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

Abstract

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

Reductive aminase and its preparation method and application

technical field

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.

Background technique

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 .

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.

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.

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.

Contents of the invention

The object of the present invention is to provide a novel reductive aminase (RedAm) and its preparation method and application.

In a first aspect of the present invention, an in vitro reductive amination method is provided, comprising the steps of:

(i) Reductive amination reaction of (S1) ketone substrate or aldehyde substrate and (S2) amine substrate in the presence of reductive amination enzyme AcRedAm.

In another preferred example, the method includes:

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:

in

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 is H or methyl (CH3);

Or R1 and R2 together with the connected C atoms form a substituted or unsubstituted 4-10 membered heterocyclic ring;

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.

In another preferred example, the 4-10 membered heterocycles include saturated or unsaturated heterocycles, or benzo 5-7 heterocycles.

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.

In another preferred embodiment, the ketone substrate or aldehyde substrate is selected from the following group:

In another preference, the amine substrate is selected from the following group:

In another preferred example, the reductive amination product represented by formula I is a chiral amine compound.

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) .

In another preferred embodiment, the reductive amination enzyme AcRedAm is from or derived from Aspergillus calidoustus.

In another preferred embodiment, the reductive amination enzyme AcRedAm is selected from the following group:

(a) a polypeptide having the amino acid sequence shown in SEQ ID NO:1;

(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) a derivative polypeptide containing the polypeptide sequence described in (a) or (b) in its sequence;

(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.

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).

In another preferred example, the polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO:1.

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.

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.

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.

In another preferred example, the reductive aminase includes wild type and mutant type.

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.

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.

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.

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.

In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of Q237N, Q237S, Q237A, and Q237G.

In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of W207C and Q237A.

In another preferred example, the mutant reductive amination enzyme is a double-mutant or triple-mutant reductive aminating enzyme.

In another preferred example, the mutant reductive aminase has a mutation selected from the group consisting of W207S/Y214C.

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.

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.

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.

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.

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.

In another preferred example, the reductive amination enzyme includes an immobilized enzyme.

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.

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.

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 ).

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.

In another preferred example, the vectors include expression vectors, shuttle vectors, and integration vectors.

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.

In another preferred example, the cells are prokaryotic cells or eukaryotic cells.

In another preferred embodiment, the host cells are eukaryotic cells, such as yeast cells or plant cells.

In another preferred example, the host cell is a Saccharomyces cerevisiae cell.

In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

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.

In another preferred example, the reaction product of the reductive amination reaction includes isomers or non-isomers.

In another preferred example, the reaction product is in S configuration, R configuration or a combination thereof.

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:

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.

In an eighth aspect of the present invention, a reaction system for performing reductive amination reaction is provided, the reaction system comprising:

(S0) the reductive amination enzyme described in the second aspect of the present invention or the wild-type reductive amination enzyme AcRedAm;

(S1) a ketone substrate or an aldehyde substrate;

(S2) an amine substrate; and

(S3) Optional NADPH or NADPH regeneration module.

In another preferred example, the NADPH regeneration module includes: glucose dehydrogenase (GDH) and glucose.

In another preferred example, the NADPH regeneration module reacts glucose and NADP+ under the catalysis of glucose dehydrogenase (GDH), thereby generating gluconolactone and NADPH.

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.

Description of drawings

Figure 1 shows the results of SDS-PAGE electrophoresis analysis after purification of the reductive aminase AcRedAm.

Figure 2 shows the resulting graph of the optimum pH required for the reaction of reductive aminase AcRedAm.

Figure 3 shows the result graph of the optimal temperature required for the reaction of reductive aminase AcRedAm.

Figure 4 shows the Tm value (4a) and temperature tolerance (4b) results graph of reductive aminase AcRedAm.

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).

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.

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.

Figure 8 shows the results of relative activity and enantioselectivity of rasagiline synthesized by double-point combination mutants in reductive aminase AcRedAm.

Figure 9 shows the results of optimization of the scale-up reaction conditions for the synthesis of rasagiline.

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.

Figure 10 shows the results of the synthesis of rasagiline by reductive aminase AcRedAm and its mutant AcQ237A.

Figure 11 shows the HPLC analysis profile of the synthesis of rasagiline by reductive aminase AcRedAm.

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.

Figure 12 shows the HPLC analysis pattern of rasagiline synthesized by reductive aminase AcRedAm mutant.

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.

Figure 13 shows the map of the recombinant plasmid pET28a-AcRedAm of the present invention.

Detailed ways

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.

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).

Reductive aminase of the present invention

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).

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.

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 .

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.

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 .

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.

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.

The polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.

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.

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.

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.

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.

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.

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.

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). 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). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

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.

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.

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.

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.

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.

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.

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.

Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

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.

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.

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.

application

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.

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.

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.

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.

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.

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%.

Preferably, the molar ratio of the ketone to the amine substrate is 1:1˜1:50.

Preferably, the concentration of the ketone is 5 mM/L, and the concentration of the amine is 5-250 mM/L.

Preferably, the hydrophilic organic solvent is dimethylsulfoxide (DMSO).

Preferably, the final concentration of the hydrophilic organic solvent in the reaction system is 2%.

The method for synthesizing rasagiline

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.

Experiments show that the enzyme of the present invention is particularly suitable for synthesizing enantiomerically pure rasagiline through in vitro enzymatic conversion.

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%.

Preferably, the molar ratio of indanone to propargylamine is 1:50.

Preferably, the concentration of said indanone is 5mM/L, and the concentration of said propargylamine is 250mM/L.

Preferably, the hydrophilic organic solvent is dimethylsulfoxide (DMSO).

Preferably, the final concentration of the hydrophilic organic solvent in the reaction system is 2%.

The main advantages of the present invention include:

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. 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. The reductive aminase mutant of the present invention is used to successfully realize the enzymatic synthesis of single enantiomerically pure rasagiline in vitro.

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.

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.

Materials and Reagents

General description of the source of the biological material described in the present invention:

1. Primer synthesis: The primers used in the present invention were all synthesized by BGI Corporation.

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.

The screening of embodiment 1 reductive aminase

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.

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.

MSTITLFGLGAMGKALAAKYIEKGYTTTIWNRTPSKAAPLVEKGAKLANTVGEGLASADLIILCLLDASVRQTLDQATAALNGKTVINLTNGTPSQARETSEWVISHGAQYIHGGIMAVPDMIGSPHAVLLYSGESAETFSRVEAHLSHLGTSKFLGTDPGSASLHDLALLSGMYGLFSGFFHATALVKSQPGTTATGF VQLLTPWLSAMTHYLGALAKQIDEGDYATQGSNMAMQVTGVQNIVRASEEAGVTADLIMPILGRMTRAAEAGYADDVVSAVIEFMKE (SEQ ID No: 1)

The AcRedAm gene is codon optimized for easy expression in Escherichia coli. The optimized coding sequence is shown as SEQ ID No:2.

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

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.

Embodiment 2 Reductive aminase expression and purification

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.

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.

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).

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.

Embodiment 3 Reductive amination enzyme AcRedAm enzymatic property determination

In this example, the enzymatic properties of the recombinant reductive aminase AcRedAm prepared in Example 2 were determined.

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. Determination of optimum pH and optimum temperature of reductive aminase AcRedAm:

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.

The result is shown in Figure 2. The results showed that AcRedAm was highly active at pH 9.0.

3.2. Determination of the optimal temperature of reductive aminase AcRedAm:

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.

The result is shown in Figure 3. The results showed that AcRedAm had high activity at 25°C.

3.3. Determination of Tm value of reductive aminase AcRedAm:

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.

The results are shown in Figure 4a. The results showed that the Tm value of AcRedAm was 45°C.

3.4. Determination of thermal stability of reductive aminase AcRedAm:

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.

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. Determination of substrate spectrum of reductive aminase AcRedAm:

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.

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.

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%.

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.

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%.

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%.

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.

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).

Embodiment 4 Construction and Enzyme Activity Determination of Reductive Aminase AcRedAm Mutant

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.

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:

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-R: MNNGTTGATAACGGTCTTGCCGTTCA (SEQ ID No: 4)

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-R: MNNGCCACCGTGGATGTATTGCGCG (SEQ ID No: 6)

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-R: MNNCAGCGCCAGATCGTGCAGGCTCG (SEQ ID No: 8)

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-R: MNNCGGGGTCAGCAGTTGAACAAAACCG (SEQ ID No: 10)

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: AGCGCGATGACCCANNKCTGGGTGCGC (SEQ ID No: 11)

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

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-R: MNNCGCCATGTTGCTGCCTTGGGTC (SEQ ID No: 14)

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-R: MNNCATCGCCATGTTGCTGCCTTGG (SEQ ID No: 16)

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.

According to the method of Example 2, the purified protein of the saturated mutant of the designated site of reductive aminase AcRedAm was obtained.

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.

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%.

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%.

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:

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-R:GCCGGGGTCAGCAGAGTTGAACAAAACCG (SEQ ID No: 18)

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-R:ACTCGGGGTCAGCAGAGTTGAACAAAACCG (SEQ ID No: 20)

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-R:ACACGGGGTCAGCAGAGTTGAACAAAACCGGTCG (SEQ ID No: 22)

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-R:ACCGGGGTCAGCAGTTGAACAAAACCGGTCG (SEQ ID No: 24)

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-R:ATGCGGGGTCAGCAGTTGAACAAAACCGGTCG (SEQ ID No: 26)

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-R:ATCGGGGTCAGCAGAGTTGAACAAAACCGGTCG (SEQ ID No: 28)

According to the method of Example 2, the purified protein of the saturated mutant of the designated site of reductive aminase AcRedAm was obtained.

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.

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.

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.

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.

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.

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%.

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.

sequence listing

<110> Shanghai Jiaotong University

<120> Reductive aminase and its preparation method and application

<130> P2021-3350

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 287

<212> PRT

<213> Aspergillus calidoustus

<400> 1

Met Ser Thr Ile Thr Leu Phe Gly Leu Gly Ala Met Gly Lys Ala Leu

1 5 10 15

Ala Ala Lys Tyr Ile Glu Lys Gly Tyr Thr Thr Thr Ile Trp Asn Arg

20 25 30

Thr Pro Ser Lys Ala Ala Pro Leu Val Glu Lys Gly Ala Lys Leu Ala

35 40 45

Asn Thr Val Gly Glu Gly Leu Ala Ser Ala Asp Leu Ile Ile Leu Cys

50 55 60

Leu Leu Asp Asn Ala Ser Val Arg Gln Thr Leu Asp Gln Ala Thr Ala

65 70 75 80

Ala Leu Asn Gly Lys Thr Val Ile Asn Leu Thr Asn Gly Thr Pro Ser

85 90 95

Gln Ala Arg Glu Thr Ser Glu Trp Val Ile Ser His Gly Ala Gln Tyr

100 105 110

Ile His Gly Gly Ile Met Ala Val Pro Asp Met Ile Gly Ser Pro His

115 120 125

Ala Val Leu Leu Tyr Ser Gly Glu Ser Ala Glu Thr Phe Ser Arg Val

130 135 140

Glu Ala His Leu Ser His Leu Gly Thr Ser Lys Phe Leu Gly Thr Asp

145 150 155 160

Pro Gly Ser Ala Ser Leu His Asp Leu Ala Leu Leu Ser Gly Met Tyr

165 170 175

Gly Leu Phe Ser Gly Phe Phe His Ala Thr Ala Leu Val Lys Ser Gln

180 185 190

Pro Gly Thr Thr Ala Thr Gly Phe Val Gln Leu Leu Thr Pro Trp Leu

195 200 205

Ser Ala Met Thr His Tyr Leu Gly Ala Leu Ala Lys Gln Ile Asp Glu

210 215 220

Gly Asp Tyr Ala Thr Gln Gly Ser Asn Met Ala Met Gln Val Thr Gly

225 230 235 240

Val Gln Asn Ile Val Arg Ala Ser Glu Glu Ala Gly Val Thr Ala Asp

245 250 255

Leu Ile Met Pro Ile Leu Gly Arg Met Thr Arg Ala Ala Glu Ala Gly

260 265 270

Tyr Ala Asp Val Asp Val Ser Ala Val Ile Glu Phe Met Lys Glu

275 280 285

<210> 2

<211> 864

<212>DNA

<213> Artificial Sequence

<400> 2

atgagcacca ttaccctgtt cggtctgggt gcgatgggca aggcgctggc ggcgaagtac 60

atcgagaaag gctataccac caccatttgg aaccgtaccc cgagcaaagc ggcgccgctg 120

gttgagaagg gtgcgaaact ggcgaacacc gttggtgaag gtctggcgag cgcggacctg 180

atcattctgt gcctgctgga taacgcgagc gtgcgtcaaa ccctggacca agcgaccgcg 240

gcgctgaacg gcaagaccgt tatcaacctg accaacggta ccccgagcca ggcgcgtgag 300

accagcgaat gggtgattag ccacggcgcg caatacatcc acggtggcat tatggcggtg 360

ccggatatga tcggtagccc gcacgcggtt ctgctgtata gcggcgagag cgcggaaacc 420

ttcagccgtg ttgaagcgca cctgagccac ctgggtacca gcaaatttct gggtaccgac 480

ccgggtagcg cgagcctgca cgatctggcg ctgctgagcg gcatgtacgg cctgttcagc 540

ggcttctttc atgcgaccgc gctggttaaa agccaaccgg gtaccaccgc gaccggtttt 600

gttcaactgc tgaccccgtg gctgagcgcg atgacccact acctgggtgc gctggcgaaa 660

cagattgacg agggtgatta tgcgacccaa ggcagcaaca tggcgatgca ggtgaccggt 720

gttcaaaaca tcgttcgtgc gagcgaggaa gcgggcgtta ccgcggacct gatcatgccg 780

attctgggtc gtatgacccg tgcggcggaa gcgggttatg cggacgtgga tgttagcgcg 840

gtgatcgagt ttatgaagga ataa 864

<210> 3

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (16)..(17)

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

<400> 3

aagaccgtta tcaacnnkac caacggta 28

<210> 4

<211> 26

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (2)..(3)

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

<400> 4

mnngttgata acggtcttgc cgttca 26

<210> 5

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (16)..(17)

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

<400> 5

tacatccacg gtggcnnkat ggcggtgc 28

<210> 6

<211> 25

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (2)..(3)

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

<400> 6

mnngccaccg tggatgtatt gcgcg 25

<210> 7

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (16)..(17)

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

<400> 7

cacgatctgg cgctgnnkag cggcatgt 28

<210> 8

<211> 26

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (2)..(3)

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

<400> 8

mnncagcgcc agatcgtgca ggctcg 26

<210> 9

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (16)..(17)

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

<400> 9

caactgctga ccccgnnkct gagcgcga 28

<210> 10

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (2)..(3)

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

<400> 10

mnncggggtc agcagttgaa caaaaccg 28

<210> 11

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (16)..(17)

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

<400> 11

agcgcgatga cccacnnkct gggtgcgc 28

<210> 12

<211> 25

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (2)..(3)

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

<400> 12

mnngtgggtc atcgcgctca gccac 25

<210> 13

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (16)..(17)

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

<400> 13

ggcagcaaca tggcgnnkca ggtgaccg 28

<210> 14

<211> 25

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (2)..(3)

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

<400> 14

mnncgccatg ttgctgcctt gggtc 25

<210> 15

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (16)..(17)

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

<400> 15

agcaacatgg cgatgnnkgt gaccggtg 28

<210> 16

<211> 25

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_feature

<222> (2)..(3)

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

<400> 16

mnncatcgcc atgttgctgc cttgg 25

<210> 17

<211> 27

<212>DNA

<213> Artificial Sequence

<400> 17

caactgctga ccccggcgct gagcgcg 27

<210> 18

<211> 27

<212>DNA

<213> Artificial Sequence

<400> 18

gccggggtca gcagttgaac aaaaccg 27

<210> 19

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 19

caactgctga ccccgagtct gagcgcga 28

<210> 20

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 20

actcggggtc agcagttgaa caaaaccg 28

<210> 21

<211> 36

<212>DNA

<213> Artificial Sequence

<400> 21

actgctgacc ccgtgtctga gcgcgatgac ccacta 36

<210> 22

<211> 32

<212>DNA

<213> Artificial Sequence

<400> 22

acacggggtc agcagttgaa caaaaccggt cg 32

<210> 23

<211> 37

<212>DNA

<213> Artificial Sequence

<400> 23

caactgctga ccccggtgct gagcgcgatg accact 37

<210> 24

<211> 31

<212>DNA

<213> Artificial Sequence

<400> 24

accggggtca gcagttgaac aaaaccggtc g 31

<210> 25

<211> 38

<212>DNA

<213> Artificial Sequence

<400> 25

caactgctga ccccgcatct gagcgcgatg accacta 38

<210> 26

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 26

atgcggggtc agcagttgaa caaaaccggt cg 32

<210> 27

<211> 37

<212>DNA

<213> Artificial Sequence

<400> 27

caactgctga ccccgatgct gagcgcgatg accact 37

<210> 28

<211> 31

<212>DNA

<213> Artificial Sequence

<400> 28

atcggggtca gcagttgaac aaaaccggtc g 31

Claims (10)

1. an in vitro reductive amination method, is characterized in that, comprises steps: (i) Reductive amination reaction of (S1) ketone substrate or aldehyde substrate and (S2) amine substrate in the presence of reductive amination enzyme AcRedAm. 2. The method of claim 1, wherein the method comprises: 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:

in 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 is H or methyl; Or R1 and R2 together with the connected C atoms form a substituted or unsubstituted 4-10 membered heterocyclic ring; 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. 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. 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. An isolated polynucleotide, characterized in that said polynucleotide encodes the reductive aminase according to claim 3. 6. A vector, characterized in that the vector contains the polynucleotide according to claim 5. 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. 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. A method for synthesizing rasagiline in vitro by reductive aminase, characterized in that it comprises the steps of: 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. A reaction system for reductive amination, characterized in that, the reaction system comprises: (S0) reductive aminase or wild-type reductive aminase AcRedAm described in claim 3; (S1) a ketone substrate or an aldehyde substrate; (S2) an amine substrate; and (S3) Optional NADPH or NADPH regeneration module.

Images