CN116334153A - Reductive amination enzyme and preparation method and application thereof - Google Patents

Reductive amination enzyme and preparation method and application thereof Download PDF

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CN116334153A
CN116334153A CN202111580432.0A CN202111580432A CN116334153A CN 116334153 A CN116334153 A CN 116334153A CN 202111580432 A CN202111580432 A CN 202111580432A CN 116334153 A CN116334153 A CN 116334153A
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冯雁
张凯
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Abstract

本发明提供了还原胺化酶及其制法和应用。具体地,本发明涉及一种还原胺化酶及其功能鉴定及体外定向进化。本发明的还原胺化酶是来自卡利曲霉菌(Aspergillus calidoustus)的还原胺化酶AcRedAm及其突变体(包括对产物雷沙吉兰对映选择性显著提高的还原胺化酶突变体)。本发明还提供了体外还原胺化方法,包括步骤:(i)在还原胺化酶AcRedAm存在下,使得(S1)酮底物或醛底物与(S2)胺底物进行还原胺化反应。本发明的酶和突变体可用于体外酶法合成对映纯雷沙吉兰等不同的产物。The invention provides reductive aminase and its preparation method and application. Specifically, the present invention relates to a reductive aminase and its function identification and in vitro directed evolution. The reductive amination enzyme of the present invention is the reductive amination enzyme AcRedAm from Aspergillus calidoustus and its mutant (including the reductive amination enzyme mutant whose enantioselectivity to the product rasagiline is significantly improved). The present invention also provides an in vitro reductive amination method, comprising the step of: (i) making (S1) ketone substrate or aldehyde substrate and (S2) amine substrate undergo reductive amination reaction in the presence of reductive amination enzyme AcRedAm. The enzyme and mutants of the present invention can be used in the in vitro enzymatic synthesis of different products such as enantiopure rasagiline.

Description

还原胺化酶及其制法和应用Reductive aminase and its preparation method and application

技术领域technical field

本发明属于分子生物学和生物催化领域,具体地涉及还原胺化酶及其制法和应用。本发明提供了一种新型的还原胺化酶及其突变体,以及在合成雷沙吉兰等化合物中的应用。The invention belongs to the fields of molecular biology and biocatalysis, and in particular relates to reductive aminase and its preparation method and application. The invention provides a novel reductive aminase and its mutant, as well as its application in the synthesis of compounds such as rasagiline.

背景技术Background technique

手性胺是众多天然产物、活性药物和其他高附加值化学品的重要组成部分。近年来,手性胺类药物约占FDA批准的新药的40%,其市场价值在2020年将近达到140亿美元。目前虽然许多用于制备手性胺的化学策略已经建立,但这些策略会受到诸如反应效率低、选择性低和不利环境发展等的限制。相比之下,来自可再生资源的酶可以提供出色的立体选择性和区域选择性,并在温和的水溶液条件下进行催化反应。另外,酶介导的生物催化还可以在不使用有毒试剂、不需要大量保护和去保护反应步骤的情况下进行,因此开发新型酶用作发展绿色化学的催化剂是当今生物合成催化领域的研究热点。Chiral amines are important building blocks of many natural products, active pharmaceuticals, and other high value-added chemicals. In recent years, chiral amine drugs have accounted for about 40% of new drugs approved by the FDA, and their market value will reach nearly 14 billion US dollars in 2020. Although many chemical strategies for the preparation of chiral amines have been established, these strategies suffer from limitations such as low reaction efficiency, low selectivity, and unfavorable environmental development. In contrast, enzymes from renewable resources can provide excellent stereoselectivity and regioselectivity, and perform catalytic reactions under mild aqueous conditions. In addition, enzyme-mediated biocatalysis can also be carried out without the use of toxic reagents and without the need for a large number of protection and deprotection reaction steps, so the development of new enzymes as catalysts for the development of green chemistry is a research hotspot in the field of biosynthetic catalysis .

在过去的20年中,大量用于合成手性胺的酶催化途径已经被开发,其中催化前手性酮还原胺化为胺的转氨酶(TAs)、胺脱氢酶(AmDHs)和亚胺还原酶(IRED)都引起了科学家相当大的兴趣。但相关研究表明TAs和AmDHs目前都仅限于伯胺的合成,需要随后化学步骤的烷基化来合成手性仲胺和叔胺。而IRED虽然可以催化NAD(P)H依赖的前手性亚胺还原为手性胺,但其更偏好于还原环状亚胺底物,对前手性酮胺化的转化率比较低。Over the past 20 years, a large number of enzymatic pathways for the synthesis of chiral amines have been developed, in which transaminases (TAs), amine dehydrogenases (AmDHs) and imine reductions that catalyze the reductive amination of prochiral ketones to amines have been developed. Enzymes (IREDs) have aroused considerable interest among scientists. However, related studies have shown that both TAs and AmDHs are currently limited to the synthesis of primary amines, requiring subsequent chemical steps of alkylation to synthesize chiral secondary and tertiary amines. Although IRED can catalyze the reduction of NAD(P)H-dependent prochiral imines to chiral amines, it prefers to reduce cyclic imine substrates, and the conversion rate of prochiral ketone amination is relatively low.

值得注意的是,Turner等人在2017年首次详细报道了来自米曲霉(Aspergillusoryzae)中的一种NADPH依赖性酶AspRedAm。该酶作为IRED的一个亚家族被命名为还原胺化酶(RedAm),其可以在水性介质中催化更广泛的酮和胺的分子间还原胺化反应。与多步化学路线和其他生物催化剂(包括TA和AmDH)相比,还原胺化酶RedAm直接催化酮和胺分子缩合生成手性胺表现出相当高的效率,具有很高的原子经济节约性。特别是,AspRedAm能够直接催化1-茚酮和炔丙胺反应生成抗帕金森病药物(R)-雷沙吉兰,并且在某些情况下,AspRedAm在底物酮与胺的比例低至1:1的情况下仍显示出高反应活性。随后,该研究小组还报道了真菌来源的两种新型耐热RedAms,它们可以利用廉价的铵盐作为胺供体来生产伯胺并在温和条件下进行连续流动的生物转化。通过利用NADPH辅因子再生系统和多种胺作为氨基供体,RedAm催化的还原胺化过程可以最大限度地提高原子经济性,从而有助于环境可持续发展。这些研究结果表明,还原胺化酶(RedAm)具有突出的工业优势,和巨大的应用潜力。It is worth noting that Turner et al first reported in detail a NADPH-dependent enzyme AspRedAm from Aspergillus oryzae in 2017. As a subfamily of IRED, this enzyme is named reductive amination enzyme (RedAm), which can catalyze a wider range of intermolecular reductive amination reactions of ketones and amines in aqueous media. Compared with multi-step chemical routes and other biocatalysts (including TA and AmDH), the reductive amination enzyme RedAm directly catalyzed the condensation of ketone and amine molecules to generate chiral amines exhibited considerable efficiency with high atom economy. In particular, AspRedAm can directly catalyze the reaction of 1-indanone and propargylamine to generate the antiparkinsonian drug (R)-rasagiline, and in some cases, AspRedAm has a substrate ketone-to-amine ratio as low as 1: 1 still showed high reactivity. Subsequently, the research group also reported two novel heat-tolerant RedAms from fungi, which can utilize cheap ammonium salts as amine donors to produce primary amines and perform continuous-flow biotransformation under mild conditions. By utilizing the NADPH cofactor regeneration system and a variety of amines as amino donors, the RedAm-catalyzed reductive amination process can maximize atom economy, thereby contributing to environmental sustainability. These research results show that reductive aminase (RedAm) has outstanding industrial advantages and great application potential.

目前,研究报道的还原胺化酶(RedAm)数量有限,难以满足对工业化应用的定制需求,因此本领域迫切需要开发新型还原胺化酶。At present, the number of reductive amination enzymes (RedAm) reported in research is limited, and it is difficult to meet the customization requirements for industrial applications. Therefore, there is an urgent need to develop new reductive amination enzymes in this field.

发明内容Contents of the invention

本发明的目的就是提供一种新型还原胺化酶(RedAm)及其制法和应用。The object of the present invention is to provide a novel reductive aminase (RedAm) and its preparation method and application.

在本发明的第一方面,提供了一种体外还原胺化方法,包括步骤:In a first aspect of the present invention, an in vitro reductive amination method is provided, comprising the steps of:

(i)在还原胺化酶AcRedAm存在下,使得(S1)酮底物或醛底物与(S2)胺底物进行还原胺化反应。(i) Reductive amination reaction of (S1) ketone substrate or aldehyde substrate and (S2) amine substrate in the presence of reductive amination enzyme AcRedAm.

在另一优选例中,所述方法包括:In another preferred example, the method includes:

在还原胺化酶AcRedAm存在下,使式Z1所示的酮底物或醛底物和式Z2所示的胺底物进行还原胺化反应,从而形成式I所示的还原胺化产物:In the presence of reductive amination enzyme AcRedAm, the ketone substrate or aldehyde substrate shown in formula Z1 and the amine substrate shown in formula Z2 are subjected to reductive amination reaction, thereby forming the reductive amination product shown in formula I:

Figure BDA0003426841710000021
Figure BDA0003426841710000021

其中in

R1为取代或未取代的烷基,取代或未取代的芳基、取代或未取代的(C1-C6亚烃基)-苯基,其中所述的取代指出一个或多个H原子被选自下组的取代基所取代:C1-C3烷基、C2-C4烯基、C2-C4炔基、C3-C6环烷基、卤素、或其组合;R1 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted (C1-C6 alkylene)-phenyl group, wherein the substitution indicates that one or more H atoms are selected from the following Substituents of the group are substituted by: C1-C3 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, halogen, or a combination thereof;

R2为H或甲基(CH3);R2 is H or methyl (CH3);

或者R1和R2与相连的C原子共同构成取代或未取代的4-10元的杂环;Or R1 and R2 together with the connected C atoms form a substituted or unsubstituted 4-10 membered heterocyclic ring;

R3为H、取代或未取代的C1-C8烷基,取代或未取代的C2-C8烯基、取代或未取代的C2-C8炔基、取代或未取代的C3-C8环烷基、取代或未取代的6-10芳基、取代或未取代的(C1-C6亚烃基)-苯基、取代或未取代的(C3-C6亚环烷基)-苯基,其中,所述的取代指出一个或多个H原子被选自下组的取代基所取代:C1-C3烷基、C2-C4烯基、C2-C4炔基、C3-C6环烷基、卤素、或其组合。R3 is H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C2-C8 alkenyl, substituted or unsubstituted C2-C8 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted Or unsubstituted 6-10 aryl, substituted or unsubstituted (C1-C6 alkylene)-phenyl, substituted or unsubstituted (C3-C6 cycloalkylene)-phenyl, wherein the substitution Indicates that one or more H atoms are substituted by a substituent selected from the group consisting of C1-C3 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, halogen, or combinations thereof.

在另一优选例中,所述的4-10元的杂环包括饱和或不饱和的杂环,或苯并的5-7杂环。In another preferred example, the 4-10 membered heterocycles include saturated or unsaturated heterocycles, or benzo 5-7 heterocycles.

在另一优选例中,所述的取代指一个或多个H原子被选自下组的取代基所取代:C1-C3烷基、C3-C6环烷基、卤素、C2-C6酯基、或其组合。In another preferred example, the substitution means that one or more H atoms are replaced by substituents selected from the group consisting of: C1-C3 alkyl, C3-C6 cycloalkyl, halogen, C2-C6 ester group, or a combination thereof.

在另一优选例中,所述的酮底物或醛底物选自下组:In another preferred embodiment, the ketone substrate or aldehyde substrate is selected from the following group:

Figure BDA0003426841710000031
Figure BDA0003426841710000031

在另一优选例中,所述的胺底物选自下组:In another preference, the amine substrate is selected from the following group:

Figure BDA0003426841710000032
Figure BDA0003426841710000032

在另一优选例中,所述的式I所示的还原胺化产物为手性胺化合物。In another preferred example, the reductive amination product represented by formula I is a chiral amine compound.

在另一优选例中,所述的式I所示的还原胺化产物选自下组:雷沙吉兰(9e)、N-乙基环己胺(4b)、N-环丙基环己胺(4f)、N-苄基环己胺(4i)、苄基丙-2-炔基胺(6e)、N-苄基苯胺(6h)、N-环丙基-4氟苯丙胺(8f)。In another preferred example, the reductive amination product represented by formula I is selected from the group consisting of rasagiline (9e), N-ethylcyclohexylamine (4b), N-cyclopropylcyclohexylamine Amine (4f), N-benzylcyclohexylamine (4i), benzylprop-2-ynylamine (6e), N-benzylaniline (6h), N-cyclopropyl-4-fluoroamphetamine (8f) .

在另一优选例中,所述的还原胺化酶AcRedAm来自或衍生自卡利曲霉菌(Aspergillus calidoustus)。In another preferred embodiment, the reductive amination enzyme AcRedAm is from or derived from Aspergillus calidoustus.

在另一优选例中,所述的还原胺化酶AcRedAm选自下组:In another preferred embodiment, the reductive amination enzyme AcRedAm is selected from the following group:

(a)具有SEQ ID NO:1所示氨基酸序列的多肽;(a) a polypeptide having the amino acid sequence shown in SEQ ID NO:1;

(b)将SEQ ID NO:1所示氨基酸序列的多肽经过一个或多个(如1-10个)氨基酸残基的取代、缺失或添加而形成的、或是添加信号肽序列后形成的、并具有还原胺化活性的衍生多肽;(b) The polypeptide of the amino acid sequence shown in SEQ ID NO: 1 is formed by substituting, deleting or adding one or more (such as 1-10) amino acid residues, or is formed after adding a signal peptide sequence, and a derivative polypeptide having reductive amination activity;

(c)序列中含有(a)或(b)中所述多肽序列的衍生多肽;(c) a derivative polypeptide containing the polypeptide sequence described in (a) or (b) in its sequence;

(d)氨基酸序列与SEQ ID NO:1所示氨基酸序列的同源性≥85%或≥90%(较佳地≥95%),并具有还原胺化活性的衍生多肽。(d) A derivative polypeptide whose amino acid sequence is ≥85% or ≥90% (preferably ≥95%) homologous to the amino acid sequence shown in SEQ ID NO: 1 and has reductive amination activity.

在另一优选例中,所述的序列(c)为由(a)或(b)添加了标签序列、信号序列或分泌信号序列后所形成的融合蛋白。In another preferred example, the sequence (c) is a fusion protein formed by adding a tag sequence, signal sequence or secretion signal sequence to (a) or (b).

在另一优选例中,所述的多肽为SEQ ID NO:1所示氨基酸序列的多肽。In another preferred example, the polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO:1.

在本发明的第二方面,提供了一种分离的或纯化的还原胺化酶,所述的还原胺化酶具有SEQ ID No:1所示的序列,并且具有至少1个氨基酸突变。In the second aspect of the present invention, an isolated or purified reductive amination enzyme is provided, the reductive amination enzyme has the sequence shown in SEQ ID No: 1 and has at least one amino acid mutation.

在另一优选例中,所述的还原胺化酶具有以下活性:催化(s1)酮底物或醛底物与(s2)胺底物进行还原胺化反应。In another preferred example, the reductive amination enzyme has the following activity: catalyzing the reductive amination reaction between (s1) ketone substrate or aldehyde substrate and (s2) amine substrate.

在另一优选例中,所述的还原胺化酶催化式Z1所示的酮底物或醛底物和式Z2所示的胺底物进行还原胺化反应,从而形成式I所示的还原胺化产物。In another preferred example, the reductive amination enzyme catalyzes the reductive amination reaction between the ketone substrate or aldehyde substrate represented by formula Z1 and the amine substrate represented by formula Z2, thereby forming the reduced aminated product.

Figure BDA0003426841710000041
Figure BDA0003426841710000041

在另一优选例中,所述的还原胺化酶包括野生型和突变型。In another preferred example, the reductive aminase includes wild type and mutant type.

在另一优选例中,所述的突变型的还原胺化酶在选自下组的位点具有突变:SEQID No:1所示序列的第207位、第214位、237位或其组合。In another preferred example, the mutant reductive aminase has a mutation at a position selected from the group consisting of position 207, position 214, position 237 of the sequence shown in SEQ ID No: 1 or a combination thereof.

在另一优选例中,上述位点中的原有氨基酸残基被另一种氨基酸残基替换,优选被选自下组的氨基酸替换:丙氨酸、丝氨酸、甘氨酸或半胱氨酸。In another preferred example, the original amino acid residue in the above position is replaced by another amino acid residue, preferably an amino acid selected from the group consisting of alanine, serine, glycine or cysteine.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:W207I、W207K、W207N、W207A、W207R、W207G、W207S、W207V、W207L、W207H、W207M、W207C、W207P、W207E、W207T、W207Q。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of W207I, W207K, W207N, W207A, W207R, W207G, W207S, W207V, W207L, W207H, W207M, W207C, W207P , W207E, W207T, W207Q.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:Y214M、Y214T、Y214S、Y214N、Y214G、Y214H、Y214V、Y214R、Y214Q、Y214A、Y214L、Y214E、Y214F、Y214K、Y214I、Y214D、Y214W、Y214C。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of Y214M, Y214T, Y214S, Y214N, Y214G, Y214H, Y214V, Y214R, Y214Q, Y214A, Y214L, Y214E, Y214F , Y214K, Y214I, Y214D, Y214W, Y214C.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:Q237N、Q237S、Q237A、Q237G。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of Q237N, Q237S, Q237A, and Q237G.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:W207C、Q237A。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of W207C and Q237A.

在另一优选例中,所述的突变型的还原胺化酶为双突变或三突变的还原胺化酶。In another preferred example, the mutant reductive amination enzyme is a double-mutant or triple-mutant reductive aminating enzyme.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:W207S/Y214C。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of W207S/Y214C.

在另一优选例中,所述的突变型的还原胺化酶进一步地在选自下组的位点具有突变:SEQ ID No:1所示序列的第117位、第90位、172位、236位或其组合。In another preferred example, the mutant reductive aminase further has a mutation at a position selected from the group consisting of the 117th, 90th, 172nd, 172nd, 236 bits or a combination thereof.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:I117L、I117Y、I117C、I117A、I117N、I117E、I117T、I117V或其组合。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of I117L, I117Y, I117C, I117A, I117N, I117E, I117T, I117V or combinations thereof.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:L90I、L90C、L90Y、L90M、L90H、L90F、L90A、L90T、L90V或其组合。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of L90I, L90C, L90Y, L90M, L90H, L90F, L90A, L90T, L90V or combinations thereof.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:L172E、L172I、L172C、L172M、L172V或其组合。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of L172E, L172I, L172C, L172M, L172V or a combination thereof.

在另一优选例中,所述的突变型的还原胺化酶具有选自下组的突变:M236L、M236I、M236S、M236C、M236A、M236V、M236T、M236G或其组合。In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of M236L, M236I, M236S, M236C, M236A, M236V, M236T, M236G or combinations thereof.

在另一优选例中,所述的还原胺化酶包括固定化酶。In another preferred example, the reductive amination enzyme includes an immobilized enzyme.

在本发明的第三方面,提供了一种分离的多核苷酸,所述的多核苷酸编码本发明第二方面所述的还原胺化酶。In the third aspect of the present invention, there is provided an isolated polynucleotide encoding the reductive aminase described in the second aspect of the present invention.

本发明还提供了一种编码野生型还原胺化酶AcRedAm的经密码子优化的多核苷酸,所述的多核苷酸的序列如SEQ ID NO:2所示。The present invention also provides a codon-optimized polynucleotide encoding wild-type reductive aminase AcRedAm, the sequence of which is shown in SEQ ID NO:2.

在另一优选例中,本发明第三方面中编码突变型还原胺化酶ACREDAM的多核苷酸的序列与SEQ ID NO:2基本相同,不同点仅在于编码特定位点的突变氨基酸的密码子存在不同(例如,对于W207S/Y214C突变型还原胺化酶AcRedAm而言,则对应于207位氨基酸的密码子为编码氨基酸S的密码子,对应于214位氨基酸的密码子为编码C的密码子)。In another preferred example, the sequence of the polynucleotide encoding the mutant reductive aminase ACREDAM in the third aspect of the present invention is basically the same as SEQ ID NO: 2, the only difference is the codon encoding the mutated amino acid at a specific site There are differences (for example, for the W207S/Y214C mutant reductive aminase AcRedAm, the codon corresponding to amino acid 207 is the codon encoding amino acid S, and the codon corresponding to amino acid 214 is the codon encoding C ).

在本发明的第四方面,提供了一种载体,所述的载体含有本发明第三方面所述的多核苷酸。In the fourth aspect of the present invention, there is provided a vector containing the polynucleotide described in the third aspect of the present invention.

在另一优选例中,所述载体包括表达载体、穿梭载体、整合载体。In another preferred example, the vectors include expression vectors, shuttle vectors, and integration vectors.

在本发明的第五方面,提供了一种遗传工程化的宿主细胞,所述的宿主细胞含有本发明第四方面所述的载体,或其基因组中整合本发明第三方面所述的多核苷酸。In the fifth aspect of the present invention, a genetically engineered host cell is provided, the host cell contains the vector described in the fourth aspect of the present invention, or its genome integrates the polynucleoside described in the third aspect of the present invention acid.

在另一优选例中,所述的细胞为原核细胞或真核细胞。In another preferred example, the cells are prokaryotic cells or eukaryotic cells.

在另一优选例中,所述的宿主细胞为真核细胞,如酵母细胞或植物细胞。In another preferred embodiment, the host cells are eukaryotic cells, such as yeast cells or plant cells.

在另一优选例中,所述的宿主细胞为酿酒酵母细胞。In another preferred example, the host cell is a Saccharomyces cerevisiae cell.

在另一优选例中,所述的宿主细胞原核细胞,如大肠杆菌。In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

在本发明的第六方面,提供了一种本发明第二方面所述的还原胺化酶或野生型的还原胺化酶AcRedAm的用途,它被用于催化还原胺化反应,或被用于制备催化还原胺化反应的催化制剂。In the sixth aspect of the present invention, there is provided a use of the reductive amination enzyme described in the second aspect of the present invention or the wild-type reductive amination enzyme AcRedAm, which is used to catalyze a reductive amination reaction, or is used to Preparation of catalytic preparations for catalytic reductive amination reactions.

在另一优选例中,所述的还原胺化反应的反应产物包括异构体、或非异构体。In another preferred example, the reaction product of the reductive amination reaction includes isomers or non-isomers.

在另一优选例中,所述的反应产物为S构型、R构型或其组合。In another preferred example, the reaction product is in S configuration, R configuration or a combination thereof.

在本发明的第七方面,提供了一种还原胺化酶体外合成雷沙吉兰的方法,包括步骤:In the seventh aspect of the present invention, a method for synthesizing rasagiline in vitro by reductive amination enzyme is provided, comprising the steps of:

在本发明第二方面所述的还原胺化酶或野生型还原胺化酶AcRedAm存在下,催化1-茚酮和炔丙胺进行反应,从而生成雷沙吉兰。In the presence of the reductive amination enzyme described in the second aspect of the present invention or the wild-type reductive amination enzyme AcRedAm, the reaction between 1-indanone and propargylamine is catalyzed to generate rasagiline.

在本发明的第八方面,提供了一种进行还原胺化反应的反应体系,所述的反应体系包括:In an eighth aspect of the present invention, a reaction system for performing reductive amination reaction is provided, the reaction system comprising:

(S0)本发明第二方面所述的还原胺化酶或野生型还原胺化酶AcRedAm;(S0) the reductive amination enzyme described in the second aspect of the present invention or the wild-type reductive amination enzyme AcRedAm;

(S1)酮底物或醛底物;(S1) a ketone substrate or an aldehyde substrate;

(S2)胺底物;和(S2) an amine substrate; and

(S3)任选的NADPH或NADPH再生模块。(S3) Optional NADPH or NADPH regeneration module.

在另一优选例中,所述的NADPH再生模块包括:葡萄糖脱氢酶(GDH)、葡萄糖。In another preferred example, the NADPH regeneration module includes: glucose dehydrogenase (GDH) and glucose.

在另一优选例中,所述的NADPH再生模块将葡萄糖和NADP+,在葡萄糖脱氢酶(GDH)催化下进行反应,从而生成葡萄糖酸内酯和NADPH。In another preferred example, the NADPH regeneration module reacts glucose and NADP+ under the catalysis of glucose dehydrogenase (GDH), thereby generating gluconolactone and NADPH.

应理解,在本发明范围内中,本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合,从而构成新的或优选的技术方案。限于篇幅,在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

附图说明Description of drawings

图1显示了还原胺化酶AcRedAm纯化后SDS-PAGE电泳分析结果图。Figure 1 shows the results of SDS-PAGE electrophoresis analysis after purification of the reductive aminase AcRedAm.

图2显示了还原胺化酶AcRedAm反应所需的最适pH结果图。Figure 2 shows the resulting graph of the optimum pH required for the reaction of reductive aminase AcRedAm.

图3显示了还原胺化酶AcRedAm反应所需的最适温度结果图。Figure 3 shows the result graph of the optimal temperature required for the reaction of reductive aminase AcRedAm.

图4显示了还原胺化酶AcRedAm的Tm值(4a)和温度耐受性(4b)结果图。Figure 4 shows the Tm value (4a) and temperature tolerance (4b) results graph of reductive aminase AcRedAm.

图5显示了还原胺化酶AcRedAm底物筛选所用的酮(5a)和胺底物(5b)以及还原胺化生成不同产物胺(5c)结果图。Figure 5 shows the ketone (5a) and amine substrates (5b) used in the substrate screening of reductive amination enzyme AcRedAm and the results of reductive amination to produce different product amines (5c).

图6显示了还原胺化酶AcRedAm中L90位点(6a)、I117位点(6b)、L172位点(6c)和M236位点(6d)饱和突变体合成雷沙吉兰的相对活性及对映选择性结果图。Figure 6 shows the relative activity of the saturated mutants at the L90 site (6a), the I117 site (6b), the L172 site (6c) and the M236 site (6d) in the reductive aminase AcRedAm to synthesize rasagiline Mapping selectivity results plot.

图7显示了还原胺化酶AcRedAm中W207位点(7a)、Y214位点(7b)和Q237位点(7d)饱和突变体合成雷沙吉兰的相对活性及对映选择性结果图。Fig. 7 shows the relative activity and enantioselectivity results of synthesizing rasagiline by saturation mutants at W207 site (7a), Y214 site (7b) and Q237 site (7d) in reductoaminase AcRedAm.

图8显示了还原胺化酶AcRedAm中双点组合突变体合成雷沙吉兰的相对活性及对映选择性结果图。Figure 8 shows the results of relative activity and enantioselectivity of rasagiline synthesized by double-point combination mutants in reductive aminase AcRedAm.

图9显示了针对合成雷沙吉兰放大反应条件优化结果图。Figure 9 shows the results of optimization of the scale-up reaction conditions for the synthesis of rasagiline.

其中,A显示了1-茚酮底物浓度的优化结果;B显示了炔丙胺底物浓度的优化结果;C显示了葡萄糖脱氢酶GDH浓度的优化结果;D显示了还原胺化酶AcRedAm浓度的优化结果。Among them, A shows the optimization result of 1-indanone substrate concentration; B shows the optimization result of propargylamine substrate concentration; C shows the optimization result of glucose dehydrogenase GDH concentration; D shows the reductive amination enzyme AcRedAm concentration optimization results.

图10显示了还原胺化酶AcRedAm及其突变体AcQ237A合成雷沙吉兰反应结果图。Figure 10 shows the results of the synthesis of rasagiline by reductive aminase AcRedAm and its mutant AcQ237A.

图11显示了还原胺化酶AcRedAm合成雷沙吉兰的HPLC分析图谱。Figure 11 shows the HPLC analysis profile of the synthesis of rasagiline by reductive aminase AcRedAm.

其中,A为1-茚酮标准品图谱;B为炔丙胺标准品图谱;C为(R)-雷沙吉兰标准品图谱;D为(S)-雷沙吉兰标准品图谱;E为还原胺化酶AcRedAm催化反应产物图谱。Wherein, A is a 1-indanone standard spectrum; B is a propargylamine standard spectrum; C is a (R)-rasagiline standard spectrum; D is a (S)-rasagiline standard spectrum; E is Reductive aminase AcRedAm catalyzed reaction product map.

图12显示了还原胺化酶AcRedAm突变体合成雷沙吉兰的HPLC分析图谱。Figure 12 shows the HPLC analysis pattern of rasagiline synthesized by reductive aminase AcRedAm mutant.

其中,A为(R)-雷沙吉兰标准品图谱;B为(S)-雷沙吉兰标准品图谱;C为突变体Q237A催化反应产物图谱;D为突变体Q237G催化反应产物图谱;E为突变体Q237S催化反应产物图谱。Among them, A is the spectrum of the standard product of (R)-rasagiline; B is the spectrum of the standard product of (S)-rasagiline; C is the spectrum of the catalytic reaction product of the mutant Q237A; D is the spectrum of the catalytic reaction product of the mutant Q237G; E is the spectrum of the catalytic reaction product of mutant Q237S.

图13显示了本发明的重组质粒pET28a-AcRedAm图谱。Figure 13 shows the map of the recombinant plasmid pET28a-AcRedAm of the present invention.

具体实施方式Detailed ways

本发明人经过广泛而深入的研究,首次意外地开发了一种还原胺化酶AcRedAm。具体地,本发明人开发了来自卡利曲霉菌(Aspergillus calidoustus)的还原胺化酶,并对其进行定向进化改造,获得最终还原胺化酶活性改善的突变体,例如对产物雷沙吉兰对映选择性显著提高的还原胺化酶突变体AcW207C、AcQ237A和AcW207S/Y214C。本发明的还原胺化酶AcRedAm及其突变体可用于体外酶法合成多种有用的化合物(包括对映纯的雷沙吉兰)。在此基础上完成了本发明。After extensive and in-depth research, the present inventors unexpectedly developed a reductive amination enzyme AcRedAm for the first time. Specifically, the inventors have developed a reductive amination enzyme from Aspergillus calidoustus, and carried out directed evolution transformation to obtain a mutant with improved final reductive amination enzyme activity, such as the product rasagiline Reductive aminase mutants AcW207C, AcQ237A and AcW207S/Y214C with significantly improved enantioselectivity. The reductive aminase AcRedAm and its mutants of the present invention can be used for in vitro enzymatic synthesis of various useful compounds (including enantiopure rasagiline). The present invention has been accomplished on this basis.

具体地,本发明人通过对还原胺化酶的序列和结构的研究,基于蛋白结构相似性、保守位点分析和宿主来源多样等原则,结合数据库进行筛选。随后,将筛选的基因在大肠杆菌表达系统中进行功能表达,纯化后获得纯化的本发明的还原胺化酶AcRedAm。经实验证明,来源于Aspergillus calidoustus的还原胺化酶AcRedAm具有广泛的底物谱,能够催化一系列酮和胺底物生成相应的初级胺和二级胺,并且能够直接催化1-茚酮和炔丙胺生成雷沙吉兰,转化率为44%,ee值为44%(S)。Specifically, the inventors conducted screening based on the principles of protein structure similarity, conserved site analysis, and diverse host sources through the study of the sequence and structure of reductive aminase, combined with databases. Subsequently, the screened gene is functionally expressed in an Escherichia coli expression system, and the purified reductive amination enzyme AcRedAm of the present invention is obtained after purification. Experiments have proved that the reductive amination enzyme AcRedAm from Aspergillus calidoustus has a broad substrate spectrum, can catalyze a series of ketone and amine substrates to generate corresponding primary and secondary amines, and can directly catalyze 1-indanone and alkyne Propylamine produces rasagiline with a conversion rate of 44% and an ee value of 44% (S).

本发明的还原胺化酶Reductive aminase of the present invention

如本文所用,如本文所用,术语“还原胺化酶AcRedAm”、“本发明的酶”、“本发明的还原胺化酶”或“本发明的胺化酶”,可互换使用,均指还原胺化酶AcRedAm。应理解,该术语包括野生型和突变型的还原胺化酶AcRedAm(例如衍生自还原胺化酶AcRedAm的衍生多肽)。As used herein, as used herein, the terms "reductive amination enzyme AcRedAm", "enzyme of the present invention", "reductive amination enzyme of the present invention" or "amination enzyme of the present invention", are used interchangeably and refer to Reductive aminase AcRedAm. It is to be understood that the term includes wild-type and mutant forms of the reductive aminase AcRedAm (eg, derivative polypeptides derived from the reductive aminase AcRedAm).

本发明提供的新型还原胺化酶及其编码的基因,来源于Aspergilluscalidoustus,该还原胺化酶被命名为AcRedAm蛋白。所述蛋白的野生型氨基酸序列为SEQID NO:1,核苷酸序列为SEQ ID NO:2。The novel reductive amination enzyme and the coded gene provided by the present invention are derived from Aspergillus calidoustus, and the reductive amination enzyme is named as AcRedAm protein. The wild-type amino acid sequence of the protein is SEQ ID NO:1, and the nucleotide sequence is SEQ ID NO:2.

在本发明中,本发明的还原胺化酶也可以是经过取代、缺失或添加一个或多个氨基酸其与SEQ ID NO:1所示蛋白具有相同功能(即具有还原胺化功能)的衍生蛋白。In the present invention, the reductive amination enzyme of the present invention may also be a derivative protein having the same function as the protein shown in SEQ ID NO: 1 (that is, having a reductive amination function) through substitution, deletion or addition of one or more amino acids .

本发明的酶还包括还原胺化酶AcRedAm的突变体。通常,所述突变体是通过用另一种氨基酸残基取代野生型还原胺化酶AcRedAm的一个或多个位置上的氨基酸残基而获得的还原胺化酶突变体;优选的取代位置为由SEQ ID NO:1表示的还原胺化酶AcRedAm氨基酸序列的第207、214、237位或它们的相应位置。本发明的突变型的还原胺化酶AcRedAm可催化1-茚酮和炔丙胺生成雷沙吉兰的活性比野生型提高,对映选择性比野生型显著提高。The enzymes of the present invention also include mutants of the reductive amination enzyme AcRedAm. Typically, the mutant is a reductive aminase mutant obtained by substituting another amino acid residue for an amino acid residue at one or more positions of the wild-type reductive aminase AcRedAm; preferred substitution positions are given by The 207th, 214th, and 237th positions of the amino acid sequence of the reductive aminase AcRedAm represented by SEQ ID NO: 1 or their corresponding positions. The mutant reductive aminase AcRedAm of the present invention can catalyze 1-indanone and propargylamine to generate rasagiline, and its activity is higher than that of the wild type, and its enantioselectivity is significantly higher than that of the wild type.

进一步地,所述的还原胺化酶突变体是通过用另一种氨基酸残基取代与野生型还原胺化酶AcRedAm表现出至少90%同源性的氨基酸序列的还原胺化酶的一个或多个位置上的氨基酸残基而获得的还原胺化酶突变体;优选的取代位置为由SEQ ID NO:1表示的还原胺化酶AcRedAm氨基酸序列的第207、214、237位或它们的相应位置,突变体催化1-茚酮和炔丙胺生成雷沙吉兰的活性比野生型提高,对映选择性比野生型显著提高。Further, the reductive amination mutant is obtained by substituting another amino acid residue for one or more parts of the reductive amination enzyme that exhibits at least 90% homology with the wild-type reductive amination enzyme AcRedAm. The reductive aminase mutant obtained by amino acid residues at the positions; the preferred substitution position is the 207th, 214th, 237th or their corresponding positions of the reductive aminase AcRedAm amino acid sequence represented by SEQ ID NO:1 , the activity of the mutant to catalyze the generation of rasagiline from 1-indanone and propargylamine is higher than that of the wild type, and the enantioselectivity is significantly higher than that of the wild type.

更进一步地,用于取代原有氨基酸残基的所述另一种氨基酸残基优选为半胱氨酸(氨基酸缩写名称为C)、甲硫氨酸(氨基酸缩写名称为M)、丙氨酸(氨基酸缩写名称为A)、丝氨酸(氨基酸缩写名称为S)或甘氨酸(氨基酸缩写名称为G)。Furthermore, the other amino acid residue used to replace the original amino acid residue is preferably cysteine (the abbreviated name of the amino acid is C), methionine (the abbreviated name of the amino acid is M), alanine (abbreviated name for amino acid is A), serine (abbreviated name for amino acid is S) or glycine (abbreviated name for amino acid is G).

在本发明中,在该还原胺化酶的突变位点对应的编码的核苷酸编码,应理解为本发明所述的“另一种氨基酸残基”所编码的核苷酸编码。In the present invention, the nucleotide code corresponding to the mutation site of the reductive aminase should be understood as the nucleotide code coded by "another amino acid residue" in the present invention.

在本发明中,还提供了一些优选的还原胺化酶突变体及其编码的基因,它们的出发还原胺化酶的基因序列为序列表中的SEQ ID NO:2,出发氨基酸序列为序列表中的SEQID NO:1。In the present invention, some preferred reductive aminase mutants and the genes encoded thereof are also provided. The gene sequence of their starting reductive aminase is SEQ ID NO: 2 in the sequence listing, and the starting amino acid sequence is the sequence listing SEQ ID NO: 1 in.

一些优选的突变类型选自下组:第207位色氨酸被半胱氨酸取代的突变体,第214位酪氨酸被甲硫氨酸取代的突变体,第237位谷氨酰胺被丙氨酸取代的突变体,第237位谷氨酰胺被丝氨酸取代的突变体,第237位谷氨酰胺被甘氨酸取代的突变体,第207位色氨酸和第214位酪氨酸分别被丝氨酸和半胱氨酸取代的突变体、或其组合。Some preferred mutation types are selected from the group consisting of: mutants in which tryptophan is replaced by cysteine at position 207, mutants in which tyrosine is replaced by methionine at position 214, glutamine at position 237 is replaced by alanine Amino acid substituted mutants, mutants in which glutamine at position 237 was replaced by serine, mutants in which glutamine at position 237 was replaced by glycine, tryptophan at position 207 and tyrosine at position 214 were replaced by serine and Cysteine-substituted mutants, or combinations thereof.

如本文所用,“分离的多肽”是指所述多肽基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化所述多肽。基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。所述多肽的纯度还可以用氨基酸序列进行进一步分析。As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify the polypeptides using standard protein purification techniques. Substantially pure polypeptides yield a single major band on non-reducing polyacrylamide gels. The purity of the polypeptide can also be further analyzed by amino acid sequence.

本发明的活性多肽可以是重组多肽、天然多肽、合成多肽。本发明的多肽可以是天然纯化的产物,或是化学合成的产物,或使用重组技术从原核或真核宿主(例如,细菌、酵母、植物)中产生。根据重组生产方案所用的宿主,本发明的多肽可以是还原胺化的,或可以是非还原胺化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。The active polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the present invention may be purified from nature, or chemically synthesized, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, plants). Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be reductively aminated, or may be non-reductively aminated. Polypeptides of the invention may or may not include an initial methionine residue.

本发明还包括所述多肽的片段、衍生物和类似物。如本文所用,术语“片段”、“衍生物”和“类似物”是指基本上保持所述多肽相同的生物学功能或活性的多肽。The invention also includes fragments, derivatives and analogs of said polypeptides. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the same biological function or activity of the polypeptide.

本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的多肽,或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物,例如聚乙二醇)融合所形成的多肽,或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列,或与抗原IgG片段的形成的融合蛋白)。根据本文的教导,这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with Formation of fusion proteins of antigen IgG fragments). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

所述的多肽优选序列为SEQ ID NO:1所示的多肽,该术语还包括具有与所示多肽具有相同功能的、SEQ ID NO:1序列的变异形式及衍生多肽。这些变异形式包括(但并不限于):一个或多个(通常为1-50个,较佳地1-30个,更佳地1-20个,最佳地1-10个)氨基酸的缺失、插入和/或取代,以及在C末端和/或N末端添加一个或数个(通常为20个以内,较佳地为10个以内,更佳地为5个以内)氨基酸。例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能。又比如,在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明还原胺化酶AcRedAm的活性片段和活性衍生物。本发明还提供所述多肽的类似物。这些类似物与天然人EGFRvA多肽的差别可以是氨基酸序列上的差异,也可以是不影响序列的修饰形式上的差异,或者兼而有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到,如通过辐射或暴露于诱变剂而产生随机诱变,还可通过定点诱变法或其他已知分子生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物,以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解,本发明的多肽并不限于上述例举的代表性的多肽。The preferred sequence of the polypeptide is the polypeptide shown in SEQ ID NO:1, and this term also includes variant forms and derivative polypeptides of the sequence of SEQ ID NO:1 that have the same function as the polypeptide shown. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the reductive amination enzyme AcRedAm of the present invention. The invention also provides analogs of said polypeptides. The difference between these analogs and the natural human EGFRvA polypeptide may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

修饰(通常不改变一级结构)形式包括:体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括还原胺化,如那些在多肽的合成和加工中或进一步加工步骤中进行还原胺化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行还原胺化的酶(如哺乳动物的还原胺化酶或去还原胺化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸,磷酸丝氨酸,磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include reductive amination, such as those resulting from reductive amination modifications of polypeptides during synthesis and processing or as further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs reductive amination, such as mammalian reductive amination enzyme or dereductive amination enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

本发明蛋白的氨基端或羧基端还可含有一个或多个多肽片段,作为蛋白标签。任何合适的标签都可以用于本发明。例如,所述的标签可以是FLAG、HA、HA1、c-Myc、Poly–His、Poly-Arg、Strep-TagII、AU1、EE、T7、4A6、ε、B、gE、以及Ty1。这些标签可用于对蛋白进行纯化。The amino-terminal or carboxy-terminal of the protein of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable label can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins.

为了使翻译的蛋白分泌表达(如分泌到细胞外),还可在所述还原胺化酶AcRedAm的氨基酸氨基末端添加上信号肽序列,如pelB信号肽等。信号肽在多肽从细胞内分泌出来的过程中可被切去。In order to secrete and express the translated protein (such as secreted out of the cell), a signal peptide sequence, such as pelB signal peptide, can also be added to the amino acid terminal of the reductive amination enzyme AcRedAm. The signal peptide can be cleaved during the secretion of the polypeptide from the cell.

本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NOs.:1所示的编码区序列相同或者是简并的变异体。如本文所用,“简并的变异体”在本发明中是指编码具有SEQ ID NO:1的蛋白质,但与SEQ ID NO:2所示的编码区序列有差别的核酸序列。A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NOs.: 1 or a degenerate variant. As used herein, a "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein having SEQ ID NO:1 but differs from the sequence of the coding region shown in SEQ ID NO:2.

编码SEQ ID NO:1的成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列;成熟多肽的编码序列和各种附加编码序列;成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。A polynucleotide encoding the mature polypeptide of SEQ ID NO: 1 includes: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequence.

术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

本发明还涉及上述多核苷酸的变异体,其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码的多肽的功能。The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

本发明还涉及与上述的序列杂交且两个序列之间具有至少50%,较佳地至少70%,更佳地至少80%相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中,“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2×SSC,0.1%SDS,60℃;或(2)杂交时加有变性剂,如50%(v/v)甲酰胺,0.1%小牛血清/0.1%Ficoll,42℃等;或(3)仅在两条序列之间的相同性至少在90%以上,更好是95%以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与SEQ ID NO:1所示的成熟多肽有相同的生物学功能和活性。The present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) to the polynucleotides of the present invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:1.

本发明还涉及与上述的序列杂交的核酸片段。如本文所用,“核酸片段”的长度至少含15个核苷酸,较好是至少30个核苷酸,更好是至少50个核苷酸,最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)以确定和/或分离编码还原胺化酶AcRedAm蛋白的多聚核苷酸。The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and/or isolate the polynucleotide encoding the reductive aminase AcRedAm protein.

本发明中的多肽和多核苷酸优选以分离的形式提供,更佳地被纯化至均质。The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

本发明的还原胺化酶AcRedAm核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法,可根据本发明所公开的有关核苷酸序列,尤其是开放阅读框序列来设计引物,并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板,扩增而得有关序列。当序列较长时,常常需要进行两次或多次PCR扩增,然后再将各次扩增出的片段按正确次序拼接在一起。The full-length nucleotide sequence of the reductive aminase AcRedAm of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

一旦获得了有关的序列,就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体,再转入细胞,然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

此外,还可用人工合成的方法来合成有关序列,尤其是片段长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

目前,已经可以完全通过化学合成来得到编码本发明蛋白(或其片段,或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外,还可通过化学合成将突变引入本发明蛋白序列中。At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。特别是很难从文库中得到全长的cDNA时,可优选使用RACE法(RACE-cDNA末端快速扩增法),用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择,并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

本发明也涉及包含本发明的多核苷酸的载体,以及用本发明的载体或还原胺化酶AcRedAm蛋白编码序列经基因工程产生的宿主细胞,以及经重组技术产生本发明所述多肽的方法。The present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the reductive aminase AcRedAm protein coding sequence, and a method for producing the polypeptide of the present invention through recombinant technology.

通过常规的重组DNA技术,可利用本发明的多聚核苷酸序列可用来表达或生产重组的还原胺化酶AcRedAm多肽。一般来说有以下步骤:The polynucleotide sequence of the present invention can be used to express or produce recombinant reductive aminase AcRedAm polypeptide by conventional recombinant DNA technology. Generally speaking, there are the following steps:

(1).用本发明的编码还原胺化酶AcRedAm多肽的多核苷酸(或变异体),或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞;(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the reductive aminase AcRedAm polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2).在合适的培养基中培养的宿主细胞;(2). Host cells cultured in a suitable medium;

(3).从培养基或细胞中分离、纯化蛋白质。(3). Isolate and purify protein from culture medium or cells.

本发明中,还原胺化酶AcRedAm多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定,任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。In the present invention, the reductive aminase AcRedAm polynucleotide sequence can be inserted into the recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors well known in the art. Any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

本领域的技术人员熟知的方法能用于构建含还原胺化酶AcRedAm编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。这些启动子的代表性例子有:大肠杆菌的lac或trp启动子;λ噬菌体PL启动子;真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding reductive aminase AcRedAm and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other promoters known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

此外,表达载体优选地包含一个或多个选择性标记基因,以提供用于选择转化的宿主细胞的表型性状,如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP),或用于大肠杆菌的四环素或氨苄青霉素抗性。In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

包含上述的适当DNA序列以及适当启动子或者控制序列的载体,可以用于转化适当的宿主细胞,以使其能够表达蛋白质。Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

宿主细胞可以是原核细胞,如细菌细胞;或是低等真核细胞,如酵母细胞;或是高等真核细胞,如哺乳动物细胞。代表性例子有:大肠杆菌,链霉菌属;鼠伤寒沙门氏菌的细菌细胞;真菌细胞如酵母;植物细胞;果蝇S2或Sf9的昆虫细胞;CHO、COS、293细胞、或Bowes黑素瘤细胞的动物细胞等。The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces spp; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells animal cells, etc.

在另一优选例中,合适的宿主细胞包括革兰氏阳性细菌如枯草芽孢杆菌,革兰氏阴性细菌如大肠杆菌,放线菌如链霉菌,酵母如酿酒酵母,真菌如曲霉菌,它们的细胞均是常用的重组载体的宿主细胞。In another preferred embodiment, suitable host cells include Gram-positive bacteria such as Bacillus subtilis, Gram-negative bacteria such as Escherichia coli, actinomycetes such as Streptomyces, yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus, their All the cells are commonly used host cells for recombinant vectors.

本发明的多核苷酸在高等真核细胞中表达时,如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子,通常大约有10到300个碱基对,作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer.

本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after exponential growth and treated with CaCl2 using procedures well known in the art. Another way is to use MgCl2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于:常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

应用application

本发明还提供了本发明还原胺化酶AcRedAm的应用,尤其是在催化(S1)酮底物或醛底物与(S2)胺底物进行还原胺化反应方面的应用。The present invention also provides the application of the reductive amination enzyme AcRedAm of the present invention, especially the application in catalyzing the reductive amination reaction of (S1) ketone substrate or aldehyde substrate and (S2) amine substrate.

一种优选的例子是用于药用化合物或中间体的合成。代表性地,本发明的还原胺化酶AcRedAm可用于制备雷沙吉兰等化合物。A preferred example is for the synthesis of pharmaceutical compounds or intermediates. Typically, the reductive amination enzyme AcRedAm of the present invention can be used to prepare compounds such as rasagiline.

还原胺化酶(RedAm)催化生成的雷沙吉兰产物中(R)-雷沙吉兰是一种有效的抗帕金森病药物,(S)-雷沙吉兰也已被证明具有显着的心脏保护活性。本发明的还原胺化酶AcRedAm可催化生成单一对映纯雷沙吉兰。Among the rasagiline products catalyzed by reductive aminase (RedAm), (R)-rasagiline is an effective anti-Parkinson's disease drug, and (S)-rasagiline has also been proved to have significant cardioprotective activity. The reductive amination enzyme AcRedAm of the present invention can catalyze the generation of single enantiopure rasagiline.

由于本发明的某些突变的还原胺化酶AcRedAm对产物雷沙吉兰对映选择性显著提高(如还原胺化酶突变体AcW207C、AcQ237A和AcW207S/Y214C),因此可用于体外酶法合成对映纯雷沙吉兰。Because the reductive amination enzyme AcRedAm of some mutations of the present invention significantly improves the enantioselectivity of product rasagiline (such as reductive amination enzyme mutants AcW207C, AcQ237A and AcW207S/Y214C), it can be used for in vitro enzymatic synthesis Antipure rasagiline.

典型地,本发明提供了一种使用本发明的还原胺化酶在体外合成相应初级胺和二级胺的方法。在本发明方法中,以相应的酮和胺为原料,在还原胺化酶的催化作用下可直接生成相应的产物胺。Typically, the invention provides a method for the in vitro synthesis of corresponding primary and secondary amines using a reductive amination enzyme of the invention. In the method of the present invention, corresponding ketones and amines are used as raw materials, and the corresponding product amines can be directly generated under the catalysis of reductive amination enzyme.

优选地,在本发明中,采用所述还原胺化酶AcRedAm或突变体,在优化后的条件下,还原胺化酶AcRedAm催化反应的转化率为20%-96%。Preferably, in the present invention, using the reductive amination enzyme AcRedAm or the mutant, under optimized conditions, the conversion rate of the reaction catalyzed by the reductive amination enzyme AcRedAm is 20%-96%.

优选地,所述酮和胺底物的摩尔比为1:1~1:50。Preferably, the molar ratio of the ketone to the amine substrate is 1:1˜1:50.

优选地,所述酮的浓度为5mM/L,所述胺的浓度为5~250mM/L。Preferably, the concentration of the ketone is 5 mM/L, and the concentration of the amine is 5-250 mM/L.

优选地,所述亲水性有机溶剂为二甲基亚砜(DMSO)。Preferably, the hydrophilic organic solvent is dimethylsulfoxide (DMSO).

优选地,所述反应体系中的亲水性有机溶剂的终浓度为2%。Preferably, the final concentration of the hydrophilic organic solvent in the reaction system is 2%.

合成雷沙吉兰的方法The method for synthesizing rasagiline

在本发明中,还提供了一种使用本发明还原胺化酶体外合成雷沙吉兰的方法。典型地,该方法包括:以1-茚酮和炔丙胺为原料,在还原胺化酶的催化作用下可直接生成雷沙吉兰。In the present invention, a method for synthesizing rasagiline in vitro using the reductive amination enzyme of the present invention is also provided. Typically, the method comprises: taking 1-indanone and propargylamine as raw materials, and directly generating rasagiline under the catalysis of reductive amination enzyme.

实验表明,本发明的酶特别适合用于通过体外酶法转化合成对映纯的雷沙吉兰。Experiments show that the enzyme of the present invention is particularly suitable for synthesizing enantiomerically pure rasagiline through in vitro enzymatic conversion.

优选地,所述还原胺化酶AcRedAm是其上述的突变体,在合适的条件下,还原胺化酶AcRedAm突变体催化1-茚酮和炔丙胺生成雷沙吉兰的活性是野生型的1.2-1.3倍,ee值提高至99%。Preferably, the reductive amination enzyme AcRedAm is its above-mentioned mutant, and under suitable conditions, the activity of the reductive amination enzyme AcRedAm mutant to catalyze 1-indanone and propargylamine to generate rasagiline is 1.2% of that of the wild type. -1.3 times, ee value increased to 99%.

优选地,所述1茚酮和炔丙胺的摩尔比为1:50。Preferably, the molar ratio of indanone to propargylamine is 1:50.

优选地,所述1茚酮的浓度为5mM/L,所述炔丙胺的浓度为250mM/L。Preferably, the concentration of said indanone is 5mM/L, and the concentration of said propargylamine is 250mM/L.

优选地,所述亲水性有机溶剂为二甲基亚砜(DMSO)。Preferably, the hydrophilic organic solvent is dimethylsulfoxide (DMSO).

优选地,所述反应体系中的亲水性有机溶剂的终浓度为2%。Preferably, the final concentration of the hydrophilic organic solvent in the reaction system is 2%.

本发明的主要优点包括:The main advantages of the present invention include:

1.利用本发明所述的还原胺化酶在体外酶法合成相应的初级胺和二级胺,对底物酮和胺的转化率可达20%-96%。1. Using the reductive amination enzyme of the present invention to enzymatically synthesize corresponding primary amines and secondary amines in vitro, the conversion rate of substrate ketones and amines can reach 20%-96%.

2.利用本发明所述的还原胺化酶在体外酶法合成雷沙吉兰,对底物1-茚酮和炔丙胺的转化率可达70%;其突变体在相同条件下,催化1-茚酮和炔丙胺生成雷沙吉兰的活性是野生型的1.2-1.3倍,ee值提高至99%。2. Utilize the reductive aminase of the present invention to synthesize rasagiline in vitro, the conversion rate of substrate 1-indanone and propargylamine can reach 70%; its mutants can catalyze 1 The activity of indanone and propargylamine to generate rasagiline is 1.2-1.3 times that of the wild type, and the ee value is increased to 99%.

3.利用本发明所述的还原胺化酶突变体成功实现了在体外酶法合成单一对映纯雷沙吉兰。3. The reductive aminase mutant of the present invention is used to successfully realize the enzymatic synthesis of single enantiomerically pure rasagiline in vitro.

4.本发明获得的还原胺化酶及其突变体可用于体外酶法合成雷沙吉兰,是区别于现有生产技术的一种新的生产方法,生产过程简化、消耗低、绿色环保。4. The reductive aminase and its mutants obtained in the present invention can be used for in vitro enzymatic synthesis of rasagiline, which is a new production method different from the existing production technology, with simplified production process, low consumption and environmental protection.

下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数是重量百分比和重量份数。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer suggested conditions. Percentages and parts are by weight unless otherwise indicated.

材料和试剂Materials and Reagents

本发明所述的生物材料的来源的一般性说明:General description of the source of the biological material described in the present invention:

1.引物合成:本发明中所使用的引物均由华大基因公司合成制备。1. Primer synthesis: The primers used in the present invention were all synthesized by BGI Corporation.

2.实验中所使用的PrimeSTAR Max DNA聚合酶购自TakaRa公司;使用的DNA胶回收试剂盒及质粒小提试剂和均购自Axygen公司。2. The PrimeSTAR Max DNA polymerase used in the experiment was purchased from TakaRa; the DNA gel recovery kit and plasmid extraction reagent used were purchased from Axygen.

实施例1还原胺化酶的筛选The screening of embodiment 1 reductive aminase

基于对于还原胺化酶的结构分析,并基于蛋白结构相似性、保守位点分析和宿主来源多样等原则,对于不同物种的数据库进行筛选,并对候选对象进行克隆和验证,从而获得了一种还原胺化酶。Based on the structural analysis of reductive aminase, and based on the principles of protein structure similarity, conserved site analysis, and diverse host sources, the databases of different species were screened, and the candidate objects were cloned and verified, thus obtaining a Reductive aminase.

具体地,在本实施例中,本发明基于筛选获得的还原胺化酶为来源于Aspergilluscalidoustus的还原胺化酶AcRedAm及其编码序列。其氨基酸序列如SEQ ID No:1所示。Specifically, in this embodiment, the reductive amination enzyme obtained based on screening in the present invention is the reductive amination enzyme AcRedAm derived from Aspergillus calidoustus and its coding sequence. Its amino acid sequence is shown in SEQ ID No:1.

MSTITLFGLGAMGKALAAKYIEKGYTTTIWNRTPSKAAPLVEKGAKLANTVGEGLASADLIILCLLDNASVRQTLDQATAALNGKTVINLTNGTPSQARETSEWVISHGAQYIHGGIMAVPDMIGSPHAVLLYSGESAETFSRVEAHLSHLGTSKFLGTDPGSASLHDLALLSGMYGLFSGFFHATALVKSQPGTTATGFVQLLTPWLSAMTHYLGALAKQIDEGDYATQGSNMAMQVTGVQNIVRASEEAGVTADLIMPILGRMTRAAEAGYADVDVSAVIEFMKE(SEQ IDNo:1)MSTITLFGLGAMGKALAAKYIEKGYTTTIWNRTPSKAAPLVEKGAKLANTVGEGLASADLIILCLLDASVRQTLDQATAALNGKTVINLTNGTPSQARETSEWVISHGAQYIHGGIMAVPDMIGSPHAVLLYSGESAETFSRVEAHLSHLGTSKFLGTDPGSASLHDLALLSGMYGLFSGFFHATALVKSQPGTTATGF VQLLTPWLSAMTHYLGALAKQIDEGDYATQGSNMAMQVTGVQNIVRASEEAGVTADLIMPILGRMTRAAEAGYADDVVSAVIEFMKE (SEQ ID No: 1)

对AcRedAm基因经过密码子优化后,以便于在大肠杆菌中表达。优化后的编码序列如SEQ ID No:2所示。The AcRedAm gene is codon optimized for easy expression in Escherichia coli. The optimized coding sequence is shown as SEQ ID No:2.

ATGAGCACCATTACCCTGTTCGGTCTGGGTGCGATGGGCAAGGCGCTGGCGGCGAAGTACATCGAGAAAGGCTATACCACCACCATTTGGAACCGTACCCCGAGCAAAGCGGCGCCGCTGGTTGAGAAGGGTGCGAAACTGGCGAACACCGTTGGTGAAGGTCTGGCGAGCGCGGACCTGATCATTCTGTGCCTGCTGGATAACGCGAGCGTGCGTCAAACCCTGGACCAAGCGACCGCGGCGCTGAACGGCAAGACCGTTATCAACCTGACCAACGGTACCCCGAGCCAGGCGCGTGAGACCAGCGAATGGGTGATTAGCCACGGCGCGCAATACATCCACGGTGGCATTATGGCGGTGCCGGATATGATCGGTAGCCCGCACGCGGTTCTGCTGTATAGCGGCGAGAGCGCGGAAACCTTCAGCCGTGTTGAAGCGCACCTGAGCCACCTGGGTACCAGCAAATTTCTGGGTACCGACCCGGGTAGCGCGAGCCTGCACGATCTGGCGCTGCTGAGCGGCATGTACGGCCTGTTCAGCGGCTTCTTTCATGCGACCGCGCTGGTTAAAAGCCAACCGGGTACCACCGCGACCGGTTTTGTTCAACTGCTGACCCCGTGGCTGAGCGCGATGACCCACTACCTGGGTGCGCTGGCGAAACAGATTGACGAGGGTGATTATGCGACCCAAGGCAGCAACATGGCGATGCAGGTGACCGGTGTTCAAAACATCGTTCGTGCGAGCGAGGAAGCGGGCGTTACCGCGGACCTGATCATGCCGATTCTGGGTCGTATGACCCGTGCGGCGGAAGCGGGTTATGCGGACGTGGATGTTAGCGCGGTGATCGAGTTTATGAAGGAATAA(SEQ ID No:2)ATGAGCACCATTACCCTGTTCGGTCTGGGTGCGATGGGCAAGGCGCTGGCGGCGAAGTACATCGAGAAAGGCTATACCACCACCATTTGGAACCGTACCCCGAGCAAAGCGGCGCCGCTGGTTGAGAAGGGTGCGAAACTGGCGAACACCGTTGGTGAAGGTCTGGCGAGCGCGGACCTGATCATTCTGTGCCTGCTGGATAACGCGAGC GTGCGTCAAACCCTGGACCAAGCGACCGCGGCGCTGAACGGCAAGACCGTTATCAACCTGACCAACGGTACCCCGAGCCAGGCGCGTGAGACCAGCGAATGGGTGATTAGCCACGGCGCGCAATACATCCACGGTGGCATTATGGCGGTGCCGGATATGATCGGTAGCCCGCACGCGGTTCTGCTGTATAGCGGCGAGAGCGCGGAAACCTTCAG CCGTGTTGAAGCGCACCTGAGCCACCTGGGTACCAGCAAAATTTCTGGGTACCGACCCGGGTAGCGCGAGCCTGCACGATCTGGCGCTGCTGAGCGGCATGTACGGCCTGTTCAGCGGCTTCTTTCATGCGACCGCGCTGGTTAAAAGCCAACCGGGTACCACCGACCGGTTTTGTTCAACTGCTGACCCCGTGGCTGAGCGCGATGACCCACT ACCTGGGTGCGCTGGCGAAACAGATTGACGAGGGTGATTATGCGACCCAAGGCAGCAACATGGCGATGCAGGTGACCGGTGTTAAAACATCGTGCGAGCGAGGAAGCGGGCGTTACCGCGGACCTGATCATGCCGATTCTGGGTCGTATGACCCGTGCGGCGGAAGCGGGTTATGCGGACGTGGATGTTAGCGCGGTGATCGAGTTTAT GAAGGAATAA (SEQ ID No: 2)

通过全基因合成方法合成SEQ ID No:2所示的序列,两端添加了酶切位点NdeⅠ和XhoⅠ,将该全长序列克隆入至市售的pET28a(+)质粒的NdeⅠ、XhoⅠ酶切位点。The sequence shown in SEQ ID No: 2 was synthesized by the whole gene synthesis method, and the restriction sites NdeI and XhoI were added at both ends, and the full-length sequence was cloned into the NdeI and XhoI restriction enzymes of the commercially available pET28a(+) plasmid site.

实施例2还原胺化酶表达与纯化Embodiment 2 Reductive aminase expression and purification

将上述筛选基因的重组表达质粒热休克转化至大肠杆菌BL21(DE3)感受态细胞,进行基因表达及蛋白纯化。培养重组菌至OD为0.6-0.8时,添加IPTG至终浓度为0.5mM,在低温18℃、220rpm条件下诱导培养过夜。The recombinant expression plasmids of the above screening genes were heat-shock transformed into Escherichia coli BL21 (DE3) competent cells for gene expression and protein purification. When the recombinant bacteria were cultured to an OD of 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and cultured overnight at a low temperature of 18°C and 220 rpm.

离心收集菌体,将细胞重悬于100mM Tris-HCl缓冲液中(pH8.0,300mM NaCl,30mM咪唑)。250mL的培养细胞最终重悬于40mL的缓冲液中,使用高压细胞破碎仪(4~6℃,700pa)进行细胞破碎,然后细胞破碎液以12000rpm高速离心30min(4℃),收集上清液再次重复12000rpm离心30min(4℃),收集上清利用Ni-NTA柱亲和纯化蛋白,使用100mM Tris-HCl缓冲液(pH8.0,300mM NaCl,50mM咪唑)洗脱杂蛋白,最后使用100mM Tris-HCl缓冲液(pH8.0,300mM NaCl,250mM咪唑)洗脱目标蛋白,洗脱目标蛋白液经过浓缩除盐得到纯化的蛋白。The cells were collected by centrifugation, and the cells were resuspended in 100 mM Tris-HCl buffer (pH 8.0, 300 mM NaCl, 30 mM imidazole). 250mL of cultured cells were finally resuspended in 40mL of buffer, and the cells were disrupted using a high-pressure cell disruptor (4-6°C, 700pa), and then the cell disruption solution was centrifuged at 12000rpm for 30min at high speed (4°C), and the supernatant was collected again. Repeat centrifugation at 12000rpm for 30min (4°C), collect the supernatant and use Ni-NTA column to affinity purify the protein, use 100mM Tris-HCl buffer (pH8.0, 300mM NaCl, 50mM imidazole) to elute the impurity protein, and finally use 100mM Tris- HCl buffer (pH8.0, 300mM NaCl, 250mM imidazole) elutes the target protein, and the eluted target protein solution is concentrated and desalted to obtain a purified protein.

纯化的蛋白保存于100mM Tris-HCl缓冲液中(pH8.0),使用12%SDS-PAGE对纯化后的蛋白进行电泳检测,使用Bradford蛋白浓度测定试剂盒(上海生工生物)测定蛋白浓度。The purified protein was stored in 100mM Tris-HCl buffer (pH8.0), and the purified protein was detected by electrophoresis using 12% SDS-PAGE, and the protein concentration was determined using the Bradford protein concentration assay kit (Shanghai Sangon Biotech).

结果如图1所示。结果表明,在30.1kDa处得到一条清晰的条带,这表明目标蛋白AcRedAm已得到纯化。The result is shown in Figure 1. The results showed that a clear band was obtained at 30.1kDa, which indicated that the target protein AcRedAm had been purified.

实施例3还原胺化酶AcRedAm酶学性质测定Embodiment 3 Reductive amination enzyme AcRedAm enzymatic property determination

在本实施例中,测定实施例2制备的重组的还原胺化酶AcRedAm的酶学性质。In this example, the enzymatic properties of the recombinant reductive aminase AcRedAm prepared in Example 2 were determined.

还原胺化酶AcRedAm及其突变体的酶活测定反应中,利用偶联葡萄糖脱氢酶(GDH)的方式提供NADPH辅因子再生。In the enzyme activity assay reaction of the reductive aminase AcRedAm and its mutants, the NADPH cofactor is regenerated by coupling glucose dehydrogenase (GDH).

3.1.还原胺化酶AcRedAm最适pH和最适温度的测定:3.1. Determination of optimum pH and optimum temperature of reductive aminase AcRedAm:

分别在不同pH(7.0、8.0、9.0、10.0)下探究AcRedAm的酶活,反应体系为:1mg/mLAcRedAm纯酶、0.7mg/mL GDH(阿拉丁)、30mM D-葡萄糖、1mM NADP+、5mM环己酮、5mM环丙胺(在调节至pH 9.0的缓冲液中)和2%(v/v)DMSO。使用Tris-HCl缓冲液将最终反应体积补足至500μL。反应在25℃下以220rpm的速度振荡孵育24小时。然后,加入30μL 10M NaOH以淬灭反应。反应混合物用500μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用GC-FID进行分析。以最高酶活定义为100%,计算不同pH反应条件下的相对酶活。The enzyme activity of AcRedAm was explored at different pH (7.0, 8.0, 9.0, 10.0). The reaction system was: 1mg/mL AcRedAm pure enzyme, 0.7mg/mL GDH (Aladdin), 30mM D-glucose, 1mM NADP+, 5mM cyclo Hexanone, 5 mM cyclopropylamine (in buffer adjusted to pH 9.0) and 2% (v/v) DMSO. Make up the final reaction volume to 500 µL using Tris-HCl buffer. Reactions were incubated at 25°C for 24 hours with shaking at 220 rpm. Then, 30 μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 500 μL of methyl tert-butyl ether. The upper organic phases were combined, dried over anhydrous MgSO4 , and analyzed using GC-FID. The highest enzyme activity was defined as 100%, and the relative enzyme activity under different pH reaction conditions was calculated.

结果如图2所示。结果表明,AcRedAm在pH 9.0时具有高活性。The result is shown in Figure 2. The results showed that AcRedAm was highly active at pH 9.0.

3.2.还原胺化酶AcRedAm最适温度的测定:3.2. Determination of the optimal temperature of reductive aminase AcRedAm:

分别在不同温度(20℃、25℃、30℃、35℃)下探究AcRedAm的酶活,反应体系为:1mg/mL AcRedAm纯酶、0.7mg/mL GDH(阿拉丁)、30mM D-葡萄糖、1mM NADP+、5mM环己酮、5mM环丙胺(在调节至pH 9.0的缓冲液中)和2%(v/v)DMSO。使用Tris-HCl缓冲液将最终反应体积补足至500μL。反应在不同温度下以220rpm的速度振荡孵育24小时。然后,加入30μL 10MNaOH以淬灭反应。反应混合物用500μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用GC-FID进行分析。以最高酶活定义为100%,计算不同温度反应条件下的相对酶活。The enzyme activity of AcRedAm was explored at different temperatures (20°C, 25°C, 30°C, 35°C). The reaction system was: 1mg/mL AcRedAm pure enzyme, 0.7mg/mL GDH (Aladdin), 30mM D-glucose, 1 mM NADP+, 5 mM cyclohexanone, 5 mM cyclopropylamine (in buffer adjusted to pH 9.0) and 2% (v/v) DMSO. Make up the final reaction volume to 500 µL using Tris-HCl buffer. Reactions were incubated at different temperatures for 24 hours with shaking at 220 rpm. Then, 30 μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 500 μL of methyl tert-butyl ether. The upper organic phases were combined, dried over anhydrous MgSO4 , and analyzed using GC-FID. The highest enzyme activity was defined as 100%, and the relative enzyme activity under different temperature reaction conditions was calculated.

结果如图3所示。结果表明,AcRedAm在25℃时具有高活性。The result is shown in Figure 3. The results showed that AcRedAm had high activity at 25°C.

3.3.还原胺化酶AcRedAm的Tm值测定:3.3. Determination of Tm value of reductive aminase AcRedAm:

使用差示扫描量热仪(DSC)测定AcRedAm的Tm值。通过记录不同温度(30-80℃)下的热容量(Cp)来研究酶的解折叠情况从而确定其Tm值。The Tm value of AcRedAm was determined using differential scanning calorimetry (DSC). The unfolding of the enzyme was studied by recording the heat capacity (Cp) at different temperatures (30-80°C) to determine its Tm value.

结果如图4a所示。结果表明,AcRedAm的Tm值为45℃。The results are shown in Figure 4a. The results showed that the Tm value of AcRedAm was 45°C.

3.4.还原胺化酶AcRedAm的热稳定性测定:3.4. Determination of thermal stability of reductive aminase AcRedAm:

在50℃条件下处理酶液不同的时间(0min、20min、40min、60min)后探究AcRedAm的酶活,反应体系为:1mg/mL AcRedAm纯酶、0.7mg/mL GDH(阿拉丁)、30mM D-葡萄糖、1mMNADP+、5mM环己酮、5mM环丙胺(在调节至pH 9.0的缓冲液中)和2%(v/v)DMSO。使用Tris-HCl缓冲液将最终反应体积补足至500μL。反应在25℃下以220rpm的速度振荡孵育24小时。然后,加入30μL 10M NaOH以淬灭反应。反应混合物用500μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用GC-FID进行分析。After treating the enzyme solution at 50°C for different times (0min, 20min, 40min, 60min), the enzyme activity of AcRedAm was explored. The reaction system was: 1mg/mL AcRedAm pure enzyme, 0.7mg/mL GDH (Aladdin), 30mM D - Glucose, 1 mM NADP+, 5 mM cyclohexanone, 5 mM cyclopropylamine (in buffer adjusted to pH 9.0) and 2% (v/v) DMSO. Make up the final reaction volume to 500 µL using Tris-HCl buffer. Reactions were incubated at 25°C for 24 hours with shaking at 220 rpm. Then, 30 μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 500 μL of methyl tert-butyl ether. The upper organic phases were combined, dried over anhydrous MgSO4 , and analyzed using GC-FID.

结果如图4b所示。结果表明,AcRedAm具有一定的热稳定性。在50℃处理20min后,AcRedAm的活性会下降大约50%。The result is shown in Fig. 4b. The results show that AcRedAm has certain thermal stability. After treatment at 50°C for 20 min, the activity of AcRedAm decreased by about 50%.

3.5.还原胺化酶AcRedAm的底物谱测定:3.5. Determination of substrate spectrum of reductive aminase AcRedAm:

反应体系为:1mg/mL AcRedAm纯酶、0.7mg/mL GDH(阿拉丁)、30mM D-葡萄糖、1mMNADP+、5mM酮底物、适当比例的胺底物(在调节至pH 9.0的缓冲液中)和2%(v/v)DMSO。使用Tris-HCl(pH 9.0)缓冲液将最终反应体积补足至500μL。反应在25℃下以220rpm的速度振荡孵育24小时。然后,加入30μL10M NaOH以淬灭反应。反应混合物用500μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用HPLC或GC-FID进行分析。The reaction system is: 1 mg/mL AcRedAm pure enzyme, 0.7 mg/mL GDH (Aladdin), 30 mM D-glucose, 1 mM NADP+, 5 mM ketone substrate, appropriate proportion of amine substrate (in buffer adjusted to pH 9.0) and 2% (v/v) DMSO. The final reaction volume was made up to 500 μL using Tris-HCl (pH 9.0) buffer. Reactions were incubated at 25°C for 24 hours with shaking at 220 rpm. Then, 30 μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 500 μL of methyl tert-butyl ether. Combine the upper organic phases, dry over anhydrous MgSO4 , and analyze using HPLC or GC-FID.

结果如图5所示。结果表明,还原胺化酶AcRedAm对胺底物e和f具有明显的偏好性,对脂肪族和芳香族的酮底物都具有适中到优秀的转化还原能力。The result is shown in Figure 5. The results show that the reductive amination enzyme AcRedAm has a clear preference for amine substrates e and f, and has moderate to excellent conversion and reduction abilities for both aliphatic and aromatic ketone substrates.

在这些反应中,该酶催化等摩尔比浓度的酮和胺的反应能够达到95%的转化率,如图5中所示4f的反应;催化酮底物1和酮底物3(环戊酮)分别和等摩尔比的e,f或者i还原胺化的转化率在20%到64%之间;催化等摩尔当量的胺底物i(苄胺)和酮底物4或者5生成二级胺的反应能达到37%到69%的转化率。In these reactions, the enzyme catalyzes the reaction of ketones and amines in equimolar ratio concentrations and can reach a conversion rate of 95%, as shown in the reaction of 4f in Figure 5; catalysis of ketone substrate 1 and ketone substrate 3 (cyclopentanone ) respectively with equimolar ratio of e, f or i reductive amination conversion between 20% to 64%; catalysis equimolar equivalent of amine substrate i (benzylamine) and ketone substrate 4 or 5 to generate secondary Amine reactions can achieve conversions ranging from 37% to 69%.

这些结果都表明本发明的酶具备催化酮底物和胺底物进行还原胺化反应的活性,因此归类为是真正的还原胺化酶。These results all indicate that the enzyme of the present invention has the activity of catalyzing the reductive amination reaction of ketone substrate and amine substrate, so it is classified as a real reductive amination enzyme.

在催化酮底物2和20摩尔当量的胺底物e或f的反应中,反应的转化率分别能到达38%和34%,其中生成产物2e的ee值为13%。其中胺底物a和酮底物4(环己酮)也可以被该酶直接催化生成环己胺,转化率达到96%。In the reaction of ketone substrate 2 and 20 molar equivalents of amine substrate e or f, the conversions of the reaction can reach 38% and 34%, respectively, and the ee value of the product 2e is 13%. Among them, the amine substrate a and the ketone substrate 4 (cyclohexanone) can also be directly catalyzed by the enzyme to generate cyclohexylamine, and the conversion rate reaches 96%.

在催化底物苯甲醛6和4摩尔当量的胺底物b,e,f,h或i生成二级胺的反应中,转化率在33%-72%之间。In the reaction between the catalytic substrate benzaldehyde 6 and 4 molar equivalents of amine substrates b, e, f, h or i to form secondary amines, the conversions ranged from 33% to 72%.

在催化一些酮底物如7,8分别和胺底物e,f的反应中,其转化率在34%-69%之间。值得注意的是,在这些反应的产物中4a,4b,4f,4i,6e,6h和8f可以用作一些相关药物合成的相关骨架前体。In the reaction of some ketone substrates such as 7 and 8 with amine substrates e and f, the conversion rate is between 34% and 69%. It is worth noting that 4a, 4b, 4f, 4i, 6e, 6h and 8f among the products of these reactions can be used as related scaffold precursors for the synthesis of some related drugs.

另外,AcRedAm可以从酮底物9(1-茚酮)和炔丙胺e起始直接合成产物雷沙吉兰9e,转化率为44%,ee值为44%(S)。In addition, AcRedAm can directly synthesize the product rasagiline 9e from the ketone substrate 9 (1-indanone) and propargylamine e, with a conversion rate of 44% and an ee value of 44% (S).

实施例4还原胺化酶AcRedAm突变体的构建及酶活测定Embodiment 4 Construction and Enzyme Activity Determination of Reductive Aminase AcRedAm Mutant

在本实施例中,针对还原胺化酶AcRedAm具有催化合成雷沙吉兰的活性特征,基于蛋白同源建模、分子对接分析通过理性设计选择位点,对该酶进行定向进化改造以获得能催化合成对映纯雷沙吉兰的突变体。In this example, the reductive aminase AcRedAm has the activity characteristic of catalyzing the synthesis of rasagiline, and based on protein homology modeling and molecular docking analysis, the site was selected through rational design, and the enzyme was subjected to directed evolution modification to obtain an active Catalyzed synthesis of mutants of enantiopure rasagiline.

以重组质粒pET28a-AcRedAm为模板(图13),以带有突变位点为简并碱基(NNK)的一对互补寡核苷酸作引物,用Primestar高保真酶进行全质粒PCR扩增,获得具有特定突变位点的重组质粒。引物序列如下:Using the recombinant plasmid pET28a-AcRedAm as a template (Figure 13), using a pair of complementary oligonucleotides with mutation sites as degenerate bases (NNK) as primers, PCR amplification of the entire plasmid was performed with Primestar high-fidelity enzymes, Obtain recombinant plasmids with specific mutation sites. The primer sequences are as follows:

对应于SEQ ID NO:2中第90位亮氨酸被其它19种氨基酸取代的突变体:A mutant corresponding to the substitution of the 90th leucine in SEQ ID NO:2 by other 19 amino acids:

L90-F:AAGACCGTTATCAACNNKACCAACGGTA(SEQ ID No:3)L90-F: AAGACCGTTATCAACNNKACCAACGGTA (SEQ ID No: 3)

L90-R:MNNGTTGATAACGGTCTTGCCGTTCA(SEQ ID No:4)L90-R: MNNGTTGATAACGGTCTTGCCGTTCA (SEQ ID No: 4)

对应于SEQ ID NO:2中第117位异亮氨酸被其它19种氨基酸取代的突变体:A mutant corresponding to the substitution of isoleucine at position 117 by other 19 amino acids in SEQ ID NO:2:

I117-F:TACATCCACGGTGGCNNKATGGCGGTGC(SEQ ID No:5)I117-F:TACATCCACGGTGGCNNKATGGCGGTGC (SEQ ID No:5)

I117-R:MNNGCCACCGTGGATGTATTGCGCG(SEQ ID No:6)I117-R: MNNGCCACCGTGGATGTATTGCGCG (SEQ ID No: 6)

对应于SEQ ID NO:2中第172位亮氨酸被其它19种氨基酸取代的突变体:A mutant corresponding to the substitution of leucine at position 172 in SEQ ID NO:2 by other 19 amino acids:

L172-F:CACGATCTGGCGCTGNNKAGCGGCATGT(SEQ ID No:7)L172-F: CACGATCTGGCGCTGNNKAGCGGCATGT (SEQ ID No: 7)

L172-R:MNNCAGCGCCAGATCGTGCAGGCTCG(SEQ ID No:8)L172-R: MNNCAGCGCCAGATCGTGCAGGCTCG (SEQ ID No: 8)

对应于SEQ ID NO:2中第207位色氨酸被其它19种氨基酸取代的突变体:A mutant corresponding to the substitution of tryptophan at position 207 in SEQ ID NO:2 by other 19 amino acids:

W207-F:CAACTGCTGACCCCGNNKCTGAGCGCGA(SEQ ID No:9)W207-F:CAACTGCTGACCCCGNNKCTGAGCGCGA (SEQ ID No:9)

W207-R:MNNCGGGGTCAGCAGTTGAACAAAACCG(SEQ ID No:10)W207-R: MNNCGGGGTCAGCAGTTGAACAAAACCG (SEQ ID No: 10)

对应于SEQ ID NO:2中第214位酪氨酸被其它19种氨基酸取代的突变体:Corresponding to the mutant in which the 214th tyrosine in SEQ ID NO:2 is replaced by other 19 kinds of amino acids:

Y214-F:AGCGCGATGACCCACNNKCTGGGTGCGC(SEQ ID No:11)Y214-F: AGCGCGATGACCCANNKCTGGGTGCGC (SEQ ID No: 11)

Y214-R:MNNGTGGGTCATCGCGCTCAGCCAC(SEQ ID No:12)Y214-R: MNNGTGGGTCATCGCGCTCAGCCAC (SEQ ID No: 12)

对应于SEQ ID NO:2中第236位甲硫氨酸被其它19种氨基酸取代的突变体:Corresponding to the mutant in which the 236th methionine in SEQ ID NO:2 is substituted by other 19 kinds of amino acids:

M236-F:GGCAGCAACATGGCGNNKCAGGTGACCG(SEQ ID No:13)M236-F: GGCAGCAACATGGCGNNKCAGGTGACCG (SEQ ID No: 13)

M236-R:MNNCGCCATGTTGCTGCCTTGGGTC(SEQ ID No:14)M236-R: MNNCGCCATGTTGCTGCCTTGGGTC (SEQ ID No: 14)

对应于SEQ ID NO:2中第237位谷氨酰胺被其它19种氨基酸取代的突变体:Corresponding to the mutant whose glutamine at position 237 in SEQ ID NO:2 is substituted by other 19 kinds of amino acids:

Q237-F:AGCAACATGGCGATGNNKGTGACCGGTG(SEQ ID No:15)Q237-F: AGCAACATGGCGATGNNKGTGACCGGTG (SEQ ID No: 15)

Q237-R:MNNCATCGCCATGTTGCTGCCTTGG(SEQ ID No:16)Q237-R: MNNCATCGCCATGTTGCTGCCTTGG (SEQ ID No: 16)

扩增体系为:重组质粒模板20ng、引物(10um)各1ul、PrimeSTAR Max DNA聚合酶25ul、补充双蒸水至50ul。扩增条件为:98℃预变性1分钟,98℃变性10秒,60℃退火30秒,72℃延伸1分钟45秒,共25个循环。反应结束后,以0.8%琼脂糖凝胶电泳检测扩增产物。产物经PCR产物纯化试剂盒纯化回收,用DpnⅠ酶(NEB公司)在37℃条件下消化回收产物2小时,降解初始模板。消化产物转化至E.coli BL21(DE3)感受态细胞,涂布到含有50ug/mL卡那霉素LB琼脂平板上,37℃过夜培养,筛选阳性克隆,测序验证。得到还原胺化酶AcRedAm指定位点的饱和突变体重组菌。The amplification system is: 20ng of recombinant plasmid template, 1ul of each primer (10um), 25ul of PrimeSTAR Max DNA polymerase, supplemented with double distilled water to 50ul. Amplification conditions were: pre-denaturation at 98°C for 1 minute, denaturation at 98°C for 10 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute and 45 seconds, a total of 25 cycles. After the reaction, the amplified products were detected by 0.8% agarose gel electrophoresis. The product was purified and recovered by a PCR product purification kit, and the recovered product was digested with DpnI enzyme (NEB Company) at 37° C. for 2 hours to degrade the initial template. The digested product was transformed into E.coli BL21 (DE3) competent cells, spread on LB agar plates containing 50ug/mL kanamycin, cultured overnight at 37°C, positive clones were screened, and verified by sequencing. Obtain the saturated mutant recombinant bacteria at the designated site of reductive aminase AcRedAm.

按照实施例2的方法得到还原胺化酶AcRedAm指定位点的饱和突变体纯化蛋白。According to the method of Example 2, the purified protein of the saturated mutant of the designated site of reductive aminase AcRedAm was obtained.

突变体酶活测定反应体系为:1mg/mL突变体纯酶、0.7mg/mL GDH(阿拉丁)、30mMD-葡萄糖、1mM NADP+、5mM 1-茚酮、250mM炔丙胺(在调节至pH 9.0的缓冲液中)和2%(v/v)DMSO。使用Tris-HCl(pH 9.0)缓冲液将最终反应体积补足至500μL。反应在25℃下以220rpm的速度振荡孵育24小时。然后,加入30μL 10M NaOH以淬灭反应。反应混合物用500μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用HPLC或GC-FID进行分析。The mutant enzyme activity assay reaction system was: 1mg/mL mutant pure enzyme, 0.7mg/mL GDH (Aladdin), 30mM D-glucose, 1mM NADP+, 5mM 1-indanone, 250mM propargylamine (adjusted to pH 9.0 buffer) and 2% (v/v) DMSO. The final reaction volume was made up to 500 μL using Tris-HCl (pH 9.0) buffer. Reactions were incubated at 25°C for 24 hours with shaking at 220 rpm. Then, 30 μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 500 μL of methyl tert-butyl ether. Combine the upper organic phases, dry over anhydrous MgSO4 , and analyze using HPLC or GC-FID.

结果如图6所示。结果表明,L90位点是影响AcRedAm正确折叠的关键位点,该位点饱和突变体中只有10个突变体能够可溶性表达纯化获得,并且纯化得到的突变体对产物雷沙吉兰对映选择性的影响很小。I117,L172和M236位点的饱和突变体中大部分突变体的活性降低,这些位点是影响AcRedAm催化活性的关键位点,其中突变体L172V表现出对(R)-雷沙吉兰具有适中的对映选择性,ee值为40%。The result is shown in Figure 6. The results show that the L90 site is the key site that affects the correct folding of AcRedAm, and only 10 mutants in the saturated mutants of this site can be soluble expressed and purified, and the purified mutants are enantioselective to the product rasagiline has little impact. The activities of most of the mutants in the saturation mutants at I117, L172 and M236 sites were reduced, these sites are the key sites affecting the catalytic activity of AcRedAm, and the mutant L172V showed moderate activity to (R)-rasagiline The enantioselectivity, ee value is 40%.

结果如图7所示。结果表明,在207和214位点的饱和突变体中,一些突变体的活性有所提高,并且大部分突变体对(S)-雷沙吉兰具有很高的对映选择性,ee值能达到>99%。突变体W207C表现出最高的活性,是野生型活性的1.2倍。在237位点的饱和突变体中,大部分突变体的活性降低了,但是一些突变体例如Q237N,Q237S,Q237G和Q237A对(R)-雷沙吉兰具有适中到优秀的对映选择性,ee值从40%到>99%。其中最好的突变体是Q237A,同野生型相比仍具有70%的活性,对(R)-雷沙吉兰具有很高的对映选择性,ee值为>99%。The result is shown in Figure 7. The results showed that among the saturation mutants at positions 207 and 214, the activity of some mutants was improved, and most of the mutants had high enantioselectivity to (S)-rasagiline, and the ee value could Achieved >99%. The mutant W207C exhibited the highest activity, which was 1.2 times that of the wild type. Among the saturation mutants at position 237, most of the mutants had reduced activity, but some mutants such as Q237N, Q237S, Q237G and Q237A had moderate to excellent enantioselectivity for (R)-rasagiline, ee values from 40% to >99%. Among them, the best mutant is Q237A, which still has 70% activity compared with the wild type, and has high enantioselectivity for (R)-rasagiline, with an ee value of >99%.

以214位点饱和突变体中筛选得到的活性提高的突变体(Y214T、Y214I、Y214G、Y214C或Y214M)质粒为模板,以带有207位点饱和突变体中活性提高的突变体(W207A、W207S、W207C、W207V、W207H或W207M)突变位点的一对互补寡核苷酸作引物,用Primestar高保真酶进行全质粒PCR扩增,获得具有特定突变位点的重组质粒。引物序列如下:Using the mutants (Y214T, Y214I, Y214G, Y214C or Y214M) plasmids with enhanced activity screened in the 214-site saturation mutants as templates, the mutants (W207A, W207S) with increased activity in the 207-site saturation mutants were used as templates. , W207C, W207V, W207H or W207M) a pair of complementary oligonucleotides at the mutation site as primers, and use Primestar high-fidelity enzymes for PCR amplification of the whole plasmid to obtain a recombinant plasmid with a specific mutation site. The primer sequences are as follows:

对应于SEQ ID NO:2中第207位色氨酸被丙氨酸取代同时第214位酪氨酸被苏氨酸、异亮氨酸、甘氨酸、半胱氨酸或甲硫氨酸其中一种取代的突变体:Corresponding to the replacement of tryptophan at position 207 by alanine in SEQ ID NO: 2 and tyrosine at position 214 by one of threonine, isoleucine, glycine, cysteine or methionine Substituted mutants:

W207A-F:CAACTGCTGACCCCGGCGCTGAGCGCG(SEQ ID No:17)W207A-F:CAACTGCTGACCCCGGCGCTGAGCGCG (SEQ ID No: 17)

W207A-R:GCCGGGGTCAGCAGTTGAACAAAACCG(SEQ ID No:18)W207A-R:GCCGGGGTCAGCAGAGTTGAACAAAACCG (SEQ ID No: 18)

对应于SEQ ID NO:2中第207位色氨酸被丝氨酸取代同时第214位酪氨酸被苏氨酸、异亮氨酸、甘氨酸、半胱氨酸或甲硫氨酸其中一种取代的突变体:Corresponding to the substitution of tryptophan at position 207 by serine and tyrosine at position 214 by one of threonine, isoleucine, glycine, cysteine or methionine in SEQ ID NO:2 mutant:

W207S-F:CAACTGCTGACCCCGAGTCTGAGCGCGA(SEQ ID No:19)W207S-F:CAACTGCTGACCCCGAGTCTGAGCGCGA (SEQ ID No: 19)

W207S-R:ACTCGGGGTCAGCAGTTGAACAAAACCG(SEQ ID No:20)W207S-R:ACTCGGGGTCAGCAGAGTTGAACAAAACCG (SEQ ID No: 20)

对应于SEQ ID NO:2中第207位色氨酸被半胱氨酸取代同时第214位酪氨酸被苏氨酸、异亮氨酸、甘氨酸、半胱氨酸或甲硫氨酸其中一种取代的突变体:Corresponding to the substitution of tryptophan at position 207 by cysteine in SEQ ID NO: 2 and tyrosine at position 214 by one of threonine, isoleucine, glycine, cysteine or methionine Substitution mutants:

W207C-F:ACTGCTGACCCCGTGTCTGAGCGCGATGACCCACTA(SEQ ID No:21)W207C-F: ACTGCTGACCCCGTGTCTGAGCGCGATGACCCACTA (SEQ ID No: 21)

W207C-R:ACACGGGGTCAGCAGTTGAACAAAACCGGTCG(SEQ ID No:22)W207C-R:ACACGGGGTCAGCAGAGTTGAACAAAACCGGTCG (SEQ ID No: 22)

对应于SEQ ID NO:2中第207位色氨酸被缬氨酸取代同时第214位酪氨酸被苏氨酸、异亮氨酸、甘氨酸、半胱氨酸或甲硫氨酸其中一种取代的突变体:Corresponding to the substitution of tryptophan at position 207 by valine in SEQ ID NO:2 and tyrosine at position 214 by one of threonine, isoleucine, glycine, cysteine or methionine Substituted mutants:

W207V-F:CAACTGCTGACCCCGGTGCTGAGCGCGATGACCCACT(SEQ ID No:23)W207V-F:CAACTGCTGACCCCGGTGCTGAGCGCGATGACCCACT (SEQ ID No: 23)

W207V-R:ACCGGGGTCAGCAGTTGAACAAAACCGGTCG(SEQ ID No:24)W207V-R:ACCGGGGTCAGCAGTTGAACAAAACCGGTCG (SEQ ID No: 24)

对应于SEQ ID NO:2中第207位色氨酸被组氨酸取代同时第214位酪氨酸被苏氨酸、异亮氨酸、甘氨酸、半胱氨酸或甲硫氨酸其中一种取代的突变体:Corresponding to the substitution of tryptophan at position 207 by histidine in SEQ ID NO: 2 and tyrosine at position 214 by one of threonine, isoleucine, glycine, cysteine or methionine Substituted mutants:

W207H-F:CAACTGCTGACCCCGCATCTGAGCGCGATGACCCACTA(SEQ ID No:25)W207H-F:CAACTGCTGACCCCGCATCTGAGCGCGATGACCCACTA (SEQ ID No: 25)

W207H-R:ATGCGGGGTCAGCAGTTGAACAAAACCGGTCG(SEQ ID No:26)W207H-R:ATGCGGGGTCAGCAGTTGAACAAAACCGGTCG (SEQ ID No: 26)

对应于SEQ ID NO:2中第207位色氨酸被甲硫氨酸取代同时第214位酪氨酸被苏氨酸、异亮氨酸、甘氨酸、半胱氨酸或甲硫氨酸其中一种取代的突变体:Corresponding to the replacement of tryptophan at position 207 by methionine in SEQ ID NO: 2 and tyrosine at position 214 by one of threonine, isoleucine, glycine, cysteine or methionine Substitution mutants:

W207M-F:CAACTGCTGACCCCGATGCTGAGCGCGATGACCCACT(SEQ ID No:27)W207M-F:CAACTGCTGACCCCGATGCTGAGCGCGATGACCCACT (SEQ ID No: 27)

W207M-R:ATCGGGGTCAGCAGTTGAACAAAACCGGTCG(SEQ ID No:28)W207M-R:ATCGGGGTCAGCAGAGTTGAACAAAACCGGTCG (SEQ ID No: 28)

按照实施例2的方法得到还原胺化酶AcRedAm指定位点的饱和突变体纯化蛋白。According to the method of Example 2, the purified protein of the saturated mutant of the designated site of reductive aminase AcRedAm was obtained.

突变体酶活测定反应体系为:1mg/mL突变体纯酶、0.7mg/mL GDH(阿拉丁)、30mMD-葡萄糖、1mM NADP+、5mM 1-茚酮、250mM炔丙胺(在调节至pH 9.0的缓冲液中)和2%(v/v)DMSO。使用Tris-HCl(pH 9.0)缓冲液将最终反应体积补足至500μL。反应在25℃下以220rpm的速度振荡孵育24小时。然后,加入30μL 10M NaOH以淬灭反应。反应混合物用500μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用HPLC或GC-FID进行分析。The mutant enzyme activity assay reaction system was: 1mg/mL mutant pure enzyme, 0.7mg/mL GDH (Aladdin), 30mM D-glucose, 1mM NADP+, 5mM 1-indanone, 250mM propargylamine (adjusted to pH 9.0 buffer) and 2% (v/v) DMSO. The final reaction volume was made up to 500 μL using Tris-HCl (pH 9.0) buffer. Reactions were incubated at 25°C for 24 hours with shaking at 220 rpm. Then, 30 μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 500 μL of methyl tert-butyl ether. Combine the upper organic phases, dry over anhydrous MgSO4 , and analyze using HPLC or GC-FID.

结果如图8所示。结果表明,组合后的双点突变体活性并没有明显提高,但是都对产物(S)-雷沙吉兰表现出很高的对映选择性,ee值>99%。其中最好的突变体W207S/Y214C活性是野生型的1.3倍。The result is shown in Figure 8. The results showed that the activity of the combined double point mutants was not significantly improved, but they all showed high enantioselectivity to the product (S)-rasagiline, with ee values>99%. The activity of the best mutant W207S/Y214C was 1.3 times that of the wild type.

实施例5还原胺化酶AcRedAm及其突变体合成雷沙吉兰的放大反应针对合成雷沙吉兰的放大反应条件进行优化,分别在不同浓度的1-茚酮(5mM-50mM),不同浓度的炔丙胺(50mM-1M),不同浓度的葡萄糖脱氢酶GDH(0.7mg/mL-2mg/mL)或不同浓度的还原胺化酶(0.25mg/mL-2.5mg/mL)条件下反应,在25℃下以220rpm的速度振荡孵育24小时。然后,加入30μL 10M NaOH以淬灭反应。反应混合物用500μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用HPLC或GC-FID进行分析。Example 5 Amplification reaction of reductive aminase AcRedAm and its mutants for synthesizing rasagiline The amplification reaction conditions for synthesizing rasagiline are optimized, respectively in different concentrations of 1-indanone (5mM-50mM), different concentrations propargylamine (50mM-1M), different concentrations of glucose dehydrogenase GDH (0.7mg/mL-2mg/mL) or different concentrations of reductive aminase (0.25mg/mL-2.5mg/mL) under the conditions of reaction, Incubate with shaking at 220 rpm for 24 hours at 25 °C. Then, 30 μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 500 μL of methyl tert-butyl ether. Combine the upper organic phases, dry over anhydrous MgSO4 , and analyze using HPLC or GC-FID.

结果如图9所示。结果表明,在1-茚酮浓度为5mM,炔丙胺浓度为250mM,酶载量(GDH浓度0.7mg/mL,还原胺化酶浓度1mg/mL)适中时能获得较高转化率。The result is shown in Figure 9. The results showed that when the concentration of 1-indanone was 5mM, the concentration of propargylamine was 250mM, and the enzyme load (GDH concentration 0.7mg/mL, reductive aminase concentration 1mg/mL) was moderate, a higher conversion rate could be obtained.

合成雷沙吉兰的放大反应体系为:1mg/mL纯酶、0.7mg/mL GDH(阿拉丁)、100mM D-葡萄糖、1mM NADP+、5mM 1-茚酮、250mM炔丙胺(在调节至pH 9.0的缓冲液中)和2%(v/v)DMSO。使用Tris-HCl(pH 9.0)缓冲液将最终反应体积补足至50mL。反应在25℃下以220rpm的速度振荡孵育180小时。分别在不同的时间点取样200ul,加入10μL 10M NaOH以淬灭反应。反应混合物用200μL甲基叔丁基醚萃取两次。合并上层有机相,用无水MgSO4干燥,并使用HPLC或GC-FID进行分析。The scale-up reaction system for the synthesis of rasagiline is: 1mg/mL pure enzyme, 0.7mg/mL GDH (Aladdin), 100mM D-glucose, 1mM NADP+, 5mM 1-indanone, 250mM propargylamine (adjusted to pH 9.0 buffer) and 2% (v/v) DMSO. The final reaction volume was made up to 50 mL using Tris-HCl (pH 9.0) buffer. Reactions were incubated for 180 hours at 25°C with shaking at 220 rpm. 200ul samples were taken at different time points, and 10μL of 10M NaOH was added to quench the reaction. The reaction mixture was extracted twice with 200 μL of methyl tert-butyl ether. Combine the upper organic phases, dry over anhydrous MgSO4 , and analyze using HPLC or GC-FID.

结果如图10-12所示。结果表明,反应60小时后还原胺化酶AcRedAm合成雷沙吉兰反应的转化率达到70%,分离产率为60%;反应120小时后突变体AcQ237A合成对映纯(R)-雷沙吉兰(ee值>99%)反应的转化率达到51%,分离产率为42%。The results are shown in Figure 10-12. The results showed that after 60 hours of reaction, the conversion rate of reductive aminase AcRedAm to synthesize rasagiline reached 70%, and the isolated yield was 60%; after 120 hours of reaction, mutant AcQ237A synthesized enantiopure (R)-rasagiline The conversion of blue (ee value>99%) reaction reached 51%, and the isolated yield was 42%.

在本发明提及的所有文献都在本申请中引用作为参考,就如同每一篇文献被单独引用作为参考那样。此外应理解,在阅读了本发明的上述讲授内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

序列表sequence listing

<110> 上海交通大学<110> Shanghai Jiaotong University

<120> 还原胺化酶及其制法和应用<120> Reductive aminase and its preparation method and application

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<221> misc_feature<221> misc_feature

<222> (2)..(3)<222> (2)..(3)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 6<400> 6

mnngccaccg tggatgtatt gcgcg 25mnngccaccg tggatgtatt gcgcg 25

<210> 7<210> 7

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (16)..(17)<222> (16)..(17)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 7<400> 7

cacgatctgg cgctgnnkag cggcatgt 28cacgatctgg cgctgnnkag cggcatgt 28

<210> 8<210> 8

<211> 26<211> 26

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (2)..(3)<222> (2)..(3)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 8<400> 8

mnncagcgcc agatcgtgca ggctcg 26mnncagcgcc agatcgtgca ggctcg 26

<210> 9<210> 9

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (16)..(17)<222> (16)..(17)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 9<400> 9

caactgctga ccccgnnkct gagcgcga 28caactgctga ccccgnnkct gagcgcga 28

<210> 10<210> 10

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (2)..(3)<222> (2)..(3)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 10<400> 10

mnncggggtc agcagttgaa caaaaccg 28mnncggggtc agcagttgaa caaaaccg 28

<210> 11<210> 11

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (16)..(17)<222> (16)..(17)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 11<400> 11

agcgcgatga cccacnnkct gggtgcgc 28agcgcgatga cccacnnkct gggtgcgc 28

<210> 12<210> 12

<211> 25<211> 25

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (2)..(3)<222> (2)..(3)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 12<400> 12

mnngtgggtc atcgcgctca gccac 25mnngtgggtc atcgcgctca gccac 25

<210> 13<210> 13

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (16)..(17)<222> (16)..(17)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 13<400> 13

ggcagcaaca tggcgnnkca ggtgaccg 28ggcagcaaca tggcgnnkca ggtgaccg 28

<210> 14<210> 14

<211> 25<211> 25

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (2)..(3)<222> (2)..(3)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 14<400> 14

mnncgccatg ttgctgcctt gggtc 25mnncgccatg ttgctgcctt gggtc 25

<210> 15<210> 15

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (16)..(17)<222> (16)..(17)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 15<400> 15

agcaacatgg cgatgnnkgt gaccggtg 28agcaacatgg cgatgnnkgt gaccggtg 28

<210> 16<210> 16

<211> 25<211> 25

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<220><220>

<221> misc_feature<221> misc_feature

<222> (2)..(3)<222> (2)..(3)

<223> n is a, c, g, or t<223> n is a, c, g, or t

<400> 16<400> 16

mnncatcgcc atgttgctgc cttgg 25mnncatcgcc atgttgctgc cttgg 25

<210> 17<210> 17

<211> 27<211> 27

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 17<400> 17

caactgctga ccccggcgct gagcgcg 27caactgctga ccccggcgct gagcgcg 27

<210> 18<210> 18

<211> 27<211> 27

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 18<400> 18

gccggggtca gcagttgaac aaaaccg 27gccggggtca gcagttgaac aaaaccg 27

<210> 19<210> 19

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 19<400> 19

caactgctga ccccgagtct gagcgcga 28caactgctga ccccgagtct gagcgcga 28

<210> 20<210> 20

<211> 28<211> 28

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 20<400> 20

actcggggtc agcagttgaa caaaaccg 28actcggggtc agcagttgaa caaaaccg 28

<210> 21<210> 21

<211> 36<211> 36

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 21<400> 21

actgctgacc ccgtgtctga gcgcgatgac ccacta 36actgctgacc ccgtgtctga gcgcgatgac ccacta 36

<210> 22<210> 22

<211> 32<211> 32

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 22<400> 22

acacggggtc agcagttgaa caaaaccggt cg 32acacggggtc agcagttgaa caaaaccggt cg 32

<210> 23<210> 23

<211> 37<211> 37

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 23<400> 23

caactgctga ccccggtgct gagcgcgatg acccact 37caactgctga ccccggtgct gagcgcgatg accact 37

<210> 24<210> 24

<211> 31<211> 31

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 24<400> 24

accggggtca gcagttgaac aaaaccggtc g 31accggggtca gcagttgaac aaaaccggtc g 31

<210> 25<210> 25

<211> 38<211> 38

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 25<400> 25

caactgctga ccccgcatct gagcgcgatg acccacta 38caactgctga ccccgcatct gagcgcgatg accacta 38

<210> 26<210> 26

<211> 32<211> 32

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 26<400> 26

atgcggggtc agcagttgaa caaaaccggt cg 32atgcggggtc agcagttgaa caaaaccggt cg 32

<210> 27<210> 27

<211> 37<211> 37

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 27<400> 27

caactgctga ccccgatgct gagcgcgatg acccact 37caactgctga ccccgatgct gagcgcgatg accact 37

<210> 28<210> 28

<211> 31<211> 31

<212> DNA<212>DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 28<400> 28

atcggggtca gcagttgaac aaaaccggtc g 31atcggggtca gcagttgaac aaaaccggtc g 31

Claims (10)

1.一种体外还原胺化方法,其特征在于,包括步骤:1. an in vitro reductive amination method, is characterized in that, comprises steps: (i)在还原胺化酶AcRedAm存在下,使得(S1)酮底物或醛底物与(S2)胺底物进行还原胺化反应。(i) Reductive amination reaction of (S1) ketone substrate or aldehyde substrate and (S2) amine substrate in the presence of reductive amination enzyme AcRedAm. 2.如权利要求1所述的方法,其特征在于,所述方法包括:2. The method of claim 1, wherein the method comprises: 在还原胺化酶AcRedAm存在下,使式Z1所示的酮底物或醛底物和式Z2所示的胺底物进行还原胺化反应,从而形成式I所示的还原胺化产物:In the presence of reductive amination enzyme AcRedAm, the ketone substrate or aldehyde substrate shown in formula Z1 and the amine substrate shown in formula Z2 are subjected to reductive amination reaction, thereby forming the reductive amination product shown in formula I:
Figure FDA0003426841700000011
Figure FDA0003426841700000011
其中in R1为取代或未取代的烷基,取代或未取代的芳基、取代或未取代的(C1-C6亚烃基)-苯基,其中所述的取代指出一个或多个H原子被选自下组的取代基所取代:C1-C3烷基、C2-C4烯基、C2-C4炔基、C3-C6环烷基、卤素、或其组合;R1 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted (C1-C6 alkylene)-phenyl group, wherein the substitution indicates that one or more H atoms are selected from the following Substituents of the group are substituted by: C1-C3 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, halogen, or a combination thereof; R2为H或甲基;R2 is H or methyl; 或者R1和R2与相连的C原子共同构成取代或未取代的4-10元的杂环;Or R1 and R2 together with the connected C atoms form a substituted or unsubstituted 4-10 membered heterocyclic ring; R3为H、取代或未取代的C1-C8烷基,取代或未取代的C2-C8烯基、取代或未取代的C2-C8炔基、取代或未取代的C3-C8环烷基、取代或未取代的6-10芳基、取代或未取代的(C1-C6亚烃基)-苯基、取代或未取代的(C3-C6亚环烷基)-苯基,其中,所述的取代指出一个或多个H原子被选自下组的取代基所取代:C1-C3烷基、C2-C4烯基、C2-C4炔基、C3-C6环烷基、卤素、或其组合。R3 is H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C2-C8 alkenyl, substituted or unsubstituted C2-C8 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted Or unsubstituted 6-10 aryl, substituted or unsubstituted (C1-C6 alkylene)-phenyl, substituted or unsubstituted (C3-C6 cycloalkylene)-phenyl, wherein the substitution Indicates that one or more H atoms are substituted by a substituent selected from the group consisting of C1-C3 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C6 cycloalkyl, halogen, or combinations thereof.
3.一种分离的或纯化的还原胺化酶,其特征在于,所述的还原胺化酶具有SEQ ID No:1所示的序列,并且具有至少1个氨基酸突变。3. An isolated or purified reductive amination enzyme, characterized in that the reductive amination enzyme has the sequence shown in SEQ ID No: 1 and has at least one amino acid mutation. 4.如权利要求3所述的还原胺化酶,其特征在于,所述的突变型的还原胺化酶在选自下组的位点具有突变:SEQ ID No:1所示序列的第207位、第214位、237位或其组合。4. The reductive aminase according to claim 3, wherein the mutant reductive aminase has a mutation at a site selected from the group consisting of the 207th sequence shown in SEQ ID No: 1 bit, 214th bit, 237th bit, or a combination thereof. 5.一种分离的多核苷酸,其特征在于,所述的多核苷酸编码权利要求3所述的还原胺化酶。5. An isolated polynucleotide, characterized in that said polynucleotide encodes the reductive aminase according to claim 3. 6.一种载体,其特征在于,所述的载体含有权利要求5所述的多核苷酸。6. A vector, characterized in that the vector contains the polynucleotide according to claim 5. 7.一种遗传工程化的宿主细胞,其特征在于,所述的宿主细胞含有权利要求6所述的载体,或其基因组中整合权利要求5所述的多核苷酸。7. A genetically engineered host cell, characterized in that the host cell contains the vector according to claim 6, or the polynucleotide according to claim 5 is integrated into its genome. 8.一种权利要求3所述的还原胺化酶或野生型还原胺化酶AcRedAm的用途,其特征在于,它被用于催化还原胺化反应,或被用于制备催化还原胺化反应的催化制剂。8. the purposes of a kind of reductive amination enzyme described in claim 3 or wild-type reductive amination enzyme AcRedAm, it is characterized in that, it is used for catalytic reductive amination reaction, or is used for the preparation of catalytic reductive amination reaction Catalytic preparation. 9.一种还原胺化酶体外合成雷沙吉兰的方法,其特征在于,包括步骤:9. A method for synthesizing rasagiline in vitro by reductive aminase, characterized in that it comprises the steps of: 在权利要求3所述的还原胺化酶或野生型还原胺化酶AcRedAm存在下,催化1-茚酮和炔丙胺进行反应,从而生成雷沙吉兰。In the presence of the reductive amination enzyme or wild-type reductive amination enzyme AcRedAm described in claim 3, catalyzing the reaction of 1-indanone and propargylamine, thereby generating rasagiline. 10.一种进行还原胺化反应的反应体系,其特征在于,所述的反应体系包括:10. A reaction system for reductive amination, characterized in that, the reaction system comprises: (S0)权利要求3所述的还原胺化酶或野生型还原胺化酶AcRedAm;(S0) reductive aminase or wild-type reductive aminase AcRedAm described in claim 3; (S1)酮底物或醛底物;(S1) a ketone substrate or an aldehyde substrate; (S2)胺底物;和(S2) an amine substrate; and (S3)任选的NADPH或NADPH再生模块。(S3) Optional NADPH or NADPH regeneration module.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118956991A (en) * 2024-10-18 2024-11-15 天津凯莱英生物科技有限公司 Synthesis method of chiral amine

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118956991A (en) * 2024-10-18 2024-11-15 天津凯莱英生物科技有限公司 Synthesis method of chiral amine

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