Preparation method of 2-aminobutyric acid

Abstract

The invention discloses a preparation method of 2-aminobutyric acid, which comprises the following steps: taking threonine and ammonium formate as precursors, and utilizing threonine deaminase mutant, leucine dehydrogenase and formate dehydrogenase to carry out enzyme catalysis reaction to synthesize the 2-aminobutyric acid, wherein compared with wild type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: 256, 257, 262, 484 and 488. The threonine deaminase mutant has the advantages of relatively high enzymatic activity, stability and the like. Therefore, by adopting the threonine deaminase mutant to prepare the 2-aminobutyric acid, the synthesis conversion rate of the 2-aminobutyric acid can be further improved, and the threonine deaminase mutant is low in production cost and more suitable for industrial production.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a method for preparing 2-aminobutyric acid. Background Art

[0002] 2-Aminobutyric acid (L-2-ABA) is an unnatural amino acid. It is a key chiral precursor of several very important drugs. 2-Aminobutyric acid is converted into 2-aminobutyramide, which is a direct precursor of the anti-epileptic drugs levetiracetam (Caplan) and brivaracetam. It can also be converted into 2-aminobutanol, which is a precursor for the synthesis of the anti-tuberculosis drug ethambutol. The optical purity of these drugs is very important for the safety and efficiency of treatment. With the continuous development of the drug market, the demand for chiral drug intermediates will also increase, so it is necessary to seek more efficient, cheap and sustainable 2-aminobutyric acid synthesis routes.

[0003] At present, the production methods of 2-aminobutyric acid mainly include chemical synthesis, microbial fermentation and enzyme catalysis. The chemical synthesis method has low cost, but the product purity is not high and there may be harmful byproducts. Although the microbial fermentation method is environmentally friendly, it has a long production cycle and low yield. The enzyme catalysis method has attracted widespread attention due to its high efficiency and specificity, but the existing enzyme catalysis system often has problems such as poor stability and low catalytic efficiency. Therefore, in order to improve the production efficiency and product quality of 2-aminobutyric acid, it is necessary to develop a new enzyme catalysis system. Summary of the invention

[0004] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a method for preparing 2-aminobutyric acid, in which a threonine deaminase mutant is used to prepare 2-aminobutyric acid, and the threonine deaminase mutant has high activity and stability, thereby further improving the conversion rate of 2-aminobutyric acid synthesis.

[0005] In the first aspect of the present application, the present application proposes a method for preparing 2-aminobutyric acid. According to an embodiment of the present application, the method comprises: using threonine and ammonium formate as precursors, using a threonine deaminase mutant, leucine dehydrogenase and formate dehydrogenase, and performing an enzyme-catalyzed reaction to synthesize 2-aminobutyric acid; wherein, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: 256, 257, 262, 484, 488. In the method of the present application, a threonine deaminase mutant is used to prepare 2-aminobutyric acid, and the threonine deaminase mutant has higher activity and stability. As a result, the method of the present application can further improve the conversion rate of 2-aminobutyric acid synthesis.

[0006] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0008] Figure 1 The three-dimensional spatial structure of the protein of the wild-type threonine deaminase in Example 2 of the present application;

[0009] Figure 2 The SDS-PAGE gel electrophoresis diagram of each threonine deaminase mutant in Example 3 of the present application;

[0010] Figure 3 The SDS-PAGE gel electrophoresis of the crude enzyme solution of H19 and H25 in Example 6 of the present application;

[0011] Figure 4 This is a schematic diagram of the enzymatic conversion to produce 2-aminobutyric acid in Example 7 of the present application. DETAILED DESCRIPTION

[0012] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0013] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0014] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0015] In order to make the present application more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present application belongs.

[0016] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0017] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0018] As used herein, the term "variant" or "mutant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0019] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred to a cell or host and / or between cells or hosts. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, the vector can produce a desired expression product by cultivating a suitable cell or host that contains the vector.

[0020] In this article, the term "cell" generally refers to the use of genetic engineering technology or cell fusion technology to modify or reorganize the genetic material of the host cell to obtain a cell with a unique trait of stable inheritance. Wherein, the term "host cell" refers to a prokaryotic cell or eukaryotic cell that can be introduced into a recombinant vector. The terms "transformed" or "transfected" used herein refer to the introduction of nucleic acids (such as vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present application, and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used for the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid) and CoS cells.

[0021] The present application proposes a method for preparing 2-aminobutyric acid, which will be described in detail below.

[0022] In the first aspect of the present application, the present application proposes a method for preparing 2-aminobutyric acid. According to an embodiment of the present application, the method comprises: using threonine and ammonium formate as precursors, using a threonine deaminase mutant, leucine dehydrogenase and formate dehydrogenase, and performing an enzyme-catalyzed reaction to synthesize 2-aminobutyric acid; wherein, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: 256, 257, 262, 484, 488.

[0023] The inventors of the present application modeled the amino acid sequence of the wild-type threonine deaminase by using the Rosetta Fold2 online software tool (http: / / robetta.bakerlab.org / ) to obtain the three-dimensional spatial structure of the protein, and used multiple analysis software to analyze the conformation of the wild-type threonine deaminase, and found that the wild-type threonine deaminase had two loop regions with particularly high B factors (241-263, 482-497), and performed site-directed saturation mutation virtual screening on the loop region to obtain a large number of threonine deaminase mutants. It was found that based on the amino acid sequence of the wild-type threonine deaminase, at least one of the 256th, 257th, 262nd, 484th, and 488th positions was subjected to site-directed mutation, and the obtained threonine deaminase mutant had the advantages of high enzyme activity and stability. Therefore, the use of threonine deaminase mutants to prepare 2-aminobutyric acid can further improve the conversion rate of 2-aminobutyric acid synthesis, and the production cost is low, which is more suitable for industrial production.

[0024] Herein, unless otherwise specified, the position of the mutation site in the threonine deaminase mutant is located based on the amino acid sequence of the wild-type threonine deaminase (the specific amino acid sequence is shown in SEQ ID NO: 1). For example, "position 256" is the 256th position numbered sequentially from the N-terminus to the C-terminus in the amino acid sequence shown in the wild-type threonine deaminase, and it should be understood that "position 256" also includes positions equivalent to 256, and the determination of equivalent positions can be determined by sequence alignment.

[0025] According to an embodiment of the present application, the above-mentioned threonine deaminase mutant may further include at least one of the following technical features:

[0026] According to an embodiment of the present application, the amino acid sequence of the wild-type threonine deaminase is shown in SEQ ID NO:1.

[0027] MMADSQPLSGAPEGAEYLRAVLRAPVYEAAQVTPLQKMEKLSSRLDNVILVKREDRQPVHSFKLRGAYAMMAGLTEEQKAHGVITASAGNHAQGVAFSSARLGVKALIVMPTATADIKVDAVRGFGGEVLL HGANFDEAKAKAIELSQQQGFTWVPPFDHPMVIAGQGTLALELLQQDAHLDRVFVPVGGGGLAAGVAVLIKQLMPQIKVIAVEAEDSACLKAALDAGHPVDLPRVGLFAEGVAVKRIGDETFRL CQ EYD D IITVDSDAICAAMKDLFEDVRAVAEPSGALALAGMKKYIALHNIRGERLAHILSGANVNFHGLRYVSERCELGEQREAALLAVTIPEEKGSFLKFCQLLGGRSVTEFNYRF ADAKNACIFVGVRLSRGLEERKEILQMLNDGGYSVVDLSDDEMAKLHVRYMVGGRPSHPLQERLYSFEFPESPGALLRFLNTLGTYWNISLFHYRSHGTDYGRVLAAFELG D HEP D FETRLNELGYDCHDETNNPAFRFFLAG (SEQ ID NO: 1).

[0028] It should be noted that in the present application, "the amino acid sequence is shown in SEQ ID NO: 1" includes the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of conservatively modified forms of SEQ ID NO: 1, all of which are within the protection scope of the present application.

[0029] As used herein, "conservatively modified amino acid sequences" refer to amino acid modifications that do not significantly affect or alter the activity and other properties of the threonine deaminase comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the threonine deaminase of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Exemplarily, conservative modifications are not more than 80% of the total number, preferably not more than 90% of the total number. In this article, "conservatively modified amino acid sequences" also include amino acid modifications of natural mutations, and "natural mutations" refer to mutations caused by changes in alleles and the like caused by individual differences and other reasons in the natural mutation process of wild-type threonine deaminase.

[0030] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: position 256 and position 488.

[0031] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is the 256th position.

[0032] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is position 488.

[0033] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, D488N.

[0034] In this article, there is no need for special explanation. When the threonine deaminase mutant has multiple mutation sites, the multiple mutation sites are located at different positions, that is, D262R, D262M, D262W, and D262Y mutations cannot exist at the same time, and D488T, D488R, and D488N mutations cannot exist at the same time.

[0035] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are: C256L / M / G / K and D488R / M / K.

[0036] Herein, in the "letter-number-letter" of the mutation site, the number indicates the position of the mutation site in the wild-type threonine deaminase, the first letter indicates the amino acid of the wild-type threonine deaminase before mutation, and the second letter indicates the amino acid of the threonine deaminase mutant after mutation. For example, "C256M" means that the cysteine ​​C at position 256 of the wild-type threonine deaminase is mutated to methionine M; "C256L / M / G / K" means that the cysteine ​​C at position 256 of the wild-type threonine deaminase is mutated to one of methionine M, leucine L, glycine G, and lysine K.

[0037] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: C256M, D488R, D488N. Exemplarily, the threonine deaminase mutant has a single mutation site, which is C256M, D488R, or D488N; the threonine deaminase mutant has multiple mutation sites, which are C256M and D488R, or C256M and D488N.

[0038] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is:

[0039] 1) C256M; or

[0040] 2) D488R; or

[0041] 3) D488N; or

[0042] 4) C256M and D488R; or

[0043] 5) C256G and D488M; or

[0044] 6) C256L and D488K; or

[0045] 7) C256L and D488R; or

[0046] 8)C256K and D488M.

[0047] According to an embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is C256M, D488R, or D488N.

[0048] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is C256M.

[0049] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is D488R.

[0050] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is D488N.

[0051] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are C256M and D488R.

[0052] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are C256G and D488M.

[0053] In an optional embodiment of the present application, compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are C256L and D488 K.

[0054] According to an embodiment of the present application, the amino acid sequence of the leucine dehydrogenase is shown in SEQ ID NO:8.

[0055] MTLEIFEYLEKYDYEQVVFCQDKESGLKAIIAIHDTTLGPALGTRMWTYDSEEAAIEDALRLAKGMTYKNAAAGLNLGGAKTVIIGDPRKDKSEAMFRALGRYIQGLNGRYITAEDVGTTVDDMDIIHEETDFVTGISPSFGSSGNPSPVTAYGVYRGMKAAAKEAFGTDNLEGKVIAVQGVGN VAYHLCKHLHAEGAKLIVTDINKEAVQRAVEEFGASAVEPNEIYGVECDIYAPCALGATVNDETIPQLKAKVIAGSANNQLKEDRHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERALKRVESIYDTIAKVIEISKRDGIATYVAADRLAEERIASLKNSRSTYLRNGHDIISRR(SEQ IDNO:8).

[0056] According to an embodiment of the present application, the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO:9.

[0057] MAKVLCVLYDDPIDGYPTTYARDNLPKIDHYPGGQTLPTPKAIDFTPGTMLGSVSGELGLRKYLESNGHTLVVTSDKDGPDSVFEKELVDADIVISQPFWPAYLTPERFAKAKNLKLALTAGIGSDHVDLQSAIDRGVTVAEVTYCNSISVAEHVVMMILGLVRNYLPAHDWARKGGWNIADCVKHSYDLEAMSVGTVAAGR IGLAVLRRLAPFDVKLHYTDRHRLPESVEKELNLTWHASPTDMYPHCDVVTLNCPLHPETEHMVNEETLKLFKRGAYIVNTARGKLCDRDAIARALENGTL AGYAGDVWFPQPAPADHPWRTMAWNGMTPHMSGTSLTAQTRYAAGTREILECFFEGRPIRDEYLIVQGGNLAGVGAHSYSKGNATGGSEEAGKFKKAG(SEQ ID NO:9).

[0058] It should be noted that threonine deaminase mutants, leucine dehydrogenase and formate dehydrogenase can be purchased or obtained by expressing them in genetically engineered bacteria or by fermentation culture. The specific methods of obtaining them are not limited and are all within the scope of protection of this application.

[0059] In an optional embodiment of the present application, the threonine deaminase mutant, leucine dehydrogenase and formate dehydrogenase are all provided in the form of fermentation broth of engineered bacteria.

[0060] It should be noted that the threonine deaminase mutant, leucine dehydrogenase and formate dehydrogenase can be co-expressed in the same genetically engineered bacteria, or can be freely combined and expressed in two genetically engineered bacteria, or can be expressed separately in three genetically engineered bacteria. The specific types are not limited and are all within the scope of protection of this application.

[0061] In an optional embodiment of the present application, the threonine deaminase mutant is provided in the form of a first fermentation broth, and the fermentation broth contains the threonine deaminase mutant.

[0062] According to an embodiment of the present application, the working concentration of the threonine deaminase mutant is 1.0 g / L to 5.0 g / L, for example, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L , 2.9g / L, 3.0g / L, 3.1g / L, 3.2g / L, 3.3g / L, 3.4g / L, 3.5g / L, 3.6g / L, 3.7g / L, 3.8g / L, 3.9g / L, 4.0g / L, 4.1g / L, 4.2g / L, 4.3g / L, 4.4g / L, 4.5g / L, 4.6g / L, 4.7g / L, 4.8g / L, 4.9g / L, 5.0g / L or any two of them as the range between the endpoints.

[0063] Herein, the term "working concentration" refers to the final concentration of the fermentation broth or enzyme added to the reaction system. For example, the "working concentration of the threonine deaminase mutant" refers to the final concentration of the first fermentation broth or the threonine deaminase mutant added to the reaction system.

[0064] In an optional embodiment of the present application, the leucine dehydrogenase and formate dehydrogenase are provided in the form of a second fermentation broth, and the fermentation broth contains leucine dehydrogenase and formate dehydrogenase.

[0065] According to an embodiment of the present application, the working concentration of the leucine dehydrogenase and the formate dehydrogenase is 30 g / L to 50 g / L, for example, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L or any two of them as the range value between the endpoint values.

[0066] It should be noted that the “working concentration of leucine dehydrogenase and formate dehydrogenase” refers to the final concentration of the second fermentation broth or “leucine dehydrogenase and formate dehydrogenase” added to the reaction system.

[0067] According to an embodiment of the present application, the method further comprises nicotinamide adenine dinucleotide.

[0068] According to an embodiment of the present application, the working concentration of nicotinamide adenine dinucleotide is 0.2 g / L to 1.0 g / L, for example, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L or a range value between any two of the endpoint values.

[0069] According to an embodiment of the present application, the method further comprises: performing post-extraction treatment on the reaction solution obtained by the enzyme-catalyzed reaction.

[0070] According to an embodiment of the present application, the post-extraction process is performed by the following steps:

[0071] acidifying the reaction solution;

[0072] The acidified product is subjected to a protein precipitation removal treatment;

[0073] The product after removing the protein precipitate is subjected to a decolorization treatment.

[0074] According to an embodiment of the present application, the acidification treatment is performed using hydrochloric acid.

[0075] According to an embodiment of the present application, the protein precipitate removal process is performed by centrifugation.

[0076] According to an embodiment of the present application, a filter aid is pre-added before the protein precipitation removal process.

[0077] According to an embodiment of the present application, the decolorization treatment is carried out using an organic decolorization membrane and activated carbon.

[0078] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0079] Materials and methods

[0080] 1) Materials: Plasmids pET28a and pRSFDuet-1 were purchased from Changsha Abiwei Biotechnology Co., Ltd.; NotI, HindIII, XhoI and other restriction endonucleases, high-fidelity enzyme premix, and T4 DNA ligase were purchased from Shanghai Yisheng Biotechnology Co., Ltd.; synthetic primers, E. coli BL21 (DE3) competent cells, DNA marker, plasmid extraction kit, DNA gel recovery and purification kit, kanamycin sulfate, and isopropyl-β-d-thiogalactoside were purchased from Shanghai Sangon Biotechnology Co., Ltd.; chemical reagents were all analytical grade. The plasmid extraction procedure was referred to the instructions of the plasmid extraction kit; the DNA gel recovery procedure was referred to the instructions of the DNA gel recovery and purification kit; the DNA fragment ligation procedure was referred to the instructions of the T4 DNA ligase kit.

[0081] 2) LB medium (g / L): peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, sterilized at 121°C for 20 min.

[0082] 3) Fermentation medium (g / L): peptone 10 g / L, yeast powder 5 g / L, glycerol 10 g / L, disodium hydrogen phosphate dodecahydrate 20 g / L, potassium dihydrogen phosphate 7 g / L, ammonium chloride 5 g / L, anhydrous sodium sulfate 1 g / L and magnesium sulfate heptahydrate 1 g / L, defoamer 0.5 ml / L, sterilize at 121°C for 30 min.

[0083] 4) HPLC analysis method: Thermo Fisher ODS-2 HypersilTM (4.6×250 mm, 5 μm); flow rate 1.0 mL / min; detection wavelength 210 nm; mobile phase: methanol: 0.05 mol / L potassium dihydrogen phosphate + 0.005 mol / L sodium decane sulfonate (pH = 2.30) = 7:93.

[0084] Example 1: Construction of wild-type threonine deaminase (abbreviated as wild-type TD) genetically engineered bacteria

[0085] (1) Synthesis of wild-type td gene

[0086] According to the information disclosed in the NCBI database, threonine deaminase from Escherichia coli (GenBank: APK04219.1) was selected, and its amino acid sequence is shown in SEQ ID NO:1 in the sequence list, and its gene sequence is shown in SEQ ID NO:2 in the sequence list. The gene coding sequence was codon optimized according to the codon preference rule of Escherichia coli, and NdeI and

[0087] After the XhoI restriction site, it was sent to Nanjing GenScript Biotechnology Co., Ltd. for artificial synthesis (SEQ ID NO: 3).

[0088] The gene sequence of threonine deaminase TD is shown in SEQ ID NO: 2:

[0089]

[0090] The sequence of the td gene coding sequence after codon optimization is shown in SEQ ID NO: 3:

[0091] catatg ctcgag .

[0092] (2) Construction of wild-type TD pET28a vector

[0093] Design primers for constructing the vector. Use 1-F (5'-ctcgagcaccaccaccaccaccactga-3' (SEQ ID NO: 4)) / 1-R (5'-atgatgatgatgatggctgct-3' (SEQ ID NO: 5)) as primers, the purchased pET28a vector as template, 2×Hieff Plus PCR Master Mix (With Dye) high-fidelity enzyme premix PCR amplified the linearized vector; using 2-F (5'-agcagccatcatcatcatcatatgatggctgactctcaacca-3' (SEQ ID NO: 6)) / 2-R (5'-tggtggtggtggtgctcgagttaaccagccaagaagaatct-3' (SEQ ID NO: 7)) as primers and the td gene DNA fragment artificially synthesized by GenScript (nucleotide sequence as shown in SEQ ID NO: 3) as template, high-fidelity enzyme premix PCR amplified the td gene DNA fragment with 5' and 3' ends completely consistent with the two end sequences of the linearized vector; 1% agarose gel electrophoresis detection, and the amplified linearized vector (5.3kb) and td gene (1.6kb) DNA fragments were recovered using a DNA gel recovery and purification kit. Using Hieff Plus One Step Cloning Kit One-step rapid cloning kit performs homologous recombination between the linearized vector and td gene fragment recovered from gel. The recombination system is: 2.5μL of pET28a linearized vector, 2.5μL of td gene fragment, 2×Hieff Enzyme Premix 5 μL, and then react at 50°C for 30 min.

[0094] (3) Construction of wild-type TD genetically engineered bacteria

[0095] The homologous recombination product obtained in (2) was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. The transformant with correct sequencing was the plasmid vector TD-pET28a containing the wild-type td gene, and the transformant was also the wild-type TD genetic engineering bacteria.

[0096] Example 2: Determination of a single site-directed mutation site in threonine deaminase TD

[0097] The protein sequence of wild-type TD was modeled using the new artificial intelligence online software tool Rosetta Fold2 (http: / / robetta.bakerlab.org / ) to obtain its three-dimensional spatial structure (see Figure 1 ), and the model was visualized and processed using PyMol. The model was evaluated using PROCHECK and Verify 3D programs. Ramachandran plot was used to illustrate the two dihedral angles in the peptide plane ( The ratio of ψ to ψ) is used to indicate the allowed and unallowed conformations of amino acid residues, and the reliability of the protein model is evaluated by the Psi / Phi Ramachandran plot calculated by PROCHECK. The compatibility of the amino acid residues of the protein side chain is evaluated by Verify 3D software, and at least 80% of the amino acid residues pass the verification if the score is ≥0.2. The molecular docking program glide-dock in Schrodinger Suites software is used for molecular docking, and the B-factor distribution of the TD protein model is analyzed by Pymol. It is found that the B-factor of the two loop regions of the protein is particularly high (241st-263rd, 482nd-497th), so the loop region is subjected to site-directed saturation mutation virtual screening to obtain the mutation sites in Table 1. The binding free energy of the ligand and the protein is calculated for these mutation sites by Rosetta Flex ddG. The lower the binding free energy value, the higher the affinity between the receptor and the ligand, and the greater the possibility of increased enzyme activity.

[0098] Table 1:

[0099] Mutation site <![CDATA[ΔG (Binding) (Kcal / mol)]]> Mutation site <![CDATA[ΔG (Binding) (Kcal / mol)]]> WT -8.6 D484M --15.1 D262R -20.7 D488R -14.8 D488V -20.4 C256M -13.9 D262M -20.1 F253H -13.6 D262E -19.3 F253I -13.5 D488M -17.6 D262W -13.4 D488T -16.5 D262Y -13.1 Q257M -16.2 F253M -12.9 D262L -16.1 C256L -12.4 D488N -15.8 D262N -12.1

[0100] Example 3: Preparation and expression of single mutants of threonine deaminase

[0101] The original plasmid TD-pET28a was used as a template for site-directed mutagenesis to obtain a recombinant plasmid TD-X-pET28a carrying a mutant encoding threonine deaminase, wherein the site-directed mutagenesis method is as follows:

[0102] The primer sequences used to design the mutants are shown in Table 2 (the bold and underlined parts in the primer sequences are the bases corresponding to the mutation sites):

[0103] Table 2:

[0104]

[0105]

[0106] Using 1-F / F253H-R as primers and TD-pET28a plasmid as template, a large DNA fragment with F253H mutation site was amplified by PCR with high-fidelity enzyme premix. Using F253H-F / 2-R as primers and TD-pET28a plasmid as template, 2×Hieff Plus PCR Master Mix (With Dye) high-fidelity enzyme premix PCR amplified small DNA fragments with 5' and 3' ends that were completely consistent with the sequences at both ends of the large DNA fragment; 1% agarose gel electrophoresis was used to detect, and the amplified large and small DNA fragments were recovered using a DNA gel recovery and purification kit. Plus One Step Cloning Kit One-step rapid cloning kit performs homologous recombination on the large and small fragments of DNA recovered from the gel. The recombination system is: 2.5μL of large fragment DNA, 2.5μL of small fragment DNA, 2×Hieff Enzyme Premix 5μL, react at 50℃ for 30min.

[0107] The homologous recombination product obtained was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin sulfate. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. The correctly sequenced transformant was the plasmid vector TD-F253H-pET28a containing the single mutant td gene, which was also the single mutant TD-F253H genetic engineering bacteria, numbered H1. In the same way, the engineered bacteria H2 with F253I mutation, H3 with F253M mutation, H4 with C256M mutation, H5 with C256L mutation, H6 with Q257M mutation, H7 with D262R mutation, H8 with D262M mutation, H9 with D262E mutation, H10 with D262L mutation, H11 with D262W mutation, H12 with D262Y mutation, H13 with D262N mutation, H14 with D484M mutation, H15 with D488V mutation, H16 with D488M mutation, H17 with D488T mutation, H18 with D488R mutation, and H19 with D488N mutation were constructed.

[0108] The correctly sequenced mutants H1-H19 genetically engineered bacteria were inoculated into test tubes containing 4 mL LB medium containing 50 μg / mL kanamycin, cultured overnight at 37°C and 200 rpm, and then transferred to 50 mL LB medium and cultured at 37°C and 200 rpm until OD 600The concentration of IPTG was 0.6-0.8, and IPTG was added to a final concentration of 0.2 mM. The culture was continued at 20°C and 200 rpm for 18 h. The fermentation broth was centrifuged at 9000 rpm for 5 min, and the supernatant was removed. The bacterial precipitate was retained and resuspended with water to a bacterial concentration of 50 g / L. Ultrasonic disruption was performed for 10 min, and the precipitate was removed by centrifugation. The supernatant was retained for SDS-PAGE gel electrophoresis analysis. The results are shown in Figure 2 The results showed that the mutant proteins of H3, H5, and H15 were not expressed normally; the mutant proteins of H7, H8, H10, H11, and H12 were poorly soluble; and the mutant proteins of H1, H2, H4, H6, H9, H13, H14, H16, H17, H18, and H19 were expressed normally and had good solubility.

[0109] Example 4: Enzyme activity assay of threonine deaminase mutants

[0110] 1. Take 100 μL of enzyme solution supernatant and add it to a centrifuge tube containing 900 μL of substrate reaction solution, and place it at 30℃ for 20 minutes. After the reaction is completed, dilute it with water to a certain multiple and centrifuge it, and take the supernatant for detection by ELISA.

[0111] The relative enzyme activity results of each single mutant are shown in Table 3:

[0112] Table 3:

[0113]

[0114]

[0115] The results in Table 3 show that the threonine deaminase mutants with single point mutations of C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, and D488N have significantly increased enzyme activities compared to the wild type. The threonine deaminase mutants with single point mutations of C256M (H4), D488R (H18), and D488N (H19) have higher enzyme activities, among which the mutant H19 with D488N has the largest increase in enzyme activity, which is 1.57 times that of the wild type.

[0116] 2. The enzyme activity results of step 1 above show that the threonine deaminase mutants with single-point mutations C256M, D488R, and D488N have higher enzyme activity, indicating that the 256th and 488th amino acids have a greater effect on the enzyme activity of threonine deaminase TD. Therefore, in order to obtain a mutant with better enzyme activity, the inventors used the wild-type TD as the starting strain and performed double-point iterative saturation mutations on the 256th and 488th amino acid sites. The specific method is as follows:

[0117] Design of double-site iterative saturation mutagenesis primer 3-F at amino acid positions 256 and 488: 5'-GGTGACGAGACTTTCAGATTG NNK CAAGAGTACTTGGACGACATC-3' (SEQ ID NO: 20) (the underlined part is the saturation mutation site, N is A or T or C or G, K is G or T) and 3-R: 5'-CTCGTTCAATCTAGTCTCGAA MNN TGGTTCGTGATCTCCCAATTC-3' (SEQ ID NO: 21) (the underlined part is a saturated mutation site, N is A or T or C or G, and M is A or C). Using the original plasmid TD-pET28a as a template, 3-F and 3-R were used as mutation primers to amplify a DNA fragment containing both the 256th and 488th mutation points. Using TD-pET28a as a template, 4-F (5'-TTCGAGACTAGATTGAACGAG-3', SEQ ID NO: 22) / 4-R

[0118] (5'-CAATCTGAAAGTCTCGTCACC-3', SEQ ID NO: 23) was used as primer, and PCR was performed to amplify the linearized vector DNA fragment with the 5' and 3' ends having sequences completely consistent with the two ends of the mutant DNA fragment; the two DNA fragments obtained above were subjected to electrophoresis and gel cutting and recovery, and the purified mutant DNA fragment was homologously recombined with the linearized vector DNA fragment. The product: vector was mixed at a molar ratio of 2:1, and 2×Hieff Enzyme Premix was reacted at 50°C for 30 minutes. Then, a recombinant genetic engineering bacterium was constructed according to the method shown in (3) of Example 1 to obtain a fully saturated mutant library containing positions 256 and 488.

[0119] From the mutant library, about 1000 single colonies were picked and inoculated into a 96-well deep-well culture plate containing 700uL LB medium, wherein the LB medium contained 50ug / mL kanamycin, and after culturing at 37°C for 6h, a final concentration of 0.2mM IPTG was added, the temperature was lowered to 20°C, and the induction culture was continued for 18h. After induction, the 96-well deep-well culture plate was centrifuged at 5000rpm for 10min, and the supernatant was discarded. The culture plate was then placed in an ultra-low temperature refrigerator at -80°C for 1h, taken out and placed at room temperature for 30min to thaw, and this freeze-thaw operation was repeated 3 times. After the freeze-thaw was completed, 200uL of water was added to resuspend the bacteria to obtain an enzyme solution, and then the enzyme activity was determined according to the method shown in Example 4, and mutants with higher enzyme activity than the wild type were screened.

[0120] The 30 mutants were screened by shaking flasks. The specific process was as follows: the mutants were inoculated into 50 mL LB medium and cultured at 37°C and 200 rpm until the OD 600 The concentration of IPTG was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM, and the culture was continued at 20°C and 200 rpm for 18 h. The fermentation broth was centrifuged at 9000 rpm for 5 min, the supernatant was removed, the bacterial precipitate was retained, and the bacterial precipitate was resuspended with water to a bacterial concentration of 50 g / L. Ultrasonic disruption was performed for 10 min, and the precipitate was removed by centrifugation. The enzyme supernatant was retained for enzyme activity determination. The determination method was the same as that in Example 4. Finally, 5 mutants with higher enzyme activity than the wild type were obtained. The specific data are shown in Table 4:

[0121] Table 4:

[0122] name Mutation site Relative enzyme activity (%) WT - 100.00 H20 C256L-D488R 107.9 H21 C256M-D488R 137.2 H22 C256G-D488M 118.9 H23 C256K-D488M 108.5 H24 C256L-D488K 113.4

[0123] Example 5: Obtaining a genetically engineered bacterium (H25) that co-expresses leucine dehydrogenase and formate dehydrogenase

[0124] In this example, two strains were used to react together to produce 2-aminobutyric acid, one of which was a genetically engineered bacterium that produced the threonine deaminase mutant H19 (see Example 3 for details, hereinafter referred to as genetically engineered bacterium H19), and the other was a genetically engineered bacterium that co-expressed leucine dehydrogenase and formate dehydrogenase, numbered H25.

[0125] According to the information disclosed in the NCBI database, leucine dehydrogenase LDH (amino acid sequence shown in SEQ ID NO: 8) from Bacillus (GenBank: WP_000171355.1) and formate dehydrogenase FDH (amino acid sequence shown in SEQ ID NO: 9) from Campylobacter aquatica (GenBank: AB091484.1) were selected. The gene coding sequences of LDH and FDH were codon optimized according to the codon preference rule of Escherichia coli, and NdeI and XhoI restriction sites were designed at both ends of the coding sequence of LDH, and HindIII and NotI restriction sites were designed at both ends of the coding sequence of FDH, and then sent to Nanjing GenScript Biotechnology Co., Ltd. for artificial synthesis (SEQ ID NO: 10 and SEQ ID NO: 11).

[0126] The sequence of the LDH coding sequence after codon optimization is shown in SEQ ID NO: 10:

[0127] catatg ctcgag ;

[0128] The sequence of the FDH coding sequence after codon optimization is shown in SEQ ID NO: 11:

[0129] aagctt gcggccgc .

[0130] Primers were designed to construct a vector for co-expression of LDH and FDH. 1-F (5'-acgtcgttaactcgagtctggtaaagaaacc-3' (SEQ ID NO: 12)) / 1-R (5'-tctctagtgtcatatgtatatctccttcttatac-3' (SEQ ID NO: 13)) were used as primers, the purchased pRSFDuet-1 vector was used as template, and 2×Hieff Plus PCR Master Mix (With Dye) high-fidelity enzyme premix PCR amplified the linearized vector; 2-F (5'-aggagatatacatatgacactagagatatttgaa-3' (SEQ ID NO: 14)) / 2-R (5'-ctttaccagactcgagttaacgacgtgagataatgtca-3' (SEQ ID NO: 15)) was used as primers, and the LDH gene DNA fragment synthesized by GenScript (nucleotide sequence as shown in SEQ ID NO: 10) was used as a template, and the LDH gene DNA fragment was amplified by high-fidelity enzyme premix PCR; 1% agarose gel electrophoresis was used to detect, and the amplified linearized vector (3.8kb) and LDH gene (1.1kb) DNA fragments were recovered using a DNA gel recovery and purification kit. The two recovered DNA fragments were double-digested with NdeI and XhoI, purified with a DNA purification kit, and then connected with T4 DNA ligase. The connection system is: vector DNA 5μL, LDH gene fragment 5μL, 10×Ligase Buffer 2μL, Gold T4 DNA Ligase 1μL, ultrapure water 7μL. React at 16℃ overnight.

[0131] The overnight ligation product was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. The correctly sequenced transformant was the plasmid vector pRSFDuet-LDH containing the LDH gene, which was also the LDH genetic engineering bacteria, numbered H20.

[0132] The LDH genetically engineered bacteria H20 were inoculated into LB medium containing 50ug / mL Kana, cultured at 37°C for 12h, and the bacteria were collected. The plasmid DNA (pRSFDuet-LDH) was extracted using a bacterial plasmid extraction kit. 3-F (5'-agctgggtaagcggccgcataatgcttaagtcg-3' (SEQ ID NO: 16)) / 3-R (5'-ctttagccataagcttgtcgacctgcaggcg-3' (SEQ ID NO: 17)) were used as primers, and the pRSFDuet-LDH vector was used as a template. 2×Hieff Plus PCRMaster Mix (With Dye) high-fidelity enzyme premix PCR amplified the vector DNA; 4-F (5'-gcaggtcgacaagcttatggctaaagtacta-3' (SEQ ID NO: 18)) / 4-R (5'-taagcattatgcggccgcttacccagctttt-3' (SEQ ID NO: 19)) was used as primers, and the FDH gene DNA fragment synthesized by GenScript (nucleotide sequence as shown in SEQ ID NO: 11) was used as a template, and the FDH gene DNA fragment was amplified by high-fidelity enzyme premix PCR; 1% agarose gel electrophoresis was used to detect, and the amplified vector DNA (4.9kb) and FDH gene (1.2kb) DNA fragments were recovered using a DNA gel recovery and purification kit. The two recovered DNA fragments were double-digested with HindIII and NotI, purified with a DNA purification kit, and then connected with T4 DNA ligase. The connection system is: vector DNA 5μL, FDH gene fragment 5μL, 10×Ligase Buffer 2μL, Gold T4 DNA Ligase 1μL, ultrapure water 7μL. React at 16℃ overnight.

[0133] The overnight ligation product was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and the transformation product was spread on LB plate containing 50 μg / mL kanamycin. After overnight culture at 37°C, positive transformants were selected and sent to Shanghai Shenggong Biotechnology Service Co., Ltd. for sequencing. The correctly sequenced transformant was the plasmid vector pRSFDuet-LDH-FDH containing FDH and LDH genes. This transformant is a genetically engineered bacterium that co-expresses LDH and FDH, numbered H25.

[0134] Example 6: Bacteria fermentation culture

[0135] 1 mL of the genetically engineered bacteria H19 for producing threonine deaminase mutants prepared in Example 3 and the genetically engineered bacteria H25 for co-expressing leucine dehydrogenase / formate dehydrogenase prepared in Example 5 were activated on LB slant medium and cultured at 37° C. for 16 h; an inoculated loop of the bacterial lawn was transferred from the slant medium to 100 mL of LB seed medium and cultured at 37° C. and 160 rpm for 10 to 16 h. The seeds were inoculated at a volume ratio of 8% into a 5L fermentation tank containing 3L fermentation medium. The temperature was controlled at 37°C±1°C, the pH was maintained at 7.0±0.1 during the fermentation process, the ventilation volume was controlled at 0.5vvm~3.0vvm, the rotation speed was controlled at 400rpm~600rpm, the dissolved oxygen was maintained at 30% by adjusting the rotation speed and ventilation volume, and after fermentation for 7~8h, the temperature was lowered to a stable level of 20~30°C, and IPTG was added with a final concentration of 0.2mM to induce for about 18h. When the OD of the fermentation no longer increased, the fermentation was terminated, and the fermentation yield of the two strains was ≥6%.

[0136] The fermentation liquid was centrifuged at 6000 rpm for 20 min, the supernatant was discarded to obtain wet bacteria, the bacteria were resuspended in water to a final concentration of 100 g / L, the bacterial liquid was subjected to high-pressure homogenization to obtain cell lysate, and the lysate supernatant obtained after centrifugation was obtained to obtain the crude enzyme solution.

[0137] The crude enzyme solutions of H19 and H25 were analyzed by SDS-PAGE gel electrophoresis. Figure 3 The results showed that all proteins were expressed normally.

[0138] Example 7: Enzymatic conversion to produce 2-aminobutyric acid

[0139] The reaction substrates were weighed sequentially into 1L of water, so that the final concentrations of ammonium formate, NAD, and L-threonine in the reaction vessel were 60g / L, 0.6g / L, and 10g / L, respectively. After adjusting the pH to 7.5 with aqueous ammonia, the H19 crude enzyme solution with a final concentration of 3g / L and the H25 crude enzyme solution with a final concentration of 40g / L prepared in Example 6 were added. The reaction was carried out at 35°C and 150rpm. During the reaction, 10g / L of threonine was added every 0.5-1h (a total of 9 times), and formic acid was used to maintain the pH at 7.5. The reaction was terminated after 10h. The schematic diagram of the enzymatic conversion to produce 2-aminobutyric acid is shown in Figure 4 . The sample was taken to detect the content of 2-aminobutyric acid, and the results showed that the 2-aminobutyric acid yield was 82.94g / L and the conversion rate was 95.81%. The reaction results of the wild-type WT crude enzyme solution operated in the same way showed that the 2-aminobutyric acid yield was 43.68g / L and the conversion rate was 50.46%. The co-reaction of H19 crude enzyme solution and H25 crude enzyme solution increased the conversion rate by 45.35% compared with the co-reaction of WT crude enzyme solution and H25 crude enzyme solution.

[0140] Example 8: Purification of 2-aminobutyric acid

[0141] The enzyme reaction solution obtained in Example 7 was acidified with hydrochloric acid, and the pH was adjusted to 4.0 to precipitate protein. A filter aid was then added, and the protein precipitate was removed by centrifugation to obtain a crude solution of 2-aminobutyric acid. A 200Da organic decolorizing membrane and activated carbon were used for decolorization to obtain a decolorized clear solution of 2-aminobutyric acid. Electrodialysis was then used to obtain a low-salt 2-aminobutyric acid solution, which was further subjected to reduced pressure distillation, cooling crystallization, and centrifugal drying to obtain a pure 2-aminobutyric acid product. The pure product was tested, and the data results are as follows:

[0142]

[0143]

[0144] The results show that the 2-aminobutyric acid obtained by the post-extraction treatment has a higher purity.

[0145] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0146] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in the field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A method for preparing 2-aminobutyric acid, characterized in that: include: Using threonine and ammonium formate as precursors, threonine deaminase mutants, leucine dehydrogenase and formate dehydrogenase were used to synthesize 2-aminobutyric acid through enzyme-catalyzed reactions; Compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: 256, 257, 262, 484, 488.

2. The method according to claim 1, characterized in that The amino acid sequence of the wild-type threonine deaminase is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that Compared with the wild-type threonine deaminase, the threonine deaminase mutant has at least one of the following mutation sites: C256M, Q257M, D262R, D262M, D262W, D262Y, D484M, D488T, D488R, and D488N.

4. The method according to claim 1, characterized in that: Compared with the wild-type threonine deaminase, the mutation sites of the threonine deaminase mutant are: C256L / M / G / K and D488R / M / K.

5. The method according to claim 1, characterized in that Compared with the wild-type threonine deaminase, the mutation site of the threonine deaminase mutant is: 1) C256M; or 2) D488R; or 3) D488N; or 4) C256M and D488R; or 5) C256G and D488M; or 6) C256L and D488K; or 7) C256L and D488R; or 8)C256K and D488M.

6. The method according to claim 1, characterized in that The amino acid sequence of the leucine dehydrogenase is shown in SEQ ID NO: 8; And / or, the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO:

9.

7. The method according to claim 1, characterized in that The working concentration of the threonine deaminase mutant is 1 g / L to 5 g / L; And / or, the working concentration of the leucine dehydrogenase and formate dehydrogenase is 30 g / L to 50 g / L.

8. The method according to claim 1, characterized in that The raw materials in the method further include nicotinamide adenine dinucleotide; Optionally, the working concentration of nicotinamide adenine dinucleotide is 0.2 g / L to 1.0 g / L.

9. The method according to claim 1, characterized in that: The method further comprises: The reaction solution obtained by the enzyme catalyzed reaction is subjected to post-extraction treatment.

10. The method according to claim 9, characterized in that The post-extraction process is performed by the following steps: acidifying the reaction solution; The acidified product is subjected to a protein precipitation removal treatment; Decolorizing the product of the protein precipitation removal treatment; Optionally, the acidification treatment is carried out using hydrochloric acid; And / or, the decolorization treatment is carried out using an organic decolorization membrane and activated carbon.

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