Amine dehydrogenase and encoding nucleic acid and use thereof

Abstract

This invention provides an amine dehydrogenase obtained by mutating an amino acid dehydrogenase as shown in SEQ ID NO.2, wherein the mutation includes at least one mutation in amino acids 134, 299, and 300 of SEQ ID NO.2. This invention also provides the nucleic acid encoding this amine dehydrogenase, and its application as a catalyst in the synthesis of chiral amines via asymmetric reduction reactions using prochiral ketones and free ammonia as substrates. The amine dehydrogenase of this invention exhibits high reductive amination activity, high catalytic activity, a wide substrate adaptability, high yield, and meets the requirement of 99%+ stereoselectivity, demonstrating very broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of protein modification technology, and particularly to an amine dehydrogenase. Furthermore, this invention also relates to the nucleic acid encoding the amine dehydrogenase, and the applications of the amine dehydrogenase. Background Technology

[0002] Among the many types of amines, chiral primary amines are crucial motifs or building blocks in numerous bioactive molecules, finding wide application in the pharmaceutical field. Their synthesis methods primarily include chemical and biological approaches. The former relies on heavy metals, while the latter's conversion rate needs improvement. The latter, via amine dehydrogenase (AmDH), can utilize inexpensive ammonia as an amino donor to asymmetricly reduce and amination prochiral hydroxyketones to generate chiral primary amines, exhibiting high theoretical conversion rates and representing an ideal green synthetic route.

[0003] Amine dehydrogenases (AmDHs) are a class of enzymes that catalyze redox reactions in substances such as carbohydrates, organic acids, and amino acids. In enzymology, they belong to the redox enzyme class. The substrate oxidized in the reaction is called the hydrogen donor or electron donor, and the substrate reduced is called the hydrogen acceptor or electron acceptor. When the acceptor is oxygen, the enzyme catalyzing the reaction is called an oxidase; otherwise, it is called a dehydrogenase. Different dehydrogenases are almost always named after their substrates. Amine dehydrogenases are a type of dehydrogenase. It has been reported that chiral amines can be prepared using biological methods, but currently, very few AmDHs have been reported, and their substrate spectrum is narrow, severely limiting their industrial applications. Summary of the Invention

[0004] In view of this, the present invention aims to provide an amine dehydrogenase that can catalyze an asymmetric reduction reaction using prochiral ketones and free ammonia as substrates to synthesize chiral amines.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides an amine dehydrogenase obtained by mutating an amino acid dehydrogenase as shown in SEQ ID NO.2, said mutation comprising at least one mutation of amino acids 134, 299, and 300 of SEQ ID NO.2.

[0007] As a preferred embodiment, the mutation includes at least one of the following (1)-(3):

[0008] (1) The 134th position is mutated from V to A or G;

[0009] (2) The 299th position is mutated from V to A or G;

[0010] (3) The 300th position is mutated from S to G, A, V or F;

[0011] Preferably, the mutation is any one of the following (M1)-(M8):

[0012] (M1) SEQ ID NO.2 shows that amino acid residue V is mutated to A at position 134.

[0013] (M2) The amino acid residue at position 134 of SEQ ID NO.2 is mutated from V to G;

[0014] (M3) SEQ ID NO.2, amino acid residue 134 is mutated from V to A; and amino acid residue 299 is mutated from V to A;

[0015] (M4) SEQ ID NO.2 has an amino acid residue at position 134 mutated from V to A; and an amino acid residue at position 299 mutated from V to G;

[0016] (M5) SEQ ID NO.2: Amino acid residue at position 134 is mutated from V to A; amino acid residue at position 299 is mutated from V to A; and amino acid residue at position 300 is mutated from S to G.

[0017] (M6) SEQ ID NO.2: Amino acid residue at position 134 is mutated from V to A; amino acid residue at position 299 is mutated from V to A; and amino acid residue at position 300 is mutated from S to A.

[0018] (M7) SEQ ID NO.2 The 134th amino acid residue is mutated from V to A; the 299th amino acid residue is mutated from V to A; and the 300th amino acid residue is mutated from S to V;

[0019] (M8)SEQ ID NO.2 The 134th amino acid residue is mutated from V to A; the 299th amino acid residue is mutated from V to A; and the 300th amino acid residue is mutated from S to F.

[0020] The amine dehydrogenase of the present invention can be used in the process of catalyzing the transamination reaction of hydroxy ketones and prochiral ketones to prepare chiral amines.

[0021] In a second aspect, the present invention provides an enzyme preparation comprising the amine dehydrogenase described above.

[0022] In a third aspect, the present invention provides a nucleic acid encoding the aforementioned amine dehydrogenase.

[0023] As a preferred embodiment, the nucleic acid is obtained by performing at least one of the following mutations (1)-(3) on the nucleotide sequence shown in SEQ ID NO.1:

[0024] (1) Nucleotides at positions 401-402 are mutated from tt to ca or gt;

[0025] (2) Nucleotides at positions 896-897 are mutated from tt to ca or gt;

[0026] (3) The nucleotides at positions 898-900 are mutated from agc to ggt, gca, gtt, or ttt;

[0027] Preferably, the mutation is any one of the following (1)-(8):

[0028] (1) The nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca;

[0029] (2) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to gt;

[0030] (3) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and nucleotides at positions 896-897 are mutated from tt to ca;

[0031] (4) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and nucleotide at position 896 is mutated from t to g;

[0032] (5) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and nucleotides at positions 896-897 are mutated from tt to ca; and nucleotides at positions 898-900 are mutated from agc to ggt;

[0033] (6) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and nucleotides at positions 896-897 are mutated from tt to ca; and nucleotides at positions 898-900 are mutated from agc to gca;

[0034] (7) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and nucleotides at positions 896-897 are mutated from tt to ca; and nucleotides at positions 898-900 are mutated from agc to gtt;

[0035] (8) The nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and the nucleotides at positions 896-897 are mutated from tt to ca; and the nucleotides at positions 898-900 are mutated from agc to ttt.

[0036] In a fourth aspect, the present invention provides an expression vector comprising the above-described nucleic acid.

[0037] Preferably, the expression vector is pET28a.

[0038] A fifth aspect of the present invention provides a cell comprising the above-described nucleic acid or expression vector.

[0039] Preferably, the cells are bacteria (e.g., Escherichia coli) or fungi.

[0040] In a sixth aspect, the present invention provides a method for preparing the above-mentioned amine dehydrogenase, the method comprising introducing the above-mentioned expression vector into cells, culturing the cells, and obtaining the amine dehydrogenase.

[0041] Preferably, the cells comprise recombinant bacteria.

[0042] The preparation method further includes introducing the above-mentioned nucleic acid into a host bacterium.

[0043] Preferably, the host bacterium includes Escherichia coli, specifically Escherichia coli BL21(DE3).

[0044] Preferably, the nucleic acid is introduced into the host bacterium via the expression vector described above.

[0045] The method for preparing amine dehydrogenase of the present invention utilizes protein engineering to modify an amino acid dehydrogenase catalyzing amino acid substrates into an amine dehydrogenase (AmDH) using ketones and aldehydes as substrates. The route for synthesizing chiral amines using AmDH-catalyzed asymmetric reduction reactions with prochiral ketones and free ammonia as substrates offers advantages such as being green, efficient, low-cost, and producing products with high optical purity, making it one of the most ideal reactions for chiral amine synthesis.

[0046] A seventh aspect of the present invention provides the use of the above-described amine dehydrogenase in the preparation of compounds of Formula I; wherein the compounds of Formula I comprise those obtained by using Formula II as a substrate under the catalysis of the amine dehydrogenase.

[0047]

[0048] in,

[0049] R1 represents any one of aryl, heteroaryl, and alkyl groups;

[0050] R2 represents any one of aryl, heteroaryl, and alkyl groups;

[0051] R1 and R2 may be the same or different.

[0052] As a preferred embodiment, the aryl group includes phenyl or benzyl, or alkyl-substituted phenyl or benzyl, or halogen-substituted phenyl or benzyl, or nitro-substituted phenyl or benzyl, or alkylphenyl, wherein the alkylphenyl group has 2-4 alkyl carbon atoms;

[0053] The heteroaryl groups include pyridine, pyrimidine, thiazole, thiophene, furan, and N-substituted indole;

[0054] The alkyl group has 1-20 carbon atoms.

[0055] The amine dehydrogenase of the present invention has high reductive amination activity, high catalytic activity, wide substrate adaptability, high yield and meets the requirement of 99%+ stereoselectivity, and has a very broad application prospect. Attached Figure Description

[0056] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0057] Figure 1 The NMR spectrum is for (R)-1-(4-fluorophenyl)propylamine. A represents the proton NMR spectrum, and B represents the carbon NMR spectrum.

[0058] Figure 2 The NMR spectrum is for (R)-1-methyl-3-phenylpropanamine. A represents the proton NMR spectrum, and B represents the carbon NMR spectrum.

[0059] Figure 3 The NMR spectrum of (R)-2-aminooctane is shown. A represents the proton NMR spectrum, and B represents the carbon NMR spectrum. Detailed Implementation

[0060] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0061] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0062] This invention relates to an amine dehydrogenase obtained by mutating an amino acid dehydrogenase as shown in SEQ ID NO.2, wherein the mutation includes at least one mutation in amino acids 134, 299, and 300 of SEQ ID NO.2.

[0063] To prepare the amine dehydrogenases described above, the following raw materials were selected for preparation in this invention:

[0064] pET28a vector: from Novagen, catalog number: 69864-3.

[0065] Escherichia coli BL21(DE3): from Beijing TransGen Biotech Co., Ltd., catalog number: CD601-02.

[0066] LB medium: 10g tryptone, 5g yeast extract, 5g NaCl, 1ml 1mol / L NaOH to adjust pH to 7.4, then bring the volume to 1L with deionized water and autoclave for 20min.

[0067] TB medium: 12g tryptone, 24g yeast extract, 4mL 87% glycerol, 100mL phosphate buffer (pH 6.5) per liter, bring the volume to 1L with deionized water, and autoclave for 20min.

[0068] Example 1: Screening and Determination of Mutation Sites

[0069] Sequence analysis, mutation, and functional verification were performed on the natural amino acid dehydrogenase protein derived from *Caldalkalibacillus thermarum* (referred to as "Cal-M0" or "M0"; the amino acid sequence of the natural amino acid dehydrogenase protein is shown in SEQ ID NO.2, and its encoding gene (AmDH gene) is shown in SEQ ID NO.1). Eleven amino acid sites were specifically selected, and further analysis revealed three important amino acid sites from these eleven sites. These three amino acid sites were then mutated in different ways to obtain mutant proteins.

[0070] The nucleotide sequence of the natural amino acid dehydrogenase derived from Caldalkalibacillus thermarum is as follows (SEQ ID No. 1):

[0071]

[0072] The amino acid sequence of the natural amino acid dehydrogenase derived from Caldalkalibacillus thermarum is as follows (SEQ ID No. 2):

[0073]

[0074]

[0075] The bolded and underlined portions above represent mutation sites.

[0076] The three amino acid sites and their mutation forms are shown in Table 1.

[0077] Table 18 Mutation Forms

[0078] Mutant name mutation site M1 V134A M2 V134G M3 V134A / V299A M4 V134A / V299G M5 V134A / V299A / S300G M6 V134A / V299A / S300A M7 V134A / V299A / S300V M8 V134A / V299A / S300F

[0079] The nucleotide sequences of the mutants encoded in Table 1 are shown in Table 2.

[0080] Table 2. Nucleotide sequences corresponding to the mutants.

[0081]

[0082] The screening and optimization process for the above mutation sites is briefly described below:

[0083] Following the preparation method of Example 5, experiments were conducted using the Mo-M8 catalysts of the present invention.

[0084] The experimental results are shown in Table 3. Specifically, Cal-M0 exhibits extremely low catalytic activity towards benzylacetone (referred to as "1s"), almost failing to react. This is presumably due to the sterically hindered benzylacetone's difficulty in entering the active pocket. A model was constructed for Cal-M0 (PDB: 1leh, identity 42.5%), using sterically hindered benzylacetone as the model substrate for molecular docking prediction of acetone and hotspot amino acids. Eleven amino acid residues, A293, I296, and K296 located in the substrate channel and L40, V134, V299, F76, S300, Y304, K314, and A317 located in the active pocket, were identified as hotspot amino acids. Considering the significant steric hindrance of benzylacetone, mutating these hotspot amino acid residues to less sterically hindered residues A or G may contribute to improved catalytic activity.

[0085] First, the sterically hindered hydrophobic residues I296, L40, V134, V299, F76, and Y304 were selected for single-site A and G mutations. Among them, only V134 and V299 showed positive mutations. V134G (M2) and V134A (M1) showed that the catalytic activity of 1s was increased by 52 times and 93 times, respectively, compared with M0. No positive mutations were found in the other amino acid residues.

[0086] Based on M1, further additions of V299G (M4) and V299A (M3) revealed a further increase in catalytic activity towards substrate 1s, approximately six-fold higher than M1. This indicates that increasing the pocket size facilitates the entry and catalysis of sterically hindered molecules. However, mutant A exhibited higher activity than mutant G, suggesting that steric hindrance is not the only factor affecting enzyme activity; the hydrophobicity of amino acid residue side chains also influences catalytic activity. Based on this, we further added several other hydrophilic amino acids, S300A, K296A, and K314A, to M3 (V134A / V299A). K296A and K314 completely lost their activity, suggesting that these two sites may be involved in substrate transport and fixation during enzyme catalysis.

[0087] The hydrophilicity / hydrophobicity change of S300A further enhances the activity of the enzyme molecule. Therefore, S300 was further mutated into S300G, which has less steric hindrance, and S300V and S300F, which have greater steric hindrance and hydrophobicity. It was found that among the several mutants of S300, the greater the steric hindrance, the lower the catalytic activity. S300G showed higher catalytic activity than S300A. At this time, its catalytic activity was increased by 1014 times compared with M0. This may be because the hydrophobicity of the adjacent 299A plays a role in the transfer and fixation of substrate, while S300G makes the active pocket larger while maintaining hydrophobicity, thus exhibiting higher enzyme activity.

[0088] Example 2: Preparation of Recombinant Bacteria

[0089] I. Construction of wild-type recombinant expression vectors

[0090] The nucleotide sequence shown in SEQ ID NO.1 was inserted between the EcoRI and XhoI restriction sites of the pET28a vector to obtain the recombinant expression vector pET28a-AmDH, which was verified by sequencing.

[0091] II. Construction of mutant recombinant expression vectors

[0092] The nucleotide sequences encoding mutants M1-M8 (see Table 2) were inserted between the EcoRI and XhoI restriction sites of the pET28a vector to obtain the recombinant expression vectors pET28a-M1-M8, which were verified to be correct by sequencing.

[0093] III. Preparation of Recombinant Bacteria

[0094] 1. Transform Escherichia coli BL21(DE3) with the recombinant expression vector pET28a-AmDH prepared in step one to obtain wild-type recombinant bacteria.

[0095] 2. The recombinant expression vectors pET28a-M1-M8 prepared in step 2 were transformed into Escherichia coli BL21(DE3) to obtain mutant recombinant bacteria, which were numbered M1-M8 respectively.

[0096] The above conversion steps are as follows:

[0097] (1) Take 50 μL of competent cells that have thawed on an ice bath, add 5 μL of sufficient plasmid containing the target gene, mix gently, and place in an ice bath for 30 minutes.

[0098] (2) Heat shock in a 42°C water bath for 45 seconds, then quickly transfer the tube to an ice bath for 2 minutes. Do not shake the centrifuge tube during this process.

[0099] (3) Add 500 μl of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, and incubate at 37°C and 200 rpm for 1 hour to allow the bacteria to recover.

[0100] (4) According to the experimental requirements, take different volumes of transformed competent cells and add them to LB agar medium containing the corresponding antibiotics, and spread the cells evenly. Place the plate at 37°C until the liquid is absorbed, then invert the plate and incubate overnight at 37°C.

[0101] Example 3: Enzyme activity determination of amino acid dehydrogenase mutant protein

[0102] The wild-type recombinant bacteria and mutant recombinant bacteria M1-M8 obtained in Example 2 were cultured, proteins were extracted, and enzyme activity was detected.

[0103] 1. The recombinant bacteria were inoculated into 10 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin. The medium was cultured at 37°C with shaking at 180 rpm for 16 h to obtain a seed culture. The seed culture was then inoculated into 50 mL of TB medium (1% inoculum) containing a final concentration of 50 μg / mL kanamycin. The culture was then cultured at 37°C with shaking at 180 rpm until the bacterial culture reached its OD value. 600nm Add 0.6-0.8 μM of IPTG to a final concentration of 0.5 μM, and continue incubation at 20°C and 180 rpm for 20-24 h with shaking. Collect the cells by centrifugation at 5000 rcf for 15 min.

[0104] The recombinant bacterial cells were washed and resuspended twice with 50mM 50mL PBS buffer. After resuspending, the cells were homogenized and centrifuged at 12000rpm for 15min. The supernatant was collected as crude enzyme solution.

[0105] 2. Filter the prepared crude enzyme solution through a 0.22 μm aqueous fiber filter membrane, and reserve the filtrate. Purification was performed in the purification system. The specific purification process was as follows: First, the HisTrap HP affinity column purification system was pre-equilibrated with loading buffer. Then, NGC was used... TM The filtrate was loaded onto a HisTrap HP affinity column using a Bio-Rad chromatographic system. After protein loading, the affinity column was washed with 4% elution buffer (elution buffer formulation: 500mM imidazole, 20mM sodium dihydrogen phosphate, 0.5M sodium chloride, pH 7.4) until the baseline approached 0 to remove other contaminating proteins. Then, the recombinant protein was eluted with 60% elution buffer at a flow rate of 1 mL / min, and the resulting eluent was the purified enzyme solution.

[0106] 3. The protein solution obtained from the above steps using wild-type recombinant bacteria is named wild-type protein solution M0. The protein solutions obtained from the above steps using mutant recombinant bacteria M1-M8 are named M1-M8 protein solutions, respectively. Protein concentration is determined using the Bradford method.

[0107] Prepare the enzyme activity assay reaction system as follows: 850 μL of 2 mol / L NH4Cl / NH4OH reaction buffer solution, a final substrate concentration of 10 mmol / L, 100 μL of the protein solution to be tested (protein content approximately 0.1 mg), and a final NADH concentration of 1 mmol / L. Incubate at 30℃ for 3 min. Measure the change in absorbance before and after the reaction at 340 nm.

[0108] Calculate enzyme activity based on changes in absorbance:

[0109] Enzyme activity calculation formula: Enzyme activity (U) = EW × V × 10 3 / 6220 / 1

[0110] Where EW: change in absorbance at 340 nm over 1 minute; V: total volume of the reaction system in mL; 6220: molar extinction coefficient in L / mol / cm; l: optical path distance of the cuvette.

[0111] Formula for calculating specific enzyme activity:

[0112] Where U: enzyme activity; mg: protein content.

[0113] The results are shown in Table 3.

[0114] Table 3. Statistical results of specific enzyme activities for different substrates

[0115] Mutant name 1-Fluorophenylacetone (U / mg protein) Benzylacetone (U / mg protein) 2-Octanone (U / mg protein) M0 4.57 0.04 0.34 M1 58.80 3.70 1.68 M2 47.04 2.06 1.32 M3 101.43 24.63 28.98 M4 43.05 25.83 21.53 M5 185.23 45.36 49.14 M6 115.56 37.28 39.14 M7 71.40 14.99 14.28 M8 7.09 1.13 2.84

[0116] Example 4: Asymmetric synthesis of (R)-1-(4-fluorophenyl)propan-2-amine

[0117]

[0118] The synthetic route is as above, involving 4-fluorophenylacetone (5.0 mmol) and NAD+. +Add 5 mL of nicotinamide adenine dinucleotide (0.005 mmol), 2 mol / L NH4Cl / NH4OH (pH 10.0) solution, amine dehydrogenase mutant M5 (0.22 μmol), GDH (glucose dehydrogenase, 0.22 μmol), and glucose (5.5 mmol) to a glass tube. Stir the reaction mixture at 37 °C for 48 h. After the reaction is complete, extract the mixture with ethyl acetate (3 × 5 mL). Ethyl acetate can extract both the substrate and product to the organic phase. Dry the organic layer with Na2SO4, then separate by silica gel column chromatography (first with 50 mL of a 99:1 mixture of ethyl acetate and ethanol, then with 150 mL of a 90:10 mixture). Take 1 mL of the ethyl acetate extract (upper layer), add 100 μL of acetic anhydride, vortex for 10 s, and use for gas chromatography detection.

[0119] The yield, configuration, and ee value of the asymmetric reduction reaction product were determined by gas chromatography. Chromatographic conditions: Agilent J&W CP-Chiralsil-DEXCB column (25 m × 0.32 mm × 0.25 μm); column temperature program: initial temperature 100 °C, ramped to 200 °C at a rate of 5 °C / min, held at 200 °C for 5 min. (R)-1-(4-fluorophenyl)propylamine was obtained in 99% yield with an ee value of 99%.

[0120] Figure 1 The NMR spectrum is for (R)-1-(4-fluorophenyl)propylamine. A represents the proton NMR spectrum, and B represents the carbon NMR spectrum.

[0121] Example 5: Asymmetric synthesis of (R)-1-methyl-3-phenylpropanamine ((R)-4-phenylbutan-2-amine)

[0122]

[0123] The synthetic route is as above, using benzylacetone (5.0 mmol) and NAD+. +Nicotinamide adenine dinucleotide (0.005 mmol), 5 mL of 2 mol / L NH4Cl / NH4OH (pH 10.0) solution, amine dehydrogenase mutant M5 (0.22 μmol), GDH (glucose dehydrogenase, 0.22 μmol), and glucose (5.5 mmol) were added to a 10 mL glass tube. The reaction mixture was stirred at 37 °C for 48 h. After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 5 mL). Ethyl acetate can extract both the substrate and product in the reaction system to the organic phase. The organic layer was dried with Na2SO4 and then separated by silica gel column chromatography (first with 50 mL of a mixture of ethyl acetate and ethanol (99:1 volume ratio), then with 150 mL of a mixture of ethyl acetate and ethanol (90:10 volume ratio)). 1 mL of the ethyl acetate extract was then added to 100 μL of acetic anhydride, vortexed for 10 s, and analyzed by gas chromatography.

[0124] The yield, configuration, and ee value of the asymmetric reduction reaction product were determined by gas chromatography. Chromatographic conditions: Agilent J&W CP-Chiralsil-DEXCB column (25 m × 0.32 mm × 0.25 μm); column temperature program: initial temperature 100 °C, ramped to 200 °C at a rate of 5 °C / min, held at 200 °C for 5 min. (R)-1-methyl-3-phenylpropylamine was obtained in 99% yield with an ee value of 99%.

[0125] Figure 2 The NMR spectrum is for (R)-1-methyl-3-phenylpropanamine. A represents the proton NMR spectrum, and B represents the carbon NMR spectrum.

[0126] Example 6: Asymmetric Synthesis of (R)-2-aminooctane ((R)-octan-2-amine)

[0127]

[0128] The synthetic route is as above, involving 2-octanone (5.0 mmol) and NAD+. +Nicotinamide adenine dinucleotide (0.005 mmol), 5 mL of 2 mol / L NH4Cl / NH4OH (pH 10.0) solution, amine dehydrogenase mutant M5 (0.22 μmol), GDH (glucose dehydrogenase, 0.22 μmol), and glucose (5.5 mmol) were added to a 10 mL glass tube. The reaction mixture was stirred at 37 °C for 48 h. After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 5 mL). Ethyl acetate can extract both the substrate and product in the reaction system to the organic phase. The organic layer was dried with Na2SO4 and then separated by silica gel column chromatography (first with 50 mL of a mixture of ethyl acetate and ethanol (99:1 volume ratio), then with 150 mL of a mixture of ethyl acetate and ethanol (90:10 volume ratio)). 1 mL of the ethyl acetate extract was then added to 100 μL of acetic anhydride, vortexed for 10 s, and analyzed by gas chromatography.

[0129] The yield, configuration, and ee value of the asymmetric reduction reaction product were determined by gas chromatography. Chromatographic conditions: Agilent J&W CP-Chiralsil-DEXCB column (25 m × 0.32 mm × 0.25 μm); column temperature program: initial temperature 100 °C, ramped to 200 °C at a rate of 5 °C / min, held at 200 °C for 5 min. (R)-2-aminooctane was obtained in 99% yield with an ee value of 99%.

[0130] Figure 3 The NMR spectrum of (R)-2-aminooctane is shown. A represents the proton NMR spectrum, and B represents the carbon NMR spectrum.

[0131] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0132] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An amine dehydrogenase, characterized in that: Obtained by mutating an amino acid dehydrogenase as shown in SEQ ID NO.2, wherein the mutation is any one of the following (M1)-(M8): (M1) The amino acid residue at position 134 of SEQ ID NO.2 is mutated from V to A; (M2) The amino acid residue at position 134 of SEQ ID NO.2 is mutated from V to G; (M3) The amino acid residue at position 134 of SEQ ID NO.2 is mutated from V to A; and the amino acid residue at position 299 is mutated from V to A; (M4) SEQ ID NO.2, amino acid residue 134 is mutated from V to A; and amino acid residue 299 is mutated from V to G; (M5) SEQ ID NO.2: Amino acid residue 134 is mutated from V to A; amino acid residue 299 is mutated from V to A; and amino acid residue 300 is mutated from S to G. (M6) SEQ ID NO.2: Amino acid residue 134 is mutated from V to A; amino acid residue 299 is mutated from V to A; and amino acid residue 300 is mutated from S to A. (M7) SEQ ID NO.2, amino acid residue 134 is mutated from V to A; amino acid residue 299 is mutated from V to A; and amino acid residue 300 is mutated from S to V; (M8) SEQ ID NO.2 The 134th amino acid residue is mutated from V to A; the 299th amino acid residue is mutated from V to A; and the 300th amino acid residue is mutated from S to F.

2. An enzyme preparation, characterized in that: The enzyme preparation includes the amine dehydrogenase according to claim 1.

3. The nucleic acid encoding the amine dehydrogenase of claim 1.

4. The nucleic acid according to claim 3, characterized in that: The nucleic acid is a mutation performed on the nucleotide sequence shown in SEQ ID NO.1 by any one of the following (1)-(8): (1) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; (2) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to gt; (3) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and nucleotides at positions 896-897 are mutated from tt to ca; (4) Nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and nucleotide at position 896 is mutated from t to g; (5) The nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and the nucleotides at positions 896-897 are mutated from tt to ca; and the nucleotides at positions 898-900 are mutated from agc to ggt; (6) The nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and the nucleotides at positions 896-897 are mutated from tt to ca; and the nucleotides at positions 898-900 are mutated from agc to gca; (7) The nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and the nucleotides at positions 896-897 are mutated from tt to ca; and the nucleotides at positions 898-900 are mutated from agc to gtt; (8) The nucleotides at positions 401-402 of SEQ ID NO.1 are mutated from tt to ca; and the nucleotides at positions 896-897 are mutated from tt to ca; and the nucleotides at positions 898-900 are mutated from agc to ttt.

5. An expression vector comprising the nucleic acid of claim 3 or 4.

6. A cell comprising the nucleic acid of claim 3 or 4 or the expression vector of claim 5.

7. A method for preparing the amine dehydrogenase according to claim 1, characterized in that: The preparation method includes introducing the expression vector according to claim 5 into cells, culturing the cells, and obtaining the amine dehydrogenase.

8. The use of the amine dehydrogenase of claim 1 in the preparation of the compound represented by formula II; wherein the compound represented by formula II comprises a product obtained by catalysis of the amine dehydrogenase using formula I as a substrate; Formula I Formula II in, R1 represents any one of aryl, heteroaryl, and alkyl groups; R2 represents any one of aryl, heteroaryl, and alkyl groups; R1 and R2 may be the same or different.

9. The application according to claim 8, characterized in that: The aryl group includes phenyl or benzyl, or alkyl-substituted phenyl or benzyl, or halogen-substituted phenyl or benzyl, or nitro-substituted phenyl or benzyl, or alkylphenyl, wherein the alkylphenyl group has 2-4 alkyl carbon atoms; The heteroaryl groups include pyridine, pyrimidine, thiazole, thiophene, furan, and N-substituted indole; The alkyl group has 1-20 carbon atoms.

Images