An amine dehydrogenase mutant and its application in synthesis of chiral amine alcohol compounds

Abstract

This invention discloses an amine dehydrogenase mutant and its application in the synthesis of chiral amine alcohols. The amine dehydrogenase mutant disclosed in this invention is derived from *Bacillus stearothermophilus* (…). Geobacillus stearothermophilus )amine dehydrogenase ( Gs The protein obtained by mutation of LDH. The amine dehydrogenase mutant of the present invention can catalyze the synthesis of chiral amine alcohols, represented by 4-hydroxy-2-butanone, from hydroxy ketone substrates. It is easy to operate, has the advantages of high product optical purity, and stereoselectivity of up to 99%. It has good industrial application prospects in the biocatalytic preparation of chiral amine alcohols.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to amine dehydrogenase mutants and their application in chiral amine alcohol compounds. Background Technology

[0002] Chiral amine alcohols are precursors for the synthesis of many important drugs and are used to synthesize a variety of biologically active compounds. Among them, (R)-3-amino-1-butanol is an important chiral drug intermediate containing a chiral amine group, which has wide applications in organic synthesis and pharmaceuticals. It can be used to synthesize antitumor drugs, anti-AIDS drugs, and β-lactam derivatives.

[0003] Currently, the synthesis of (R)-3-amino-1-butanol is mainly achieved through chemical methods. Existing literature reports that chiral (R)-alanine is used as a starting material. After amino protection, diazomethane is used to elongate the carbon chain, converting it into a β-amino acid ester. Deprotection followed by reduction yields the target product. The disadvantages of this method are the difficulty in obtaining high-chiral-purity (R)-alanine and the hazardous use of diazomethane, making it unsuitable for large-scale industrial production. Another method involves reacting crotonate with (R)-(+)-α-phenylethylamine as a substrate to generate a set of epimers with two chiral centers. Separation by silica gel column chromatography yields a single isomer, which is then reduced by esterification and debenzylation to obtain (R)-3-amino-1-butanol. This route has fewer steps and readily available starting materials, but suffers from poor reaction selectivity, difficult separation and purification, and expensive reducing agents, making it unsuitable for large-scale production. Using ethyl 3-butyrate as a starting material, ethyl 3-(S)-butyrate was obtained via bioreduction. In subsequent reaction steps, it reacted with phthalimide under Witsunobu reagent catalysis, undergoing Walden transformation to successfully change the 3-(S) configuration to the desired 3-(R) configuration intermediate, yielding the final target compound. However, the lengthy synthetic route, the use of the relatively expensive Witsunobu reagent, and the costly and hazardous lithium aluminum hydride, among other factors, make industrial-scale application difficult.

[0004] In summary, the chemical synthesis of (R)-3-aminobutanol suffers from drawbacks such as raw material shortages, high costs, high-temperature and high-pressure reaction conditions, environmental pollution, and low safety.

[0005] Therefore, finding green methods to synthesize chiral amine alcohols, especially (R)-3-amino-1-butanol, has important industrial application value for promoting the green upgrading of existing chemical processes and strengthening environmental protection. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to efficiently prepare chiral amine alcohol compounds.

[0007] To address the above technical problems, this invention first provides an amine dehydrogenase mutant (also known as the GsAmDH mutant protein) with substitution mutations at positions 43, 68, 114, 134, 146, 187, 261, 291, and 294 of the amino acid sequence shown in SEQ ID NO: 1. The wild-type amine dehydrogenase originates from *Geobacillus stearothermophilus*, and its sequence is uniprot: P13154 (GsLDH). The amino acid sequence shown in SEQ ID No. 1 is obtained by mutating the wild-type sequence at two sites, K68S and D261L (referred to as GsAmDH). Optionally, a tagged sequence, such as a histidine tag, can be added for ease of manipulation.

[0008] The mutant amine dehydrogenase is a protein obtained by mutating any one, two, three, four, five, six, seven, eight, or all of the following nine amino acid sequences as shown in SEQ ID No. 1:

[0009] X1. Mutate the threonine residue at position 43 of GsAmDH to a methionine residue;

[0010] X2. Mutate the lysine residue at position 68 of GsAmDH to a serine residue;

[0011] X3. Mutate the glutamic acid residue at position 114 of GsAmDH to a valine residue, a cysteine ​​residue, or a leucine residue.

[0012] X4. Mutate the threonine residue at position 134 of GsAmDH to a glycine residue or a cysteine ​​residue.

[0013] X5. Mutate the proline residue at position 146 of GsAmDH to a valine residue.

[0014] X6. Mutate the histidine residue at position 187 of GsAmDH to an aspartic acid residue.

[0015] X7. Mutate the aspartic acid residue at position 261 of GsAmDH to a leucine residue.

[0016] X8. Mutate the valine residue at position 291 of GsAmDH to a cysteine ​​residue;

[0017] X9. Mutate the valine residue at position 294 of GsAmDH to a cysteine ​​residue or an isoleucine residue.

[0018] In one embodiment of the present invention, the GsAmDH mutant protein is:

[0019] GsAmDH-T43M, which means mutating the 43rd position of GsAmDH to methionine;

[0020] GsAmDH-E114V means that the 114th position of GsAmDH is mutated to valine.

[0021] GsAmDH-E114L, which means mutating the 114th position of GsAmDH to leucine;

[0022] GsAmDH-E114C, which involves mutating the 114th position of GsAmDH to cysteine;

[0023] GsAmDH-T134C involves mutating the 134th position of GsAmDH to cysteine.

[0024] GsAmDH-T134G means that the 134th position of GsAmDH is mutated to glycine.

[0025] GsAmDH-P146V means that the 146th position of GsAmDH is mutated to valine;

[0026] GsAmDH-H187D, which involves mutating the 187th position of GsAmDH to aspartic acid;

[0027] GsAmDH-V291C involves mutating the 291st position of GsAmDH to cysteine.

[0028] GsAmDH-V294C involves mutating the 294th position of GsAmDH to cysteine.

[0029] GsAmDH-V294I, which involves mutating the 294th position of GsAmDH to isoleucine;

[0030] GsAmDH-T43M / E114V, which means mutating the 43rd and 114th positions of GsAmDH to methionine and valine, respectively;

[0031] GsAmDH-T43M / T134C involves mutating positions 43 and 134 of GsAmDH to methionine and cysteine, respectively.

[0032] GsAmDH-T43M / P146V, which means mutating the 43rd and 146th positions of GsAmDH to methionine and valine, respectively;

[0033] GsAmDH-T43M / H187D, which means mutating the 43rd and 187th positions of GsAmDH to methionine and aspartic acid, respectively;

[0034] GsAmDH-T43M / V291C involves mutating positions 43 and 291 of GsAmDH to methionine and cysteine, respectively.

[0035] GsAmDH-T43M / V294C involves mutating positions 43 and 294 of GsAmDH to methionine and cysteine, respectively.

[0036] GsAmDH-E114V / T134C, which means mutating positions 114 and 134 of GsAmDH to valine and cysteine, respectively;

[0037] GsAmDH-E114V / P146V means that positions 114 and 146 of GsAmDH are mutated to valine.

[0038] GsAmDH-E114V / H187D, which means mutating positions 114 and 187 of GsAmDH to valine and aspartic acid, respectively;

[0039] GsAmDH-E114V / V291C, which involves mutating positions 114 and 291 of GsAmDH to valine and cysteine, respectively;

[0040] GsAmDH-E114V / V294C, which involves mutating positions 114 and 294 of GsAmDH to valine and cysteine, respectively.

[0041] GsAmDH-T134C / P146V, which involves mutating positions 134 and 146 of GsAmDH to cysteine ​​and valine, respectively.

[0042] GsAmDH-T134C / H187D involves mutating positions 134 and 187 of GsAmDH to cysteine ​​and aspartic acid, respectively.

[0043] GsAmDH-T134C / V291C involves mutating positions 134 and 291 of GsAmDH to cysteine.

[0044] GsAmDH-T134C / V294C involves mutating positions 134 and 294 of GsAmDH to cysteine.

[0045] GsAmDH-P146V / H187D, which involves mutating positions 146 and 187 of GsAmDH to valine and aspartic acid, respectively.

[0046] GsAmDH-P146V / V291C involves mutating positions 146 and 291 of GsAmDH to valine and cysteine, respectively.

[0047] GsAmDH-P146V / V294C involves mutating positions 146 and 294 of GsAmDH to valine and cysteine, respectively.

[0048] GsAmDH-H187D / V291C involves mutating positions 187 and 291 of GsAmDH to aspartic acid and cysteine, respectively.

[0049] GsAmDH-H187D / V294C involves mutating positions 187 and 294 of GsAmDH to aspartic acid and cysteine, respectively.

[0050] GsAmDH-V291C / V294C involves mutating positions 291 and 294 of GsAmDH to cysteine.

[0051] GsAmDH-E114V / T134C / P146V means that positions 114, 134, and 146 of GsAmDH are mutated to valine, cysteine, and valine, respectively.

[0052] GsAmDH-E114V / T134C / H187D, which means mutating positions 114, 134, and 187 of GsAmDH to valine, cysteine, and aspartic acid, respectively.

[0053] GsAmDH-E114V / T134C / V294C, which means mutating positions 114, 134, and 294 of GsAmDH to valine, cysteine, and cysteine, respectively.

[0054] GsAmDH-E114V / P146V / H187D, which means mutating positions 114, 146, and 187 of GsAmDH to valine, valine, and aspartic acid, respectively.

[0055] GsAmDH-E114V / P146V / V294C, that is, the 114th, 146th and 294th positions of GsAmDH are mutated to valine, valine and cysteine ​​respectively;

[0056] GsAmDH-E114V / H187D / V294C, which means mutating positions 114, 187, and 294 of GsAmDH to valine, aspartic acid, and cysteine, respectively.

[0057] GsAmDH-T134C / P146V / H187D, that is, mutating positions 134, 146, and 187 of GsAmDH to cysteine, valine, and aspartic acid, respectively.

[0058] GsAmDH-T134C / P146V / V294C, which means mutating positions 134, 146, and 294 of GsAmDH to cysteine, valine, and cysteine, respectively.

[0059] GsAmDH-T134C / H187D / V294C, which means mutating positions 134, 187, and 294 of GsAmDH to cysteine, aspartic acid, and cysteine, respectively.

[0060] GsAmDH-P146V / H187D / V294C, which means mutating positions 146, 187, and 294 of GsAmDH to valine, aspartic acid, and cysteine, respectively.

[0061] GsAmDH-E114V / T134C / P146V / H187D, that is, mutating positions 114, 134, 146, and 187 of GsAmDH to valine, cysteine, valine, and aspartic acid, respectively.

[0062] GsAmDH-E114V / T134C / P146V / V294C, that is, mutating positions 114, 134, 146, and 294 of GsAmDH to valine, cysteine, valine, and cysteine, respectively;

[0063] GsAmDH-E114V / T134C / H187D / V294C, that is, mutating positions 114, 134, 187, and 294 of GsAmDH to valine, cysteine, aspartic acid, and cysteine, respectively;

[0064] GsAmDH-E114V / P146V / H187D / V294C, that is, mutating positions 114, 146, 187, and 294 of GsAmDH to valine, valine, aspartic acid, and cysteine, respectively;

[0065] GsAmDH-T134C / P146V / H187D / V294C, that is, mutating positions 134, 146, 187, and 294 of GsAmDH to cysteine, valine, aspartic acid, and cysteine, respectively.

[0066] GsAmDH-E114V / T134C / P146V / H187D / V294C, that is, the 114th, 134th, 146th, 187th and 294th positions of GsAmDH are mutated to valine, cysteine, valine, aspartic acid and cysteine ​​respectively.

[0067] The present invention correspondingly provides a nucleic acid molecule encoding the above-mentioned mutant amine dehydrogenase, its expression cassette, a recombinant vector containing the encoding gene or its expression cassette, and a recombinant microorganism containing the gene or the expression cassette or the recombinant vector.

[0068] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0069] The expression cassette of the nucleic acid molecule refers to DNA capable of expressing the mutant amine dehydrogenase in a host cell. This DNA may include not only a promoter to initiate transcription of the mutant amine dehydrogenase encoding gene, but also a terminator to terminate transcription of the mutant amine dehydrogenase. Furthermore, the expression cassette may also include an enhancer sequence.

[0070] The recombinant vector for the nucleic acid molecule may be a bacterial plasmid (such as an expression vector based on the T7 promoter expressed in bacteria, specifically pET-28a), bacteriophage, yeast plasmid (such as YEp series vectors), or retroviral packaging plasmid carrying the mutant amine dehydrogenase encoding gene.

[0071] The recombinant vector is a vector obtained by inserting the nucleic acid molecule into an expression vector.

[0072] The recombinant microorganism containing the nucleic acid molecule encoding the mutant amine dehydrogenase can be a yeast, bacteria, algae, or fungus carrying the gene encoding GsAmDH or the mutant GsAmDH protein, such as Escherichia coli.

[0073] The present invention also provides another method for preparing chiral amine alcohol compounds, the method comprising: using a hydroxy ketone as a substrate, and catalyzing a reaction using the mutant amine dehydrogenase, or recombinant cells of the mutant amine dehydrogenase, or lysates of the recombinant cells, to obtain the chiral amine alcohol compound.

[0074] In the above method, the recombinant cells can be obtained by introducing a recombinant vector that can express the mutant amine dehydrogenase into biological cells.

[0075] The biological cells may be microorganisms. The microorganisms may be *Escherichia coli* or other bacteria. In one embodiment of the present invention, the microorganism is *Escherichia coli* BL21(DE3).

[0076] The recombinant vector can be a recombinant plasmid obtained by replacing the small DNA fragment between the Nco I and Xho I recognition sequences in the pET28a(+) vector with the encoding gene of the mutant amine dehydrogenase.

[0077] In the reaction in which the mutant amine dehydrogenase is used as a biological enzyme to catalyze the formation of chiral amine alcohols from hydroxyketones, the reaction system may contain the substrate and the mutant amine dehydrogenase, as well as the coenzyme NAD. + and / or NH4 + .

[0078] The concentration of the substrate in the reaction system can be 1-100 mmol / L (e.g., 30 mmol / L). The concentration of the recombinant cells in the reaction system can be 50-500 g / L (e.g., 100 g / L). The concentration of the lysate in the reaction system can be 10-50 g / L (e.g., 20 g / L). The concentration of the GsAmDH or the GsAmDH mutant protein in the reaction system can be 0.1-2 g / L (e.g., 0.1 g / L). The NAD... + The concentration in the reaction system can be 0.1-2.0 mmol / L (e.g., 1.0 mmol / L). The NH4... + The concentration in the reaction system can be from 100 mmol / L to 4 mol / L.

[0079] The reaction system for the catalytic reaction can be obtained by adding the following substances to a 100 mmol / L to 4 mol / L (e.g., 1 mol / L) ammonium chloride / ammonia water buffer solution (obtained by mixing ammonium chloride and ammonia water in an equimolar ratio): the hydroxy ketone, the recombinant cells or the lysate of the recombinant cells, and NAD. + (Present as an aqueous solution of oxidized coenzyme I), glucose dehydrogenase, glucose, lysozyme, DNase I (deoxyribonuclease). The amounts of each substance added can be: the hydroxy ketone 1-100 mmol / L (e.g., 30 mmol / L), the recombinant cells 50-500 g / L (e.g., 100 g / L) or the lysate of the recombinant cells 10-50 g / L (e.g., 20 g / L), NAD... + (In the form of oxidized coenzyme I aqueous solution) 0.1-2.0 mmol / L (e.g. 1.0 mmol / L), glucose dehydrogenase powder 2 g / L, glucose 100 mmol / L, lysozyme 1 g / L, DNase I (deoxyribonuclease) 6 U / mL.

[0080] Preferably, the pH of the ammonium chloride / ammonia buffer solution is 7-11.

[0081] The glucose dehydrogenase powder is prepared by a method including the following steps: glucose dehydrogenase (uniprot: P12310The DNA fragment between the Nde I and Xho I recognition sequences of the pET24a(+) vector was replaced with a gene to obtain a recombinant vector, denoted as pET24a-GDH. pET24a-GDH was introduced into *E. coli* BL21(DE3) to obtain a recombinant bacterium, denoted as BL21(DE3) / pET24a-GDH. BL21(DE3) / pET24a-GDH was cultured, and the resulting bacterial cells were lysed and centrifuged. The supernatant was then placed in a freeze dryer to obtain the glucose dehydrogenase powder.

[0082] The reaction temperature can be 30-45℃, specifically 40℃; the reaction time is determined by the complete reaction, generally 0.5-48 hours, specifically 24 hours. The lysate of the recombinant cells can be obtained by lysing the recombinant cells.

[0083] The chiral amine alcohol compound may be 3-amino-1-butanol, 2-amino-1-butanol, 2-amino-1-pentanol, tert-leucine alcohol, 2-aminocyclohexanol, 4-aminocyclohexanol, phenylglycine alcohol, 3-amino-3-phenylpropanol, 2-amino-2-(4-fluorophenyl)ethanol, 2-amino-2-(4-chlorophenyl)ethanol, or 2-amino-2-(4-bromophenyl)ethanol.

[0084] Further, the chiral amine alcohol compounds are (R)-3-amino-1-butanol, (S)-2-amino-1-butanol, (S)-2-amino-1-pentanol, (S)-tert-leucine alcohol, (1S,2R)-2-aminocyclohexanol, cis-4-aminocyclohexanol, (S)-phenylglycine alcohol, (R)-3-amino-3-phenylpropanol, (S)-2-amino-2-(4-fluorophenyl)ethanol, (S)-2-amino-2-(4-chlorophenyl)ethanol, and (S)-2-amino-2-(4-bromophenyl)ethanol.

[0085] The hydroxy ketone may be 4-hydroxy-2-butanone, 1-hydroxy-2-pentanone, 1-hydroxy-3,3-dimethylbutane-2-one, 2-hydroxycyclohexanone, 4-hydroxycyclohexanone, 2-hydroxyacetophenone, 1-hydroxy-3-phenylprop-2-one, 4-fluoro-2-hydroxyacetophenone, 4-chloro-2-hydroxyacetophenone, or 4-bromo-2-hydroxyacetophenone.

[0086] This invention utilizes the double mutant GsAmDH of the amine dehydrogenase from *Geobacillus stearothermophilus* to catalyze the synthesis of chiral amine alcohols, represented by 4-hydroxy-2-butanone, from hydroxy ketone substrates. Furthermore, directed evolution techniques and methods were applied to modify the amine dehydrogenase GsAmDH from *Geobacillus stearothermophilus*, obtaining its mutant. This mutant can catalyze the synthesis of chiral amine alcohols, represented by 4-hydroxy-2-butanone, from hydroxy ketone substrates with high yield and high ee value. This invention is convenient to operate and produces products with high optical purity, showing promising industrial applications in the biocatalytic preparation of chiral amine alcohols. Attached Figure Description

[0087] Figure 1 This is an asymmetric reductive amination reaction catalyzed by the GsAmDH-GDH cascade reaction.

[0088] Figure 2 A schematic diagram of primer design for constructing a single-point saturated mutant library of amine dehydrogenases.

[0089] Figure 3 The HPLC chromatogram shows the catalytic reduction of 4-hydroxy-2-butanone to (R)-3-amino-1-butanol by amine dehydrogenase GsAmDH or its mutant.

[0090] Figure 4 The results are from the liquid chromatography of chiral amino alcohol compound standards.

[0091] Figure 5 This represents the detection results of amine dehydrogenase GsAmDH or its mutants catalyzing hydroxy ketone substrates. "c." after the percentage sign indicates conversion rate, and "dr" indicates the proportion of diastereomers.

[0092] Figure 6 Amine dehydrogenase GsAmDH or its mutant catalyzes the amplification reaction of 4-hydroxy-2-butanone to (R)-3-amino-1-butanol. Detailed Implementation

[0093] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0094] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0095] 2×High-fidelity Master Mix: Qingke Biotechnology Co., Ltd., Product No.: TP001;

[0096] 4-Hydroxy-2-Butanone: Shanghai Aladdin Biochemical Technology Co., Ltd., Product No.: H106330;

[0097] 1-Hydroxy-2-Butanone: Shanghai Bide Pharmaceutical Technology Co., Ltd., Product No.: BD59017;

[0098] 1-Hydroxy-2-pentanone: Shanghai Bide Pharmaceutical Technology Co., Ltd., Product No.: BD01004105;

[0099] 1-Hydroxy-3,3-dimethyl-2-one: Shanghai Bid Pharmaceutical Technology Co., Ltd., Product No.: BD447193;

[0100] 2-Hydroxycyclohexanone: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: H923258;

[0101] 4-Hydroxycyclohexanone: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: H839473;

[0102] 2-Hydroxyacetophenone: Shanghai Mairui Chemical Technology Co., Ltd., Product No.: M20409;

[0103] 1-Hydroxy-3-phenylprop-2-one: Shanghai Aichun Biotechnology Co., Ltd., Product No.: 27793356;

[0104] 4-Fluoro-2-hydroxyacetophenone: Guangzhou Yiyi Information Technology Co., Ltd., Product No.: SY028417;

[0105] 4-Chloro-2-hydroxyacetophenone: Shanghai Kaiwei Chemical Biotechnology Co., Ltd., Product No.: JZ0018QM;

[0106] 4-Bromo-2-hydroxyacetophenone: Shanghai Kaiwei Chemical Biotechnology Co., Ltd., Product No.: JZ00EJD1.

[0107] Example 1: Preparation of engineered bacteria containing the GsAmDH gene mutant of amine dehydrogenase

[0108] I. Preparation of engineered strains containing the amine dehydrogenase gene GsAmDH

[0109] To explore novel amine dehydrogenase candidate enzymes with potential reductive amination capabilities for hydroxyketone substrates, two representative amine dehydrogenases (leucine amine dehydrogenase and phenylalanine amine dehydrogenase) were used as templates. Through phylogenetic analysis, sequence alignment, and literature review, a double-site mutation (K68S / D261L) was performed on the amine dehydrogenase GsLDH (uniprot: P13154) from *Geobacillus stearothermophilus* to obtain a double mutant, named GsAmDH. After the complete gene of the corresponding gene was synthesized, it was constructed into the pET28a expression vector using Nco I and Xho I to obtain the expression vector pET28a-GsAmDH for expressing amine dehydrogenase. This vector was then introduced into *Escherichia coli* BL21(DE3) to obtain a recombinant strain, denoted as BL21(DE3) / pET28a-GsAmDH, which is the amine dehydrogenase GsAmDH genetically engineered strain.

[0110] pET28a(+) was introduced into Escherichia coli BL21(DE3) to obtain recombinant strain BL21(DE3) / pET28a, which served as the control strain.

[0111] II. Preparation of further mutant engineered strains of the GsAmDH gene for amine dehydrogenase

[0112] Based on previous literature reports of amine dehydrogenases from different sources and the analysis of molecular docking results, saturation mutants were constructed from amino acid residues surrounding the active pocket that have a significant impact on substrate recognition and catalysis. Based on this, mutant libraries and mutants were constructed as shown in Tables 1 and 2, and primers were designed as shown in Tables 1 and 2.

[0113] Table 1 Primer sequences used for mutant library construction

[0114]

[0115] Note: Table 1 contains primers for multiple single-stranded DNAs. Primers GsAmDH-F1, GsAmDH-F2, GsAmDH-F3, GsAmDH-F4, GsAmDH-F5, GsAmDH-F6, GsAmDH-F7, GsAmDH-F8, and GsAmDH-F9 are mixed in a molar ratio of 12:6:1:1 according to the NDT:VMA:ATG:TGG order in the table. R represents A or G, Y represents C or T, M represents A or C, K represents G or T, S represents G or C, W represents A or T, H represents A, T, or C, B represents G, T, or C, V represents G, A, or C, D represents G, A, or T, and N represents A, T, G, or C.

[0116] Table 2. Mutants and primer sequences used

[0117]

[0118]

[0119] Among them, the primer design for constructing the amine dehydrogenase single-point saturation mutant library is as follows: Figure 2 As shown. The specific construction method is as follows:

[0120] Two rounds of Mega-PCR reaction

[0121] The T43 mutant library, K68 mutant library, E114 mutant library, T134 mutant library, P146 mutant library, H187 mutant library, D261 mutant library, V291 mutant library and V294 mutant library were constructed using the recombinant vector pET28a-GsAmDH obtained in step one as a template.

[0122] The GsAmDH-T43M / E114V, GsAmDH-T43M / T134C, GsAmDH-T43M / P146V, GsAmDH-T43M / H187D, GsAmDH-T43M / V291C, and GsAmDH-T43M / V294C mutants were constructed using the recombinant vector pET28a-GsAmDH-T43M as a template, and two rounds of Mega-PCR reactions were performed using the primer pairs corresponding to each mutant. The GsAmDH-E114V / T134C, GsAmDH-E114V / P146V, GsAmDH-E114V / H187D, GsAmDH-E114V / V291C, and GsAmDH-E114V / V294C mutants were constructed using the recombinant vector pET28a-GsAmDH-E114V as a template. Two rounds of Mega-PCR reactions were performed using the primer pairs corresponding to each mutant, namely GsAmDH-F11, GsAmDH-F12, GsAmDH-F13, GsAmDH-F14, and GsAmDH-F15. The GsAmDH-T134C / P146V, GsAmDH-T134C / H187D, GsAmDH-T134C / V291C, and GsAmDH-T134C / V294C were constructed using the recombinant vector pET28a-GsAmDH-T134C as a template. Two rounds of Mega-PCR reactions were performed using the primer pairs corresponding to each mutant, GsAmDH-F12, GsAmDH-F13, GsAmDH-F14, and GsAmDH-F15, respectively. The GsAmDH-P146V / H187D, GsAmDH-P146V / V291C, and GsAmDH-P146V / V294C strains were constructed using the recombinant vector pET28a-GsAmDH-P146V as a template. Two rounds of Mega-PCR reactions were performed using the primer pairs corresponding to each mutant, GsAmDH-F13, GsAmDH-F14, and GsAmDH-F15, respectively. The GsAmDH-H187D / V291C and GsAmDH-H187D / V294C strains were constructed using the recombinant vector pET28a-GsAmDH-H187D as a template. Two rounds of Mega-PCR reactions were performed using the primer pairs corresponding to each mutant, GsAmDH-F14 and GsAmDH-F15, respectively. GsAmDH-V291C / V294C was constructed using the recombinant vector pET28a-GsAmDH-V291C as a template, and two rounds of Mega-PCR were performed using the primers corresponding to the mutant GsAmDH-F15.The GsAmDH-E114V / T134C / P146V, GsAmDH-E114V / T134C / H187D, and GsAmDH-E114V / T134C / V294C were constructed using the recombinant vector pET28a-GsAmDH-E114V / T134C as a template. Two rounds of Mega-PCR reactions were performed using the primer pairs corresponding to each mutant, GsAmDH-F12, GsAmDH-F13, and GsAmDH-F15, respectively. Both GsAmDH-E114V / P146V / H187D and GsAmDH-E114V / P146V / V294C were constructed using the recombinant vector pET28a-GsAmDH-E114V / P146V as a template. Two rounds of Mega-PCR reactions were performed on GsAmDH-F13 and GsAmDH-F15 using the corresponding primer pairs for each mutant, respectively. For GsAmDH-E114V / H187D / V294C, two rounds of Mega-PCR reactions were performed on GsAmDH-F15 using the recombinant vector pET28a-GsAmDH-E114V / H187D as a template and the corresponding primer pairs for the mutant. Both GsAmDH-T134C / P146V / H187D and GsAmDH-T134C / P146V / V294C were constructed using the recombinant vector pET28a-GsAmDH-T134C / P146V as a template. Two rounds of Mega-PCR reactions were performed on GsAmDH-F13 and GsAmDH-F15 using the primers corresponding to each mutant, respectively. For GsAmDH-T134C / H187D / V294C, two rounds of Mega-PCR reactions were performed on GsAmDH-F15 using the recombinant vector pET28a-GsAmDH-T134C / H187D as a template and the primers corresponding to the mutant. GsAmDH-P146V / H187D / V294C was constructed using the recombinant vector pET28a-GsAmDH-P146V / H187D as a template, and two rounds of Mega-PCR were performed on GsAmDH-F15 using the primers corresponding to the mutants. GsAmDH-E114V / T134C / P146V / H187D and GsAmDH-E114V / T134C / P146V / V294C were also constructed using the recombinant vector pET28a-GsAmDH-E114V / T134C / P146V as a template, and two rounds of Mega-PCR were performed on GsAmDH-F13 and GsAmDH-F15 using the primers corresponding to each mutant, respectively. GsAmDH-E114V / T134C / H187D / V294C was constructed using the recombinant vector pET28a-GsAmDH-E114V / T134C / H187D as a template, and two rounds of Mega-PCR were performed on GsAmDH-F15 using the primers corresponding to the mutant.The GsAmDH-E114V / P146V / H187D / V294C gene was constructed using the recombinant vector pET28a-GsAmDH-E114V / P146V / H187D as a template. Two rounds of Mega-PCR reactions were performed on GsAmDH-F15 using primers corresponding to the mutant. The GsAmDH-T134C / P146V / H187D / V294C variant was constructed using the recombinant vector GsAmDH-T134C / P146V / H187D as a template, and two rounds of Mega-PCR were performed on GsAmDH-F15 using the primers corresponding to the mutant. The GsAmDH-E114V / T134C / P146V / H187D / V294C variant was also constructed using the recombinant vector GsAmDH-E114V / T134C / P146V / H187D as a template, and two rounds of Mega-PCR were performed on GsAmDH-F15 using the primers corresponding to the mutant. When constructing the same mutant library or mutant, the templates for the first and second rounds of PCR were the same.

[0123] The PCR reaction system and procedure are as follows:

[0124] The first-round PCR system consisted of 50 μL: 1 μL template (150 ng / μL); 25 μL 2× High-fidelity Master Mix; 22 μL ddH2O; 1 μL front primer (10 μmol / L); and 1 μL back primer (10 μmol / L). The specific sequences of the primers used are shown in Tables 1 and 2, with the front and back primers in the same primer pair labeled "F" and "R," respectively.

[0125] First round PCR procedure: pre-denaturation at 98℃ for 2 minutes; denaturation at 98℃ for 10 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 30 or 50 seconds, cycle 30 times; final extension at 72℃ for 5 minutes.

[0126] The second round of PCR consisted of 50 μL: 1 μL template (150 ng / μL); 25 μL 2× High-fidelity Master Mix; 22 μL ddH2O; and 2 μL of the first round of PCR product.

[0127] Second round PCR procedure: pre-denaturation at 98℃ for 2 minutes; denaturation at 98℃ for 10 seconds, annealing at 60℃ for 15 seconds, extension at 72℃ for 2 minutes, cycle 30 times; final extension at 72℃ for 5 minutes.

[0128] Obtaining engineered strains of GsAmDH gene mutant of amine dehydrogenase

[0129] After the second round of PCR, 2 μL of DpnI enzyme was added to each reaction system, digested at 37°C for 2 hours, and 1 μL was electroporated into E. coli BL21(DE3) competent cells. The cells were then incubated upside down at 37°C for 12-16 hours. Once transformants had grown, plasmids were extracted from the transformants and sequenced. Transformants with non-directional or directional mutations in the target nucleotides were obtained, which were the engineered strains of the GsAmDH gene mutant amine dehydrogenase. The recombinant plasmids containing the target nucleotides of these strains with non-directional or directional mutations were the expression vectors for expressing the GsAmDH gene mutant amine dehydrogenase.

[0130] Example 2: Expression of the GsAmDH mutant of amine dehydrogenase and preparation of whole cells, crude enzyme powder and pure enzyme solution

[0131] The recombinant strains BL21(DE3) / pET28a-GsAmDH, BL21(DE3) / pET28a, and various amine dehydrogenase GsAmDH gene mutant strains prepared in Example 1 were induced to express enzymes, and whole cells, crude enzyme powder, and pure enzyme solutions of each strain were obtained. The operation steps for each strain are as follows:

[0132] Pick the strain into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and incubate overnight at 37°C with shaking at 220 rpm for 12 h. Then, inoculate the strain into 500 mL of TB liquid medium containing 50 μg / mL kanamycin at a volume percentage of 1%, and incubate at 37°C until OD500. 600 When the concentration is around 0.8, add a final concentration of 0.1 mmol / L LPTG, induce expression at 20℃ and 220 rpm for 15 h, centrifuge at 4℃ and 4,000 rpm for 10 min, collect the precipitated bacterial cells (i.e., whole cells), add phosphate buffer (50 mmol / L, pH 7.4) at a volume ratio of 1:5 (wet weight of bacterial cells to buffer) to obtain a bacterial suspension, sonicate the bacterial cells at 8,000 rpm under ice bath, centrifuge at 4℃ for 30 min, and collect the supernatant, which is the crude enzyme solution.

[0133] Take 20 mL of the above crude enzyme solution and freeze it at -80℃ for 24 h. Then dry it in a vacuum freeze dryer to obtain crude enzyme powder.

[0134] The crude enzyme solution was bound to a pre-equilibrated HisTrap HP column. Five column volumes were washed at 2 mL / min, and five column volumes were eluted at 2 mL / min. The eluent (the pure enzyme solution) was collected. Pre-equilibration buffer and wash buffer A (50 mmol / L phosphate buffer, 0.5 mol / L NaCl, 20 mmol / L imidazole, pH 7.4); elution buffer B (50 mmol / L phosphate buffer, 0.5 mol / L NaCl, 500 mmol / L imidazole, pH 7.4).

[0135] Example 3: The amine dehydrogenase GsAmDH mutant catalyzes the production of (R)-3-amino-1-butanol from 4-hydroxy-2-butanone.

[0136] The crude enzyme powder or whole-cell catalysis of 4-hydroxy-2-butanone to (R)-3-amino-1-butanol using the amine dehydrogenase mutant prepared in Example 2 was used, as shown in the example. Figure 1 As shown, BL21(DE3) / pET28a was used as a control.

[0137] The reaction system for the catalytic reaction of the GsAmDH mutant was obtained by adding the following substances to a 1 mol / L ammonium chloride / ammonia buffer solution (ammonium chloride and ammonia were mixed in an equimolar ratio, pH 9.0): 30 mmol / L substrate 4-hydroxy-2-butanone, 20 g / L crude enzyme powder of amine dehydrogenase GsAmDH or its mutant, or 100 g / L whole cells, and 1 mmol / L NAD. + (Present as an aqueous solution of oxidized coenzyme I), 2 g / L crude GDH enzyme powder, 100 mmol / L glucose, 1 g / L lysozyme (Beijing Solarbio Science & Technology Co., Ltd., CAS: 12650-88-3, specific activity 20000 U / mg), 6 U / mL DNase I (deoxyribonuclease, Beijing Dingguo Changsheng Biotechnology Co., Ltd., CAS: 9003-98-9, specific activity 2000 U / mg).

[0138] The above-mentioned crude GDH enzyme powder is glucose dehydrogenase powder, which can catalyze the reaction of the substrate glucose and NAD. + The enzyme is added to generate gluconic acid and NADH, in order to achieve the regeneration of the coenzyme NADH. The enzyme powder is prepared according to the following method:

[0139] GDH amino acids (uniprot: P12310The DNA fragment between the NdeI and XhoI recognition sequences of the pET24a(+) vector was replaced with the corresponding gene sequence to obtain the recombinant vector, denoted as pET24a-GDH. pET24a-GDH was introduced into *E. coli* BL21(DE3) to obtain the recombinant bacteria, denoted as BL21(DE3) / pET24a-GDH. BL21(DE3) / pET24a-GDH was cultured, and the resulting bacterial cells were lysed and centrifuged. The supernatant was then placed in a freeze dryer to obtain crude GDH enzyme powder.

[0140] The specific enzyme activity of crude GDH enzyme powder was 2.14 U / mg. The enzyme activity determination method is as follows:

[0141] Enzyme activity was calculated by monitoring the decrease in absorbance of NADH at 340 nm. Enzyme activity was defined as the activity at pH 9.0, temperature 40 °C, and substrate concentration of 1 mmol / L NAD. + Under conditions of 10 mmol / L glucose, the amount of enzyme required to consume 1 μmol / L NADH per minute. Enzyme activity calculation formula: Enzyme activity (U) = EW × V × 10 3 / (6220×0.3), where EW represents the change in absorbance at 340nm per minute, V represents the volume of the reaction liquid in mL, 6220 is the molar extinction coefficient of the coenzyme in L / (mol×cm), and 0.3 is the optical path distance in cm.

[0142] The reaction system catalyzed by the above GsAmDH mutant was reacted at 40℃ for 24 hours. After the reaction, the conversion rate was calculated and stereoselectivity was analyzed, as follows:

[0143] Boil the reaction solution obtained from the above reaction for 5 minutes, centrifuge at 12,000 rpm for 10 minutes, remove the precipitate, retain the supernatant, and derivatize with a derivatizing agent (derivatizing agent preparation method: N...). α-(2,4-dinitro-5-fluorophenyl)-L-propanamide (Marfey's reagent) was dissolved in acetonitrile to prepare a 14 mM solution, which was stored in the dark. Derivatization method: 150 μL of the above reaction solution + 50 μL of 1 mol / L ammonium chloride / ammonia buffer (pH 9.0) + 500 μL of acetonitrile, then filtered. 50 μL of the filtered mixture + 30 μL of Marfey's reagent (14 mmol / L, dissolved in acetonitrile) + 40 μL of NaHCO3 (1 mol / L) + 100 μL of DMSO were mixed thoroughly. The mixture was reacted at 80 °C and 1,000 rpm for 10 min, then 10 μL of HCl (4 mol / L) was added to terminate the reaction. HPLC analysis was then performed. HPLC detection conditions: Zorbax SB-C18 column (4.6*150mm, 5μm), detection wavelength 340nm, column temperature: 25℃, flow rate: 1mL / min, sample loading volume: 10μL. The gradient elution program is shown in Table 3.

[0144] Table 3 Gradient elution program for HPLC

[0145]

[0146]

[0147] Note: The percentage in Table 3 represents the volume percentage content.

[0148] The HPLC detection results are shown in Table 4.

[0149] Conversion rate = A1 / A2 × 100%; A1: Peak area of ​​(R)-3-amino-1-butanol obtained by liquid chromatography analysis; A2: Peak area of ​​standard (R)-3-amino-1-butanol obtained by liquid chromatography analysis.

[0150] No (R)-3-amino-1-butanol was generated in the reaction products of BL21(DE3) / pET28a. The HPLC chromatogram of the reaction catalyzed by amine dehydrogenase GsAmDH or its mutant to reduce 4-hydroxy-2-butanone to (R)-3-amino-1-butanol is shown below. Figure 3 As shown in the figure. The results showed that the amine dehydrogenase GsAmDH or its mutants could asymmetrically reduce 4-hydroxy-2-butanone to (R)-3-amino-1-butanol with a conversion rate of 35-99% and a stereoselectivity greater than 99% (R).

[0151] Table 4. Results of detection of 4-hydroxy-2-butanone catalyzed by amine dehydrogenase GsAmDH and its mutants.

[0152]

[0153]

[0154] Note: The reaction system contains 30 mmol / L of substrate 4-hydroxy-2-butanone and 0.1 g / mL of wet bacterial cells of amine dehydrogenase GsAmDH or its mutant; ee (stereoselectivity) = (A R -A S ) / (A S +A R )×100%; A S Peak area values ​​of (S)-3-amino-1-butanol obtained by liquid chromatography analysis; A R Peak area value of (R)-3-amino-1-butanol obtained by liquid chromatography analysis.

[0155] Example 4. Enzymatic characterization of the amine dehydrogenase GsAmDH mutant catalyzing the production of (R)-3-amino-1-butanol from 4-hydroxy-2-butanone.

[0156] Based on the experimental results in Table 4, the amine dehydrogenase mutants with higher activity, namely GsAmDH-V294C (named mh96), GsAmDH-T134C / V294C (named mh102), and GsAmDH-E114V / T134C / P146V / H187D / V294C (named mh174), were selected for enzymatic characterization. At the same time, a control GsAmDH was set up.

[0157] Enzyme activity is defined as the amount of enzyme required to produce or consume 1 μmol / L NADH in one minute. The formula for calculating enzyme activity is: Enzyme activity (U) = EW × V × 10⁻⁶. 3 / (6220×0.3), where EW represents the change in absorbance at 340nm per minute, V represents the volume of the reaction liquid in mL, 6220 is the molar extinction coefficient of the coenzyme in L / (mol×cm), and 0.3 is the optical path distance in cm.

[0158] The reaction system was prepared by adding the following substances to a 1 mol / L ammonium chloride / ammonia buffer solution (ammonium chloride and ammonia were mixed in an equimolar ratio, pH 9.0): 0.2 mmol / L NADH, 1-20 mmol / L 4-hydroxy-2-butanone, and an appropriate amount of the purified enzyme solution from Example 2. The reaction was carried out at 40 °C, and the absorbance of NADH was measured over 5 minutes. The initial reaction rate at different substrate concentrations was determined, and then nonlinear fitting of the Michaelis-Menten equation was performed using Origin.

[0159] The test results are shown in Table 5. The results indicate that the amine dehydrogenase GsAmDH or its mutant catalyzes the K+ of 4-hydroxy-2-butanone. m The value is 1.51-2.08 mM, k cat The value is 0.09-0.27s.-1 k cat / K m The value is 0.043-0.179s. -1 mM -1 .

[0160] Table 5 Michaelis constants for amine dehydrogenase mutants

[0161]

[0162] Example 5: Amine dehydrogenase GsAmDH mutant catalyzes other hydroxyketone substrates

[0163] The amine dehydrogenase mutants (GsAmDH and mh96, mh102, mh174) prepared using the method in Example 2 catalyzed hydroxy ketone substrates. The hydroxy ketone substrates were 4-hydroxy-2-butanone (1a), 1-hydroxy-2-butanone (2a), 1-hydroxy-2-pentanone (3a), 1-hydroxy-3,3-dimethylbutane-2-one (4a), 2-hydroxycyclohexanone (5a), 4-hydroxycyclohexanone (6a), 2-hydroxyacetophenone (7a), 1-hydroxy-3-phenylprop-2-one (8a), 4-fluoro-2-hydroxyacetophenone (9a), 4-chloro-2-hydroxyacetophenone (10a), and 4-bromo-2-hydroxyacetophenone (11a). The corresponding products generated are (R)-3-amino-1-butanol (1b), (S)-2-amino-1-butanol (2b), (S)-2-amino-1-pentanol (3b), (S)-tert-leucine alcohol (4b), (1S,2R)-2-aminocyclohexanol (5b), cis-4-aminocyclohexanol (6b), (S)-phenylglycine alcohol (7b), (R)-3-amino-3-phenylpropanol (8b), (S)-2-amino-2-(4-fluorophenyl)ethanol (9b), (S)-2-amino-2-(4-chlorophenyl)ethanol (10b), and (S)-2-amino-2-(4-bromophenyl)ethanol (11b).

[0164] The reaction system for the above catalytic reaction was obtained by adding the following substances to 1 mol / L ammonium chloride / ammonia buffer (pH 9.0): 10 mmol / L substrate, 100 g / L whole-cell concentration of amine dehydrogenase GsAmDH or its mutants (GsAmDH-V294C, GsAmDH-T134C / V294C and GsAmDH-E114V / T134C / P146V / H187D / V294C), and NAD+. + (Present in the form of oxidized coenzyme I aqueous solution) 1 mmol / L, GDH crude enzyme powder from Example 3 2 g / L, glucose 100 mmol / L, lysozyme 1 g / L, DNase I 6 U / mL.

[0165] The reaction conditions were 40°C for 24 hours.

[0166] The products generated from the catalytic substrates 4-hydroxy-2-butanone (1a), 1-hydroxy-2-butanone (2a), 1-hydroxy-2-pentanone (3a), 1-hydroxy-3,3-dimethylbutane-2-one (4a), 2-hydroxycyclohexanone (5a), 4-hydroxycyclohexanone (6a), 2-hydroxyacetophenone (7a), 1-hydroxy-3-phenylprop-2-one (8a), 4-fluoro-2-hydroxyacetophenone (9a), 4-chloro-2-hydroxyacetophenone (10a), and 4-bromo-2-hydroxyacetophenone (11a) were derivatized with Marfey's reagent (derivatization method: take 150 μL of the above reaction solution + 50 μL of 1 mol / L ammonium chloride / ammonia water buffer (pH 9.0) + 500 μL of acetonitrile, and then filter. Take 50 μL of the filtered mixture + 30 μL of Marfey's reagent (14 mmol / L, dissolved in acetonitrile) + 40 μL of... Mix NaHCO3 (1 mol / L) with 100 μL DMSO, react at 80℃ and 1,000 rpm for 10 min, then add 10 μL 4 mol / L HCl to terminate the reaction. HPLC analysis was then performed to detect the reaction.

[0167] HPLC detection conditions: Zorbax SB-C18 column (4.6*150mm, 5μm), detection wavelength 340nm; column temperature, 25℃; flow rate, 1mL / min; sample loading volume, 10μL; mobile phase A, double-distilled water containing 0.1% (v / v) trifluoroacetic acid; mobile phase B, methanol containing 0.1% (v / v) trifluoroacetic acid; the gradient elution program is shown in Table 6, and the HPLC detection of standard chiral amine alcohols is shown in Table 6. Figure 4 The results of liquid phase detection conversion are shown in Table 7 and... Figure 5 .

[0168] Table 6 Gradient elution program for HPLC

[0169]

[0170] Table 7. Detection results of hydroxyketone substrates catalyzed by amine dehydrogenase mutants.

[0171]

[0172]

[0173] Note: The conversion and ee in Table 7, from top to bottom, represent the conversion and stereoselectivity of the hydroxy ketone substrates 4-hydroxy-2-butanone (1a), 1-hydroxy-2-butanone (2a), 1-hydroxy-2-pentanone (3a), 1-hydroxy-3,3-dimethylbutane-2-one (4a), 2-hydroxycyclohexanone (5a), 4-hydroxycyclohexanone (6a), 2-hydroxyacetophenone (7a), 1-hydroxy-3-phenylprop-2-one (8a), 4-fluoro-2-hydroxyacetophenone (9a), 4-chloro-2-hydroxyacetophenone (10a), and 4-bromo-2-hydroxyacetophenone (11a) catalyzed by GsAmDH, mh96, mh102, and mh174.

[0174] As shown in Table 7, compared with the control GsAmDH, mh96, mh102, and mh174 have good catalytic activity for hydroxy ketone substrates, especially for the substrate 4-hydroxy-2-butanone, which has good catalytic activity and enantioselectivity.

[0175] Example 6: Scale-up reaction of amine dehydrogenase GsAmDH mutant mh174 catalyzing the production of (R)-3-amino-1-butanol from 4-hydroxy-2-butanone.

[0176] The amine dehydrogenase mutant mh174 prepared by the method in Example 2 catalyzed the scale-up reaction of 4-hydroxy-2-butanone to (R)-3-amino-1-butanol, increasing the concentration of the substrate 4-hydroxy-2-butanone to 100 mmol / L.

[0177] The reaction system was obtained by adding the following substances to a 1 mol / L ammonium chloride / ammonia buffer (pH 9.0): 100 mmol / L of substrate 4-hydroxy-2-butanone, 100 g / L of whole cells, and NAD+. + (In the form of oxidized coenzyme I aqueous solution) 1 mmol / L, GDH crude enzyme powder of Example 3 2 g / L, glucose 100 mmol / L, lysozyme 1 g / L, DNase I (deoxyribonuclease) 6 U / mL.

[0178] The conditions for the above asymmetric reduction reaction were 40℃ for 0, 1, 2, 3, 4, 8, 12, 18, 24, 30, 36, 42 and 48 h. After the reaction was completed, the reaction solution was boiled for 5 minutes, centrifuged at 12,000 rpm for 10 minutes, the precipitate was removed, the supernatant was retained, derivatized with Marfey's reagent and then analyzed by HPLC.

[0179] Test results are shown Figure 6 The amine dehydrogenase mutant mh174 catalyzed a 99% conversion rate of the substrate 4-hydroxy-2-butanone (100 mmol / L) after 36 h, with a stereoselectivity greater than 99%(R), which was much higher than the control GsAmDH (60% conversion rate after 36 h).

[0180] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> An amine dehydrogenase mutant and its application in the synthesis of chiral amine alcohol compounds <160> 1 <210> 1 <211> 429 <212> PRT <213> Artificial sequence <400> 1 MELFKYMETYDYEQVLFCQDKESGLKAIIAIHDTTLGPALGGTRMWMYNSEEEALEDALRLARGMTYSNAAAGLNLGGGKTVIIGDPRKDKNEAMFRAFGRFIQGLN GRYITAEDVGTTVADMDIIYQETDYVTGISPEFGSSGNPSPATAYGVYRGMKAAAKEAFGSDSLEGKVVAVQGVGNVAYHLCRHLHEEGAKLIVTDINKEVVARAVE EFGAKAVDPNDIYGVECDIFAPCALGGIINDQTIPQLKAKVIAGSALNQLKEPRHGDIIHEMGIVYAPDYVINAGGVINVADELYGYNRERAMKKIEQIYDNIEKVF AIAKRDNIPTYVAADRMAEERIETMRKAASQFLQNGHHILSRRPRPLTAARAGLRRADDGGTTTMQEQKFRILTINPGSTSTKIGVFENERAIASKKRSATRAGASAI 429

Claims

1. A mutant amine dehydrogenase, characterized in that, The mutant amine dehydrogenase is: GsAmDH-E114V, that is, the amino acid sequence shown in SEQ ID NO: 1, has a mutation at position 114 to valine; GsAmDH-E114L, that is, the amino acid sequence shown in SEQ ID NO: 1, has a mutation at position 114 to leucine; GsAmDH-E114C, that is, the amino acid sequence shown in SEQ ID NO: 1, has a mutation at position 114 to cysteine; GsAmDH-T43M / E114V, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its 43rd and 114th positions mutated to methionine and valine, respectively. GsAmDH-E114V / T134C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114 and 134 mutated to valine and cysteine, respectively. GsAmDH-E114V / P146V, that is, the amino acid sequence shown in SEQ ID NO: 1, has 114 and 146 mutated to valine; GsAmDH-E114V / H187D, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its 114th and 187th positions mutated to valine and aspartic acid, respectively. GsAmDH-E114V / V291C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114 and 291 mutated to valine and cysteine, respectively. GsAmDH-E114V / V294C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its 114th and 294th positions mutated to valine and cysteine, respectively. GsAmDH-E114V / T134C / P146V, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 134, and 146 mutated to valine, cysteine, and valine, respectively. GsAmDH-E114V / T134C / H187D, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 134, and 187 mutated to valine, cysteine, and aspartic acid, respectively. GsAmDH-E114V / T134C / V294C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 134, and 294 mutated to valine, cysteine, and cysteine, respectively. GsAmDH-E114V / P146V / H187D, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 146, and 187 mutated to valine, valine, and aspartic acid, respectively. GsAmDH-E114V / P146V / V294C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 146, and 294 mutated to valine, valine, and cysteine, respectively. GsAmDH-E114V / H187D / V294C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 187, and 294 mutated to valine, aspartic acid, and cysteine, respectively. GsAmDH-E114V / T134C / P146V / H187D, that is, the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 134, 146, and 187 mutated to valine, cysteine, valine, and aspartic acid, respectively. GsAmDH-E114V / T134C / P146V / V294C, that is, the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 134, 146, and 294 mutated to valine, cysteine, valine, and cysteine, respectively. GsAmDH-E114V / T134C / H187D / V294C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 134, 187, and 294 mutated to valine, cysteine, aspartic acid, and cysteine, respectively. GsAmDH-E114V / P146V / H187D / V294C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 146, 187, and 294 mutated to valine, valine, aspartic acid, and cysteine, respectively. The amino acid sequence GsAmDH-E114V / T134C / P146V / H187D / V294C, i.e., the amino acid sequence shown in SEQ ID NO: 1, has its positions 114, 134, 146, 187, and 294 mutated to valine, cysteine, valine, aspartic acid, and cysteine, respectively.

2. A nucleic acid molecule encoding the mutant amine dehydrogenase as described in claim 1.

3. An expression cassette containing the nucleic acid molecule as described in claim 2.

4. A recombinant vector containing the nucleic acid molecule as described in claim 2 or the expression cassette as described in claim 3.

5. Recombinant microorganisms containing the nucleic acid molecule as described in claim 2, the expression cassette as described in claim 3, or the recombinant vector as described in claim 4.

6. A method for preparing a chiral amino alcohol compound, comprising: Using hydroxyketones as substrates, chiral amine alcohol compounds are obtained by catalytic reaction using the mutant amine dehydrogenase as described in claim 1.

7. The method according to claim 6, characterized in that: The chiral amine alcohol compounds are: 3-amino-1-butanol, 2-amino-1-butanol, 2-amino-1-pentanol, tert-leucine alcohol, 2-aminocyclohexanol, 4-aminocyclohexanol, phenylglycine alcohol, 3-amino-3-phenylpropanol, 2-amino-2-(4-fluorophenyl)ethanol, 2-amino-2-(4-chlorophenyl)ethanol, and 2-amino-2-(4-bromophenyl)ethanol; The hydroxy ketones are: 4-hydroxy-2-butanone, 1-hydroxy-2-butanone, 1-hydroxy-2-pentanone, 1-hydroxy-3,3-dimethylbutane-2-one, 2-hydroxycyclohexanone, 4-hydroxycyclohexanone, 2-hydroxyacetophenone, 1-hydroxy-3-phenylprop-2-one, 4-fluoro-2-hydroxyacetophenone, 4-chloro-2-hydroxyacetophenone, and 4-bromo-2-hydroxyacetophenone.

8. The method according to claim 7, characterized in that: The chiral amine alcohol compounds are (R)-3-amino-1-butanol, (S)-2-amino-1-butanol, (S)-2-amino-1-pentanol, (S)-tert-leucine alcohol, (1S,2R)-2-aminocyclohexanol, cis-4-aminocyclohexanol, (S)-phenylglycine alcohol, (R)-3-amino-3-phenylpropanol, (S)-2-amino-2-(4-fluorophenyl)ethanol, (S)-2-amino-2-(4-chlorophenyl)ethanol, and (S)-2-amino-2-(4-bromophenyl)ethanol.

9. The method according to any one of claims 6 to 8, characterized in that: A chiral amine alcohol compound is obtained by catalytic reaction using recombinant cells expressing the mutant amine dehydrogenase as described in claim 1 or lysates of said recombinant cells.

10. The method as described in claim 9, characterized in that, The recombinant cells are obtained by introducing a recombinant vector that expresses the mutant amine dehydrogenase as described in claim 1 into biological cells; the lysate of the recombinant cells can be obtained by lysing the recombinant cells.

11. The method as described in claim 9, characterized in that, The concentration of the substrate in the reaction system is 1-100 mmol / L; The concentration of the recombinant cells in the reaction system is 50-500 g / L; The concentration of the pyrolysis product in the reaction system is 10-50 g / L; The concentration of the mutant amine dehydrogenase in the reaction system is 0.1-2 g / L.

12. The method as described in claim 11, characterized in that, The substrate was at a concentration of 30 mmol / L in the reaction system; The concentration of the recombinant cells in the reaction system was 100 g / L; The concentration of the pyrolysis product in the reaction system is 20 g / L; The concentration of the mutant amine dehydrogenase in the reaction system was 0.1 g / L.

13. The method as described in claim 9, characterized in that, The reaction system also contains the coenzyme NAD. + and / or NH4 + .

14. The method as described in claim 13, characterized in that, The NAD + The concentration of NH4 in the reaction system can be 0.1-2.0 mmol / L; + The concentration in the reaction system is 100 mmol / L to 4 mol / L.

15. The method as described in claim 9, characterized in that, The reaction system for the catalytic reaction is obtained by adding the following substances to a buffer solution containing ammonium chloride and ammonia in an equimolar ratio of 100 mmol / L to 4 mol / L: the hydroxy ketone, the recombinant cells or lysates of the recombinant cells, and NAD in the form of an aqueous solution of oxidized coenzyme I. + glucose dehydrogenase, glucose, lysozyme and DNase .

16. The method as described in claim 15, characterized in that, The amounts of each substance added are as follows: 1-100 mmol / L of the hydroxy ketone, 50-500 g / L of the recombinant cells or 10-50 g / L of the lysate of the recombinant cells, and NAD in the form of an aqueous solution of oxidized coenzyme I. + 0.1-2.0 mmol / L, glucose dehydrogenase powder 2g / L, glucose 100mmol / L, lysozyme 1g / L, DNase 6 U / mL; the pH of the ammonium chloride / ammonia buffer solution is 7-11.

17. The method as described in claim 16, characterized in that, The amounts of each substance added are: 30 mmol / L of the hydroxy ketone, 100 g / L of the recombinant cells or 20 g / L of the lysate of the recombinant cells, and NAD in the form of an aqueous solution of oxidized coenzyme I. + 1.0 mmol / L, glucose dehydrogenase powder 2 g / L, glucose 100 mmol / L, lysozyme 1 g / L, DNase 6 U / mL.

18. The method as described in claim 9, characterized in that, The reaction temperature is 30-45℃; the reaction time is determined by the time the reaction is complete.

19. The method as described in claim 18, characterized in that, The reaction temperature is 40°C; the reaction time is 0.5-48 hours.

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