weigh 0.2g of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid into a 100ml reaction flask, add 10ml of phosphate buffer (50mM, pH=8.0) and mix well, then use 30% ammonia water Adjust the pH of the solution to 8.0. Add 20ml FpDAAO crude enzyme solution, 2ml PadpkA crude enzyme solution, 8ml alcohol dehydrogenase crude enzyme solution, 50mg catalase, NADP ⁺ and isopropanol to racemize the substrate in the initial reaction system. The concentration of 3,4-tetrahydroisoquinoline-3-carboxylic acid was 5 g/L, the concentration of NADP ⁺ was 0.03 mM, and the concentration of isopropyl alcohol was 20 mM. After mixing, immediately take a sample as "0 hours". Using a water bath to control the reaction temperature to 30 ℃, magnetic stirring, the reaction was sampled for 16 hours. The content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution The concentration of isoquinoline-3-carboxylic acid in two configurations (g/L). The yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction solution was 82.1% (calculated in the same manner as in Example 3), and the ee value was 99.4%.

实施例5 FgDAAO-PfdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸Example 5 FgDAAO-PfdpkA multi-enzyme coupling preparation (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

按照实施例2的方法，分别制备源自禾谷镰刀菌(Fusarium graminearum)CS3005的D-氨基酸氧化酶粗酶液、荧光假单胞菌(Pseudomonas fluorescens)Pf0-1的哌啶酸还原酶粗酶液以及枯草芽胞杆菌(Bacillus subtilis)168的葡萄糖脱氢酶粗酶液。According to the method of Example 2, a crude D-amino acid oxidase enzyme solution derived from Fusarium graminearum CS3005 and a crude pipecolic acid reductase enzyme from Pseudomonas fluorescens Pf0-1 were prepared respectively. And Bacillus subtilis (Bacillus subtilis) 168 glucose dehydrogenase crude enzyme liquid.

称取0.1g外消旋1，2，3，4-四氢异喹啉-3-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml FgDAAO粗酶液，2.5ml PfdpkA粗酶液，7.5ml葡萄糖脱氢酶粗酶液，20mg过氧化氢酶，NADP ⁺以及D-葡萄糖，使起始反应体系中的底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的浓度为2.5g/L，NADP ⁺的浓度为0.014mM，D-葡萄糖的浓度为10mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，氨水调节反应pH在8～8.5范围内，反应18小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-3-甲酸两种构型的浓度(g/L)。反应液中(S)-1，2，3，4-四氢异喹啉-3-甲酸的反应收率为79.8％(计算方式同实施例3)，ee值为99.1％。 Weigh 0.1g of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid into a 100ml reaction flask, add 10ml of phosphate buffer (50mM, pH=8.0) and mix well, then use 30% ammonia water Adjust the pH of the solution to 8.0. Add 20ml FgDAAO crude enzyme solution, 2.5ml PfdpkA crude enzyme solution, 7.5ml glucose dehydrogenase crude enzyme solution, 20mg catalase, NADP ⁺ and D-glucose to race the substrate in the initial reaction system 1 , The concentration of 2,3,4-tetrahydroisoquinoline-3-carboxylic acid is 2.5 g/L, the concentration of NADP ⁺ is 0.014 mM, and the concentration of D-glucose is 10 mM. After mixing, immediately take a sample as "0 hours". Using a water bath to control the reaction temperature to 30 ℃, magnetic stirring, ammonia water to adjust the reaction pH in the range of 8 ~ 8.5, the reaction was sampled for 18 hours. The content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution The concentration of isoquinoline-3-carboxylic acid in two configurations (g/L). The reaction yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction solution was 79.8% (calculated in the same manner as in Example 3), and the ee value was 99.1%.

实施例6 TvDAAO-PedpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸Example 6 TvDAAO-PedpkA multi-enzyme coupling preparation (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

按照实施例2的方法，分别制备源自三角酵母(Trigonopsis variabilis)CBS 4095的D-氨基酸氧化酶粗酶液、虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶粗酶液以及乳酸杆菌(Lactobacillus kefiri)DSM20587的醇脱氢酶粗酶液。According to the method of Example 2, crude D-amino acid oxidase enzyme solution derived from Trigonopsis variabilis CBS 4095 and Pseudomonas enteromophila str. L48 piperidine acid reductase crude enzyme were prepared respectively Solution and the crude enzyme solution of alcohol dehydrogenase of Lactobacillus kefiri DSM20587.

称取0.3g外消旋1，2，3，4-四氢异喹啉-3-甲酸至100ml反应瓶中，加10ml磷酸缓冲液(50mM，pH＝8.0)混匀后，用30％氨水调节溶液pH值到8.0。加入20ml TvDAAO粗酶液，3ml PedpkA粗酶液，7ml醇脱氢酶粗酶液，40mg过氧化氢酶，NADP ⁺以及异丙醇，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的浓度为7.5g/L，NADP ⁺的浓度为0.05mM，异丙醇浓度为25mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，反应48小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-3-甲酸两种构型的浓度(g/L)。反应液中(S)-1，2，3，4-四氢异喹啉-3-甲酸的反应收率(计算方式同实施例3)为78.6％，ee值为99.2％。 Weigh 0.3g of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid into a 100ml reaction flask, add 10ml of phosphate buffer (50mM, pH=8.0) and mix well, then use 30% ammonia water Adjust the pH of the solution to 8.0. Add 20ml TvDAAO crude enzyme solution, 3ml PedpkA crude enzyme solution, 7ml alcohol dehydrogenase crude enzyme solution, 40mg catalase, NADP ⁺ and isopropanol to racemize the substrate in the initial reaction system. The concentration of 3,4-tetrahydroisoquinoline-3-carboxylic acid was 7.5 g/L, the concentration of NADP ⁺ was 0.05 mM, and the concentration of isopropyl alcohol was 25 mM. After mixing, immediately take a sample as "0 hours". Using a water bath to control the reaction temperature to 30 ℃, magnetic stirring, the reaction was sampled for 48 hours. The content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution The concentration of isoquinoline-3-carboxylic acid in two configurations (g/L). The reaction yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction solution (calculated in the same manner as in Example 3) was 78.6%, and the ee value was 99.2%.

实施例7纯酶FsDAAO-PpdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸Example 7 Pure enzyme FsDAAO-PpdpkA multi-enzyme coupling preparation (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

底物溶液的配制：用50mM的磷酸盐缓冲溶液(pH＝8.0)配制5g/L外消旋1，2，3，4-四氢异喹啉-3-甲酸溶液并用30％氨水调节溶液pH至8.0。Preparation of substrate solution: Prepare a 5 g/L racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid solution with 50 mM phosphate buffer solution (pH=8.0) and adjust the solution pH with 30% ammonia water To 8.0.

取1.6ml底物溶液加入到5mL反应管中，再加入FsDAAO纯酶液，黄素腺嘌呤二核苷酸钠盐，过氧化氢酶，PpdpkA纯酶液，NADP ⁺，葡萄糖脱氢酶纯酶液，以及D-葡萄糖，并用磷酸盐缓冲溶液(50mM，pH＝8.0)将反应总体积补到2ml，使起始反应体系中底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的浓度为4g/L，FsDAAO纯酶的起始浓度为0.1mg/ml，FAD起始浓度为100μM，纯酶PpdpkA的起始浓度为0.1mg/ml，葡萄糖脱氢酶纯酶的起始浓度为0.1mg/ml，NADP ⁺的起始浓度为0.01mM，过氧化氢酶起始浓度为0.5mg/ml，D-葡萄糖的起始浓度为15mM。混匀后，立即取样，作为“0小时”。将反应管置于30℃恒温水浴中，磁力搅拌，反应5小时。反应结束后用高效液相色谱检测反应体系中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-3-甲酸两种构型的浓度(g/L)。(S)-1，2，3，4-四氢异喹啉-3-甲酸的反应收率为88.3％(计算方式同实施例3)，ee值达99.5％。 Take 1.6ml of substrate solution into a 5mL reaction tube, and then add FsDAAO pure enzyme solution, flavin adenine dinucleotide sodium salt, catalase, PpdpkA pure enzyme solution, NADP ⁺ , glucose dehydrogenase pure enzyme Solution, and D-glucose, and the total volume of the reaction was made up to 2 ml with phosphate buffer solution (50 mM, pH=8.0) to racemize the substrate 1,2,3,4-tetrahydroisoquine in the initial reaction system The concentration of porphyrin-3-carboxylic acid is 4g/L, the initial concentration of FsDAAO pure enzyme is 0.1mg/ml, the initial concentration of FAD is 100μM, the initial concentration of pure enzyme PpdpkA is 0.1mg/ml, and glucose dehydrogenase is pure The initial concentration of enzyme was 0.1 mg/ml, the initial concentration of NADP ⁺ was 0.01 mM, the initial concentration of catalase was 0.5 mg/ml, and the initial concentration of D-glucose was 15 mM. After mixing, immediately take a sample as "0 hours". The reaction tube was placed in a constant temperature water bath at 30°C, magnetically stirred, and reacted for 5 hours. After the reaction is completed, the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction system is detected by high-performance liquid chromatography, and then the 1,2,3,4 in the reaction solution can be known -The concentration of two configurations of tetrahydroisoquinoline-3-carboxylic acid (g/L). The reaction yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was 88.3% (calculated in the same way as in Example 3), and the ee value reached 99.5%.

实施例8 FsDAAO-PpdpkA多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸Example 8 FsDAAO-PpdpkA multi-enzyme coupling preparation (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

称取0.24g外消旋1，2，3，4-四氢异喹啉-3-甲酸至100ml反应瓶中，加10ml磷酸缓冲液 (50mM，pH＝8.0)混匀后，用5M氢氧化钠溶液调节溶液pH值到8.0。加入20ml FsDAAO粗酶液，3ml PpdpkA粗酶液，7ml葡萄糖脱氢酶粗酶液，20mg过氧化氢酶，NADP ⁺以及D-葡萄糖，使底物外消旋1，2，3，4-四氢异喹啉-3-甲酸的起始浓度为6g/L，NADP ⁺的起始浓度为0.04mM，D-葡萄糖起始浓度为20mM。混匀后，立即取样，作为“0小时”。利用水浴控制反应温度为30℃，磁力搅拌，0.5M氢氧化钠溶液调节反应pH在8～8.5范围内，反应24小时取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知反应液中1，2，3，4-四氢异喹啉-3-甲酸两种构型的浓度(g/L)。(S)-1，2，3，4-四氢异喹啉-3-甲酸的反应收率为84.9％(计算方式同实施例3)，ee值为99.2％。 Weigh 0.24g of racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid into a 100ml reaction flask, add 10ml of phosphate buffer (50mM, pH=8.0) and mix well, then use 5M hydroxide The sodium solution adjusts the pH of the solution to 8.0. Add 20ml FsDAAO crude enzyme solution, 3ml PpdpkA crude enzyme solution, 7ml glucose dehydrogenase crude enzyme solution, 20mg catalase, NADP ⁺ and D-glucose to race the substrate 1,2,3,4-tetra The initial concentration of hydroisoquinoline-3-carboxylic acid was 6 g/L, the initial concentration of NADP ⁺ was 0.04 mM, and the initial concentration of D-glucose was 20 mM. After mixing, immediately take a sample as "0 hours". Using a water bath to control the reaction temperature to 30 ℃, magnetic stirring, 0.5M sodium hydroxide solution to adjust the reaction pH in the range of 8 ~ 8.5, 24 hours of reaction sampling. The content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography can be obtained by knowing the 1,2,3,4-tetrahydro in the reaction solution The concentration of isoquinoline-3-carboxylic acid in two configurations (g/L). The reaction yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was 84.9% (calculated in the same way as in Example 3), and the ee value was 99.2%.

实施例9(S)-1，2，3，4-四氢异喹啉-3-甲酸的制备与分离Example 9 Preparation and separation of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid

底物溶液及反应体系如实施例3。The substrate solution and the reaction system are as in Example 3.

反应结束后，将反应体系的pH值调至5.0-6.0。99℃水浴，待蛋白变性析出后，抽滤。取滤液于65℃条件下旋蒸，将反应体积浓缩10倍。置于冰上，冷却后，抽滤。将析出的白色晶体，小心刮下，置于烘箱中，烘干并称重。称取0.05g白色烘干晶体，用50mM磷酸盐缓冲溶液(pH＝8.0)定容至50ml，取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知分离后产品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量。(S)-1，2，3，4-四氢异喹啉-3-甲酸的分离产率为75.1％(分离产率＝实际分离得到的产物的量(mg)/理论的产物的量(mg)×100％)，ee值达99.8％。After the reaction, the pH value of the reaction system was adjusted to 5.0-6.0. 99 ℃ water bath, after protein denaturation and precipitation, suction filtration. The filtrate was taken to be rotary evaporated at 65°C, and the reaction volume was concentrated 10 times. Place on ice, after cooling, filter with suction. The precipitated white crystals were carefully scraped off, placed in an oven, dried and weighed. Weigh 0.05g of white dried crystals, dilute to 50ml with 50mM phosphate buffer solution (pH=8.0), and take a sample. The content of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the sample taken by high performance liquid chromatography is detected, and then the 1,2,3,4-tetra The content of hydrogen isoquinoline-3-carboxylic acid in two configurations. The isolated yield of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid is 75.1% (isolated yield = amount of product actually isolated (mg) / amount of theoretical product ( mg)×100%), the ee value reaches 99.8%.

对比例1 FsDAAO制备(S)-1，2，3，4-四氢异喹啉-3-甲酸Comparative Example 1 Preparation of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by FsDAAO

底物溶液的配制：用50mM的磷酸盐缓冲溶液(pH＝8.0)配制5g/L的外消旋1，2，3，4-四氢异喹啉-3-甲酸溶液并用30％氨水调节溶液pH至8.0。Preparation of substrate solution: Prepare a 5 g/L racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid solution with 50 mM phosphate buffer solution (pH=8.0) and adjust the solution with 30% ammonia water pH to 8.0.

取1.6ml底物溶液加入到5mL反应管中，再加入0.4mL FsDAAO粗酶液(粗酶液中已含有足量辅酶FAD，因此，粗酶液反应体系中不需额外添加FAD)。混匀后，取样，作为“0小时”并进行HPLC分析。将反应管置于30℃恒温水浴中，磁力搅拌，反应24小时。反应结束后用HPLC法检测反应体系中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量，即可得知反应体系中1，2，3，4-四氢异喹啉-3-甲酸两种构型的浓度(g/L)。Take 1.6ml of substrate solution into a 5mL reaction tube, and then add 0.4mL of FsDAAO crude enzyme solution (the crude enzyme solution already contains a sufficient amount of coenzyme FAD, so no additional FAD is needed in the crude enzyme solution reaction system). After mixing, take a sample as "0 hours" and perform HPLC analysis. The reaction tube was placed in a constant temperature water bath at 30°C, magnetically stirred, and reacted for 24 hours. After the reaction, the content of the two configurations of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the reaction system is detected by HPLC, and then the 1,2,3,4-tetra The concentration of two configurations of hydrogen isoquinoline-3-carboxylic acid (g/L).

FsDAAO表现出严格的R-构型立体选择性，转化率为49.9％(转化率＝[(初始外消旋底物的浓度(g/L)-残余的底物浓度(g/L))/初始外消旋底物的浓度(g/L)]×100％)，(S)-1，2，3，4-四氢异喹啉-3-甲酸的ee值达99％以上。FsDAAO exhibits strict R-configuration stereoselectivity with a conversion rate of 49.9% (conversion rate = [(initial racemic substrate concentration (g/L)-residual substrate concentration (g/L))/ The initial racemic substrate concentration (g/L)]×100%), (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid has an ee value of more than 99%.

对比例2 FsDAAO-NH ₃·BH ₃制备(S)-1，2，3，4-四氢异喹啉-3-甲酸 Comparative Example 2 Preparation of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid by FsDAAO-NH ₃ ·BH ₃

底物溶液的配制：用50mM的磷酸盐缓冲溶液(pH8.0)配制5g/L外消旋1，2，3，4-四氢异喹啉-3-甲酸溶液并用30％氨水调节溶液pH至8.0。Preparation of substrate solution: prepare 50g/L racemic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid solution with 50mM phosphate buffer solution (pH8.0) and adjust the solution pH with 30% ammonia water To 8.0.

向100mL反应器中加入24mL底物溶液，6mL FsDAAO粗酶液(粗酶液中已含有足量辅酶FAD，因此，粗酶液反应体系中不需额外添加FAD)，12mg过氧化氢酶冻干粉末和0.4g NH ₃·BH ₃。混匀后，立即取样，作为“0小时”。将反应体系置于30℃恒温水浴中，磁力搅拌，反应24小时，取样。高效液相色谱检测所取样品中1，2，3，4-四氢异喹啉-3-甲酸两种构型的含量。FsDAAO表现出严格的R-构型立体选择性，反应收率为81.2％， (S)-1，2，3，4-四氢异喹啉-3-甲酸的ee值为99.2％。 Add 24mL substrate solution to the 100mL reactor, 6mL FsDAAO crude enzyme solution (the crude enzyme solution already contains sufficient coenzyme FAD, so no additional FAD is needed in the crude enzyme solution reaction system), 12mg catalase lyophilization Powder and 0.4g NH ₃ · BH ₃ . After mixing, immediately take a sample as "0 hours". The reaction system was placed in a constant temperature water bath at 30°C, magnetically stirred, and reacted for 24 hours, and a sample was taken. The content of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the taken samples was detected by high performance liquid chromatography. FsDAAO showed strict R-configuration stereoselectivity, the reaction yield was 81.2%, and the ee value of (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid was 99.2%.

上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。The above embodiments are only for explaining the technical concept and features of the present invention, and the purpose thereof is to allow those familiar with the technology to understand the content of the present invention and implement it accordingly, but not to limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

一种多酶耦合制备(S)-1，2，3，4-四氢异喹啉-3-甲酸(I)的方法，A method for preparing (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (I) by multiple enzyme coupling,

所述方法包括如下步骤：The method includes the following steps:

以1，2，3，4-四氢异喹啉-3-甲酸外消旋体或1，2，3，4-四氢异喹啉-3-甲酸盐的外消旋体为底物，使该底物中(R)-1，2，3，4-四氢异喹啉-3-甲酸在氧化脱氢酶的催化作用下反应生成式(II)所示的亚胺酸；1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid racemate or 1,2,3,4-Tetrahydroisoquinoline-3-carboxylate racemate as substrate , The (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid in the substrate is reacted under the catalytic action of oxidative dehydrogenase to form the imidic acid represented by formula (II);

使所述式(II)所示的亚胺酸在哌啶酸还原酶与能够供给氢负离子的辅酶的存在下转化为所述(S)-1，2，3，4-四氢异喹啉-3-甲酸。The imine acid represented by the formula (II) is converted into the (S)-1,2,3,4-tetrahydroisoquinoline in the presence of pipecolic acid reductase and a coenzyme capable of supplying hydride ions -3-carboxylic acid.
如权利要求1所述的方法，其特征在于，所述1，2，3，4-四氢异喹啉-3-甲酸盐为1，2，3，4-四氢异喹啉-3-甲酸的碱金属盐或铵盐。The method according to claim 1, wherein the 1,2,3,4-tetrahydroisoquinoline-3-carboxylate is 1,2,3,4-tetrahydroisoquinoline-3 -Alkali metal salts or ammonium salts of formic acid.
如权利要求1所述的方法，其特征在于，所述氧化脱氢酶是能够选择性催化(R)-1，2，3，4-四氢异喹啉-3-甲酸的酶，且选择性大于等于80％，优选大于等于90％。The method of claim 1, wherein the oxidative dehydrogenase is an enzyme capable of selectively catalyzing (R)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and is selected The property is 80% or more, preferably 90% or more.
如权利要求1或3所述的方法，其特征在于，所述氧化脱氢酶为D-氨基酸氧化酶。The method according to claim 1 or 3, wherein the oxidative dehydrogenase is D-amino acid oxidase.
如权利要求4所述的方法，其特征在于，所述D-氨基酸氧化酶为选自如下D-氨基酸氧化酶中的一种或多种的组合：来源于三角酵母(Trigonopsis variabilis)CBS 4095的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自禾谷镰刀菌(Fusarium graminearum)CS3005的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶、来自梨孢镰刀菌(Fusarium poae)2516的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶，来自茄病镰刀菌(Fusarium solani)M-0718的D-氨基酸氧化酶或其突变体或与其氨基酸序列同源性大于80％的其它D-氨基酸氧化酶。The method of claim 4, wherein the D-amino acid oxidase is a combination of one or more selected from the following D-amino acid oxidases: derived from Trigonopsis variabilis CBS 4095 D-amino acid oxidase or its mutants or other D-amino acid oxidases with amino acid sequence homology greater than 80%, D-amino acid oxidase from Fusarium graminearum CS3005 or its mutants or its amino acids Other D-amino acid oxidases with sequence homology greater than 80%, D-amino acid oxidases from Fusarium poae 2516 or mutants thereof or other D-amino acids with amino acid sequence homology greater than 80% Oxidase, D-amino acid oxidase from Fusarium solani M-0718 or its mutants or other D-amino acid oxidases with amino acid sequence homology greater than 80%.
如权利要求4所述的方法，其特征在于，所述D-氨基酸氧化酶的使用形式为离体的D-氨基酸氧化酶，或者离体的D-氨基酸氧化酶的粗酶液或者纯酶或者固定化酶，或胞内表达D-氨基酸氧化酶的细胞。The method according to claim 4, characterized in that the use form of the D-amino acid oxidase is isolated D-amino acid oxidase, or a crude enzyme solution or pure enzyme of isolated D-amino acid oxidase or Immobilized enzymes, or cells that express D-amino acid oxidase intracellularly.
如权利要求6所述的方法，其特征在于，所述细胞为表达D-氨基酸氧化酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述D-氨基酸氧化酶基因连接在所述表达载体pET-28a(+)上。The method according to claim 6, wherein the cell is an engineered bacterium that expresses D-amino acid oxidase and contains an expression vector pET-28a(+), and the host cell of the engineered bacterium is E. coli BL21 ( DE3); wherein, the D-amino acid oxidase gene is linked to the expression vector pET-28a(+).
如权利要求1所述的方法，其特征在于，所述哌啶酸还原酶为选自如下哌啶酸还原酶中的一中或多种的组合：来源于恶臭假单胞菌(Pseudomonas putida)KT2440的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于绿脓杆菌 (Pseudomonas aeruginosa)PAO1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于荧光假单胞菌(Pseudomonas fluorescens)Pf0-1的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶、来源于虫媒假单胞菌(Pseudomonas entomophila str.)L48的哌啶酸还原酶或其突变体或与其氨基酸序列同源性大于80％的哌啶酸还原酶。The method of claim 1, wherein the pipecolic acid reductase is a combination of one or more selected from the following pipecolic acid reductases: derived from Pseudomonas putida KT2440 pipecolic acid reductase or its mutants or pipecolic acid reductase with amino acid sequence homology greater than 80%, pipecolic acid reductase derived from Pseudomonas aeruginosa PAO1 or its mutants or its Pipecolic acid reductase with amino acid sequence homology greater than 80%, pipecolic acid reductase derived from Pseudomonas fluorescens Pf0-1 or its mutants or homology with its amino acid sequence greater than 80% Pipecolic acid reductase, pipecolic acid reductase derived from Pseudomonas (Pseudomonas entomophila str.) L48 or a mutant thereof or a pipecolic acid reductase with a homology greater than 80% in amino acid sequence.
如权利要求1所述的方法，其特征在于，所述哌啶酸还原酶的使用形式为离体的哌啶酸还原酶，含有离体的哌啶酸还原酶的粗酶液或者纯酶或者固定化酶，或胞内表达哌啶酸还原酶的细胞。The method according to claim 1, characterized in that the piperidinate reductase is used in the form of isolated pipecolic acid reductase, a crude enzyme solution or pure enzyme containing the isolated pipecolic acid reductase or Immobilized enzymes, or cells expressing pipecolic acid reductase intracellularly.
如权利要求9所述的方法，其特征在于，所述细胞为表达哌啶酸还原酶且含有表达载体pET-28a(+)的工程菌，所述工程菌的宿主细胞为E.coli BL21(DE3)；其中，所述哌啶酸还原酶基因连接在所述表达载体pET-28a(+)上。The method according to claim 9, wherein the cell is an engineered bacterium expressing pipecolic acid reductase and contains an expression vector pET-28a(+), and the host cell of the engineered bacterium is E. coli BL21( DE3); wherein the pipecolic acid reductase gene is linked to the expression vector pET-28a(+).
如权利要求1所述的方法，其特征在于，所述能够供给氢负离子的辅酶为NADH和/或NADPH。The method according to claim 1, wherein the coenzyme capable of supplying hydride ions is NADH and/or NADPH.
如权利要求1所述的方法，其特征在于，使所述生成亚胺酸的反应还在黄素腺嘌呤二核苷酸(FAD)的存在下进行。The method according to claim 1, wherein the reaction for generating imidic acid is also carried out in the presence of flavin adenine dinucleotide (FAD).
如权利要求1所述的方法，其特征在于，使所述生成亚胺酸的反应还在过氧化氢酶的存在下进行。The method according to claim 1, wherein the reaction for generating imidic acid is also carried out in the presence of catalase.
如权利要求1所述的方法，其特征在于，使生成所述亚胺酸的反应在设定温度和有氧环境中进行，所述设定温度为20～70℃。The method according to claim 1, characterized in that the reaction for producing the imidic acid is performed in a set temperature and an aerobic environment, and the set temperature is 20 to 70°C.
如权利要求1或14所述的方法，其特征在于，所述方法的实施过程包括：首先构建反应体系，然后控制所述反应体系处于设定温度和有氧环境中进行反应，所述反应体系包括所述底物、所述氧化脱氢酶、所述哌啶酸还原酶、辅酶、辅酶再生***、溶剂，所述反应体系还选择性地包括pH缓冲剂和/或pH调节剂，所述辅酶包括NAD ⁺和/或NADH，或者，所述辅酶包括NADP ⁺和/或NADPH。 The method according to claim 1 or 14, wherein the implementation process of the method comprises: firstly constructing a reaction system, and then controlling the reaction system to perform the reaction in a set temperature and an aerobic environment, the reaction system Including the substrate, the oxidative dehydrogenase, the pipecolic acid reductase, coenzyme, coenzyme regeneration system, solvent, the reaction system also optionally includes a pH buffer and/or pH regulator, The coenzyme includes NAD ⁺ and/or NADH, or the coenzyme includes NADP ⁺ and/or NADPH.
如权利要求15所述的方法，其特征在于，所述辅酶再生***包括辅酶再生酶和辅酶再生底物。The method of claim 15, wherein the coenzyme regeneration system includes a coenzyme regeneration enzyme and a coenzyme regeneration substrate.
如权利要求16所述的方法，其特征在于，所述辅酶再生酶为葡萄糖脱氢酶，所述辅酶再生底物为葡萄糖；或者，所述辅酶再生酶为醇脱氢酶，所述辅酶再生底物为异丙醇。The method of claim 16, wherein the coenzyme regenerating enzyme is glucose dehydrogenase and the coenzyme regenerating substrate is glucose; or, the coenzyme regenerating enzyme is alcohol dehydrogenase and the coenzyme regenerating The substrate is isopropyl alcohol.
如权利要求17所述的方法，其特征在于，所述葡萄糖脱氢酶来源于枯草芽胞杆菌Bacillus subtilis 168；和/或，所述醇脱氢酶来源于乳酸杆菌Lactobscillus kefir DSM20587。The method according to claim 17, wherein the glucose dehydrogenase is derived from Bacillus subtilis Bacillus 168; and/or the alcohol dehydrogenase is derived from Lactobacillus Lactobscillus kefir DSM20587.
如权利要求15所述的方法，其特征在于，所述反应体系还包括过氧化氢酶。The method of claim 15, wherein the reaction system further comprises catalase.
如权利要求19所述的方法，其特征在于，所述过氧化氢酶为牛肝过氧化氢酶冻干粉。The method of claim 19, wherein the catalase is bovine liver catalase lyophilized powder.
如权利要求15所述的方法，其特征在于，所述反应体系还包括黄素腺嘌呤二核苷酸。The method of claim 15, wherein the reaction system further comprises flavin adenine dinucleotide.