Amine dehydrogenase and encoding nucleic acid and use thereof

Abstract

The present application provides an amine dehydrogenase, which is obtained by mutating an amino acid dehydrogenase as shown in SEQ ID NO. 1, and the mutation comprises at least one mutation in the amino acids at positions 293, 69, 115 and 262 of SEQ ID NO. 1. The present application also provides the use of the amine dehydrogenase in preparing chiral amines. By means of protein engineering, the present application modifies the amino acid dehydrogenase catalyzing amino acid substrates into an amine dehydrogenase (AmDH) with ketone and aldehyde as substrates. The amine dehydrogenase of the present application has high reductive amination activity, and can be applied in the process of catalyzing transamination of hydroxy ketone and prochiral ketone compounds to prepare chiral amines, including but not limited to 3-hydroxy-D-tyrosine. The high catalytic activity, high yield and 99%+ stereoselectivity meet the requirements, and the present application has the advantages of green and efficient, low cost and high optical purity of products, and is one of the most ideal reactions for the synthesis of chiral amines.

Description

Technical Field

[0001] This invention belongs to the field of protein modification technology, specifically relating to an amine dehydrogenase. Furthermore, this invention also relates to the nucleic acid encoding this amine dehydrogenase, and its application in the synthesis of chiral amines. 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] Optically pure 3-hydroxy-D-tyrosine (D-DOPA) is a chiral amine and a non-natural amino acid with diverse biological activities and potential applications. In terms of neuroprotection and cognitive improvement, animal experiments have shown that it can delay the progression of Alzheimer's disease and enhance memory function in aged mice by promoting nerve cell metabolism, increasing levels of neurotransmitters such as dopamine and serotonin, and improving brain oxygenation. Simultaneously, as a serotonin precursor, it may also alleviate depressive symptoms, improve sleep quality, and reduce anxiety by regulating serotonin levels, but further clinical validation of its psychological health regulatory effects is needed. Furthermore, this compound is a key intermediate in the synthesis of tanshinone (an active ingredient in traditional Chinese medicine for treating cardiovascular diseases), especially for the efficient preparation of high-optically pure tanshinone sodium in asymmetric synthesis. In the field of biochemical research, its unique D-configuration and stereochemical difference from natural L-amino acids provide an important research tool for revealing the stereoselective mechanisms of enzymatic reactions or the structure-activity relationship of receptor-ligand binding.

[0004] The industrial production of optically pure 3-hydroxy-D-tyrosine mostly employs chemical methods such as metal-catalyzed direct reductive amination of 3,4-dihydroxyphenylpyruvate, kinetic resolution of coupled 3-hydroxy-D-tyrosine, or asymmetric hydrogenation of enamines. However, chemical methods suffer from severe environmental pollution, numerous byproducts, complex product purification, and difficulties in catalyst recovery. In contrast to chemical synthesis of 3-hydroxy-D-tyrosine, biosynthetic routes offer advantages such as mild reaction conditions, readily available raw materials, and environmental friendliness. However, chiral resolution catalyzed by lipases and cyclohexylamine oxidases requires racemic amines as raw materials, resulting in high production costs and complex steps; transaminase-catalyzed transamination reactions require the addition of excess organic amines or amino acids as amino donors and are subject to substrate inhibition and reaction equilibrium issues, making industrial application challenging.

[0005] Dehydrogenases 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 (AmDHs) are a type of dehydrogenase. It has been reported that amine dehydrogenases can be used to prepare chiral amines using biological methods, but currently, very few types of AmDHs have been reported, and their substrate spectrum is narrow, severely limiting their industrial applications. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention provides an amine dehydrogenase that can catalyze an asymmetric reduction reaction using prochiral ketones and free ammonia as substrates to synthesize chiral amines.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] The first object of the present invention is to provide an amine dehydrogenase obtained by mutating an amino acid dehydrogenase as shown in SEQ ID NO.1, wherein the mutation includes at least one mutation of amino acids 293, 69, 115, and 262 of SEQ ID NO.1.

[0009] Preferably, the mutation includes at least one of the following mutations of SEQ ID NO.1:

[0010] A1): The 293rd position is mutated from V to any one of A, C, or G;

[0011] A2): The 69th position is mutated from K to S;

[0012] A3): The 262nd position is changed from N to L;

[0013] A4): The 115th position is mutated from E to any one of V, L, A, C, G, and F;

[0014] In other words, the mutation can be any one of A1)-A4), or any combination of two, three or four.

[0015] As a further preferred embodiment, the mutation is any one of the following (a1)-(a15):

[0016] (a1) In SEQ ID NO.1, amino acid residue 69 is mutated from K to S, amino acid residue 262 is mutated from N to L; amino acid residue 115 is mutated from E to V; amino acid residue 293 is mutated from V to G;

[0017] (a2) In SEQ ID NO.1, amino acid residue 69 is mutated from K to S, amino acid residue 262 is mutated from N to L; amino acid residue 115 is mutated from E to A; amino acid residue 293 is mutated from V to C;

[0018] (a3) In SEQ ID NO.1, amino acid residue 69 is mutated from K to S, amino acid residue 262 is mutated from N to L, and amino acid residue 293 is mutated from V to A;

[0019] (a4) In SEQ ID NO.1, amino acid residue 69 is mutated from K to S, amino acid residue 262 is mutated from N to L; amino acid residue 115 is mutated from E to L; amino acid residue 293 is mutated from V to A;

[0020] (a5) The amino acid residue at position 293 of SEQ ID NO.1 is mutated from V to A;

[0021] (a6) The amino acid residue at position 293 of SEQ ID NO.1 is mutated from V to C;

[0022] (a7) The amino acid residue at position 293 of SEQ ID NO.1 is mutated from V to G;

[0023] (a8) The amino acid residue at position 115 of SEQ ID NO.1 is mutated from E to V;

[0024] (a9) The amino acid residue at position 115 of SEQ ID NO.1 is mutated from E to L;

[0025] (a10) The amino acid residue at position 115 of SEQ ID NO.1 is mutated from E to A;

[0026] (a11) The amino acid residue at position 115 of SEQ ID NO.1 is mutated from E to C;

[0027] (a12) The amino acid residue at position 115 of SEQ ID NO.1 is mutated from E to G;

[0028] (a13) The amino acid residue at position 115 of SEQ ID NO.1 is mutated from E to F;

[0029] (a14) The 69th amino acid residue of SEQ ID NO.1 is mutated from K to S, and the 262nd amino acid residue is mutated from N to L;

[0030] (a15) In SEQ ID NO.1, amino acid residue 69 is mutated from K to S, amino acid residue 262 is mutated from N to L, and amino acid residue 115 is mutated from E to V;

[0031] As the optimal choice, the mutation is:

[0032] In SEQ ID NO.1, amino acid residue 69 is mutated from K to S, amino acid residue 262 is mutated from N to L, amino acid residue 115 is mutated from E to V, and amino acid residue 293 is mutated from V to G.

[0033] A second object of the present invention is to provide an enzyme preparation comprising the above-described amine dehydrogenase.

[0034] A third objective of this invention is to provide a nucleic acid encoding the aforementioned amine dehydrogenase.

[0035] Preferably, the nucleic acid is a mutation of SEQ ID NO.2 made by at least one of the following methods:

[0036] B1): Nucleotides 877-879 are mutated from GTT to any one of GCC, TGT, or GGG;

[0037] B2): Nucleotides at positions 205-207 are mutated from AAG to AGC;

[0038] B3): Nucleotides 784-786 are mutated from AAT to TTA;

[0039] B4): Nucleotides at positions 343-345 are mutated from GAG to any one of GTG, TTA, GCC, TGT, GGG, or TTT;

[0040] In other words, the mutation can be any one of B1)-B4), or any combination of two, three or four.

[0041] As a further preferred embodiment, the nucleic acid is a mutation of SEQ ID NO.2 performed by any one of the following (b1)-(b15):

[0042] b1) Nucleotides 205-207 are mutated from AAG to AGC, nucleotides 343-345 are mutated from GAG to GTG, nucleotides 784-786 are mutated from AAT to TTA, and nucleotides 877-879 are mutated from GTT to GGG.

[0043] b2) Nucleotides 205-207 are mutated from AAG to AGC, nucleotides 343-345 are mutated from GAG to GCC, nucleotides 784-786 are mutated from AAT to TTA, and nucleotides 877-879 are mutated from GTT to TGT;

[0044] b3) Nucleotides 205-207 are mutated from AAG to AGC, nucleotides 784-786 are mutated from AAT to TTA, and nucleotides 877-879 are mutated from GTT to GCC;

[0045] b4) Nucleotides 205-207 are mutated from AAG to AGC, nucleotides 343-345 are mutated from GAG to TTA, nucleotides 784-786 are mutated from AAT to TTA, and nucleotides 877-879 are mutated from GTT to GCC;

[0046] b5) Nucleotides 877-879 are mutated from GTT to GCC;

[0047] b6) Nucleotides 877-879 are mutated from GTT to TGT;

[0048] b7) Nucleotides 877-879 are mutated from GTT to GGG;

[0049] b8) Nucleotides 343-345 are mutated from GAG to GTG;

[0050] b9) Nucleotides 343-345 are mutated from GAG to TTA;

[0051] b10) Nucleotides 343-345 are mutated from GAG to GCC;

[0052] b11) Nucleotides 343-345 are mutated from GAG to TGT;

[0053] b12) Nucleotides 343-345 are mutated from GAG to GGG;

[0054] b13) Nucleotides 343-345 are mutated from GAG to TTT;

[0055] b14) Nucleotides 205-207 are mutated from AAG to AGC, and nucleotides 784-786 are mutated from AAT to TTA;

[0056] b15) Nucleotides 205-207 are mutated from AAG to AGC, nucleotides 343-345 are mutated from GAG to GTG, and nucleotides 784-786 are mutated from AAT to TTA;

[0057] As the most preferred embodiment, the nucleic acid is a mutation of SEQ ID NO.2 performed as follows:

[0058] Nucleotides 205-207 are mutated from AAG to AGC, nucleotides 343-345 are mutated from GAG to GTG, nucleotides 784-786 are mutated from AAT to TTA, and nucleotides 877-879 are mutated from GTT to GGG.

[0059] A fourth objective of this invention is to provide an expression vector comprising the aforementioned nucleic acid;

[0060] Preferably, the expression vector is pET28a.

[0061] A fifth object of the present invention is to provide a cell comprising the above-described nucleic acid or expression vector;

[0062] Preferably, the cells are bacteria such as Escherichia coli or fungi.

[0063] A sixth object of the present invention is to provide a method for preparing the above-mentioned amine dehydrogenase, the method comprising introducing the above-mentioned expression vector into cells, culturing the cells, collecting bacterial cells, and obtaining the amine dehydrogenase; or

[0064] The preparation method includes directly culturing the above-mentioned cells, collecting bacterial cells, and obtaining the amine dehydrogenase;

[0065] Preferably, the cells are bacteria such as Escherichia coli or fungi;

[0066] As a further preferred embodiment, the preparation method further includes the following steps: breaking the bacterial cells, centrifuging to collect the supernatant, and obtaining a crude amine dehydrogenase solution.

[0067] A seventh object of the present invention is to provide the use of the above-mentioned amine dehydrogenase as a catalyst in the preparation of compounds of formula II with configuration R;

[0068] Preferably, the compound of formula II with R configuration is prepared using the compound shown in formula I as a substrate and the above-mentioned deamination hydrogenase as a catalyst.

[0069]

[0070] Wherein, R1 represents any one of H, hydroxyl, fluorine, chloro, bromo, alkyl, methoxy, or nitro;

[0071] R2 represents any one of H, hydroxyl, fluorine, chloro, bromine, methyl, alkyl, methoxy, or nitro;

[0072] R1 and R2 may be the same or different;

[0073] Preferably, the alkyl group has 1-4 carbon atoms;

[0074] As a further preferred embodiment, both R1 and R2 are hydroxyl groups.

[0075] The eighth object of the present invention is to provide a method for catalytic synthesis of the compound of formula II in the R configuration, wherein the compound of formula II in the R configuration is prepared using the compound of formula I as a substrate and the above-mentioned deamination hydrogenase as a catalyst.

[0076] Wherein, R1 represents any one of H, hydroxyl, fluorine, chloro, bromo, alkyl, methoxy, or nitro;

[0077] R2 represents any one of H, hydroxyl, fluorine, chloro, bromine, methyl, alkyl, methoxy, or nitro;

[0078] R1 and R2 may be the same or different;

[0079] Preferably, the alkyl group has 1-4 carbon atoms;

[0080] As a further preferred embodiment, both R1 and R2 are hydroxyl groups;

[0081] Preferably, the compound shown in Formula I, coenzyme NAD, is used. + Glucose, the amine dehydrogenase according to claim 1 or 2, glucose dehydrogenase, organic solvent dimethyl sulfoxide and inorganic solvent are mixed evenly and reacted at 25-45℃ for 12h-36h to obtain the compound of formula II with R configuration; the inorganic solvent is NH4Cl / NH4OH solution.

[0082] Preferably, the inorganic solvent has a pH value of 8-10, and more preferably, the inorganic solvent has a pH value of 9.5;

[0083] Preferably, the coenzyme NAD + The concentration is 0.5-2.0 mol%, and the coenzyme NAD is further preferred. + The concentration is 0.5 mol%.

[0084] Preferably, the amount of amine dehydrogenase added is 1-4 U / ml, and the amount of glucose dehydrogenase added is 1-4 U / ml; more preferably, the amount of amine dehydrogenase added is 3 U / ml, and the amount of glucose dehydrogenase added is 3 U / ml.

[0085] As a preferred method, the reaction is carried out at 35°C for 24 hours.

[0086] This invention utilizes protein engineering to modify an amino acid dehydrogenase that catalyzes amino acid substrates into an amine dehydrogenase (AmDH) that uses ketones and aldehydes as substrates. The amine dehydrogenase of this invention exhibits high reductive amination activity and can be applied to the catalytic transamination reaction of hydroxy ketones and prochiral ketones to prepare chiral amines, including but not limited to 3-hydroxy-D-tyrosine. It boasts high catalytic activity, high yield, and meets the requirement of 99%+ stereoselectivity. It offers advantages such as being green and efficient, low cost, and producing high optical purity products, making it one of the most ideal reactions for chiral amine synthesis. Attached Figure Description

[0087] 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:

[0088] Figure 1 Image of the optimal mutant 13 recombinant bacteria plate.

[0089] Figure 2 Image of seed culture for the optimal mutant 13.

[0090] Figure 3 Image of fermentation broth for the optimal mutant 13.

[0091] Figure 4 The present invention provides the synthetic route for 3-hydroxy-D-tyrosine (D-DOPA).

[0092] Figure 5 The above are gas chromatograms of 3-hydroxy-D-tyrosine (D-DOPA) of the present invention. Figure A shows the gas chromatogram of racemic 3-hydroxy-tyrosine, and Figure B shows the gas chromatogram of optically pure 3-hydroxy-D-tyrosine (D-DOPA). Detailed Implementation

[0093] 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.

[0094] The present invention relates to an amine dehydrogenase obtained by mutating an amino acid dehydrogenase such as SEQ ID NO.1, wherein the mutation includes at least one mutation of amino acids 69, 115, 262, and 293 of SEQ ID NO.1.

[0095] The raw materials used in the preparation of this invention are as follows:

[0096] pET28a vector: purchased from Novagen, catalog number: 69864-3.

[0097] Escherichia coli BL21(DE3): Purchased from Beijing TransGen Biotechnology Co., Ltd., catalog number: CD601-02.

[0098] LB medium: Each liter contains 10g tryptone, 5g yeast extract, and 5g NaCl. Adjust the pH to 7.4 with 1ml of 1mol / L NaOH, and bring the volume to 1L with deionized water. Autoclave for 20 minutes.

[0099] TB medium: Each liter contains 12g tryptone, 24g yeast extract, 4mL 87% glycerol, and 100mL phosphate buffer (pH 6.5), and is brought to a final volume of 1L with deionized water. Autoclave for 20 minutes.

[0100] Example 1: Screening and Determination of Mutation Sites

[0101] Sequence analysis, mutation, and functional verification were performed on the natural amino acid dehydrogenase protein derived from Staphylococcus agnetis (its amino acid sequence is shown in SEQ ID NO.1, and the codon-optimized (E. coli preferred) nucleic acid sequence (AmDH gene) is shown in SEQ ID NO.2 (synthesized by Qingke Biotechnology), with codon optimization software JCat: http: / / www.jcat.de). Twelve amino acid sites were specifically selected (36, 69, 115, 135, 178, 191, 205, 228, 252, 262, 293, and 303). Through further research and analysis (using molecular docking software: Autodock for molecular docking simulation; Pymol for visualization of protein and small molecule three-dimensional structures; and Hotsport hotspot amino acid analysis), four important amino acid sites (69, 115, 262, and 293) were screened from the 12 amino acid sites. By mutating these four amino acid sites in different ways, mutant proteins were obtained.

[0102] The amino acid sequence of the natural amino acid dehydrogenase derived from Staphylococcus agnetis is as follows (SEQ ID No. 1):

[0103] The nucleotide sequence obtained after codon optimization of the above amino acid sequence is as follows (SEQ ID No. 2):

[0104]

[0105]

[0106] The bolded and underlined portions above represent mutation sites.

[0107] The four amino acid sites and their mutation modes are shown in Table 1.

[0108] Table 115 Amino Acid Mutation Modes

[0109] mutant number Mutation method a1 E115V a2 E115L a3 E115A a4 E115C a5 E115G a6 E115F a7 V293A a8 V293C a9 V293G a10 K69S\N262L a11 K69S\N262L\E115V a12 K69S\N262L\V293A a13 K69S\N262L\E115V\V293G a14 K69S\N262L\E115L\V293A a15 K69S\N262L\E115A\V293C

[0110] In the table, the letter before the number represents the amino acid before the mutation, and the letter after the number represents the amino acid after the mutation. The nucleotide sequences of the mutants encoded in Table 1 are shown in Table 2.

[0111] Table 2. Nucleotide sequences corresponding to the mutants.

[0112]

[0113]

[0114] Example 2: Preparation of Recombinant Bacteria

[0115] I. Construction of Wild-Type Recombinant Expression Vectors

[0116] 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. Sequencing confirmed its correctness; the sequencing results are shown below. Figure 1 .

[0117] II. Construction of mutant recombinant expression vectors

[0118] The nucleotide sequences encoding mutants a1-a15 (see Table 2) were inserted between the EcoRI and XhoI restriction sites of the pET28a vector to obtain recombinant expression vectors pET28a-1 to pET28a-15, respectively, and the sequencing confirmed that they were correct.

[0119] III. Preparation of Recombinant Bacteria

[0120] 1. The recombinant expression vector pET28a-AmDH obtained in step one was transformed into Escherichia coli BL21(DE3) to obtain wild-type recombinant bacteria.

[0121] 2. The recombinant expression vector pET28a-1-15 prepared in step 2 was transformed into Escherichia coli BL21(DE3) to obtain mutant recombinant bacteria, which were numbered sequentially as mutant recombinant bacteria 1-15.

[0122] Example 3: Enzyme activity determination of amino acid dehydrogenase mutant protein

[0123] The wild-type recombinant bacteria and mutant recombinant bacteria 1-15 obtained in Example 2 were cultured separately, proteins were extracted, and enzyme activity was detected.

[0124] The specific steps are as follows:

[0125] 1. The recombinant bacteria ( Figure 1 The seed culture was inoculated into 10 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37 °C with shaking at 180 rpm for 16 h to obtain the seed culture. Figure 2 The seed culture was then inoculated into 50 mL of TB medium containing a final concentration of 50 μg / mL kanamycin (1% inoculum), and cultured at 37°C and 180 rpm with shaking until the bacterial OD reached the target concentration. 600nm Add 0.6-0.8 μM IPTG to a final concentration of 0.5 μM, and continue incubation at 20℃ and 180 rpm with shaking for 20-24 hours to obtain the fermentation broth. Figure 3 Centrifuge at 5000 rcf for 15 min and collect the bacterial cells.

[0126] Figure 1 Image of the optimal mutant 13 recombinant bacteria plate.

[0127] Figure 2 Image of seed culture for the optimal mutant 13.

[0128] Figure 3 Image of fermentation broth for the optimal mutant 13.

[0129] The recombinant bacterial cells were washed and resuspended twice with 50 mL of 50 mM PBS buffer. After resuspension, the cells were homogenized and centrifuged at 12000 rpm for 15 min. The supernatant was collected as the crude enzyme solution.

[0130] The target protein containing the 6×His tag was isolated and purified using Ni-NTA affinity chromatography. First, the Ni-NTA column was equilibrated with protein loading buffer, and then the filtered crude enzyme solution was loaded onto the Ni-NTA column. Next, a gradient elution was performed using protein purification elution buffers with imidazole concentrations of 30 mM, 60 mM, 300 mM, and 500 mM. After desalting, the purified protein was obtained. The molecular weight of the target protein was analyzed by SDS-PAGE electrophoresis, and the protein concentration was determined using the Bradford method, with a concentration of 12 mg / mL.

[0131] 2. The protein solution obtained by performing the above steps on wild-type recombinant bacteria is named wild-type protein solution. The protein solutions obtained by performing the above steps on mutant recombinant bacteria 1-15 are named mutant 1-mutant 15 protein solutions, respectively. Protein concentration is determined by the Bradford method.

[0132] 3. Prepare the enzyme activity detection reaction system: final substrate concentration 10 mM, reaction buffer solution 850 μL of 2 M NH4Cl / NH3·H2O, 100 μL of the protein solution to be tested (protein content approximately 0.1 mg), and final NADH concentration 1 mM. Incubate at 30℃ for 3 min. Measure the change in absorbance before and after the reaction at 340 nm.

[0133] 4. Calculate enzyme activity based on changes in absorbance:

[0134] Enzyme activity calculation formula:

[0135] Where EW: change in absorbance at 340 nm over 1 minute; V: total volume of the reaction system in mL; 6620: molar extinction coefficient in L / mol / cm; l: optical path distance of the cuvette.

[0136] Enzyme activity = U / mg

[0137] Where U: enzyme activity; mg: protein content.

[0138] The results are shown in Table 3.

[0139] Table 3. Statistical results of enzyme activity

[0140]

[0141]

[0142] Table 3 shows that there are significant differences in enzyme activity among the different mutants, with mutant 13 exhibiting the highest activity, followed by mutant 15. Mutant 13 was sequenced by Qingke Biotechnology, and the sequencing was successful. Subsequent experiments will be conducted using mutant 13.

[0143] Example 4: Thermostability of the Amine Dehydrogenase of the Present Invention

[0144] Add 1.0 mL of purified AmDH to a 10 mL EP tube, followed by a volume of PBS buffer (50 mM, pH 7.5) to dilute the protein concentration to 1.0 mg / mL. After thorough mixing, incubate the sample in water baths at 35, 50, and 70 °C. Measure the remaining enzyme activity at regular intervals. Define the relative activity at time 0 before incubation as 100%.

[0145] Amine dehydrogenases catalyze the reductive amination of ketone substrates to produce chiral amines, consuming NADH to generate NAD in the process. + This leads to a decrease in the absorbance at 340 nm (NADH's maximum absorption peak is located at 340 nm).

[0146] Based on the Lambert-Beer law, enzyme activity was determined by spectrophotometry of the absorbance of NADH at a wavelength of 340 nm (extinction coefficient ε = 6220 M). -1 ·cm -1 The initial rate of change was used to determine the enzyme reductive amination activity assay. The standard assay system (1 mL, 37 °C) contained 50 mM 3,4-dihydroxyphenylpyruvate, 1.0 mM NADH, 2.0 M NH4Cl / NH4OH buffer (pH 10.0) and an appropriate amount of enzyme (0.05 mg).

[0147] The enzyme activity unit (1U) is defined as the amount of enzyme required to reduce and amination 1 μmol of ketone substrate (while consuming 1 μmol of NADH) within 1 min.

[0148] The experimental results are shown in Table 4.

[0149] Table 4. Thermal stability of amine dehydrogenases

[0150]

[0151]

[0152] As shown in Table 4, the amine dehydrogenase of the present invention, when placed at 55°C for 120 hours, still has an enzyme activity recovery rate of over 80%, demonstrating good thermal stability.

[0153] Example 5: pH stability of the amine dehydrogenase of the present invention

[0154] Add 1.0 mL of purified amine dehydrogenase (protein concentration 5.0 mg / mL; if the enzyme concentration after purification is too high, dilute with buffer solution (50 mM, pH 7.5 PBS); if it is too low, concentrate by centrifugation using a protein filter tube) to a 10 mL EP tube. Then add 4.0 mL of NH4Cl / NH4OH buffer solution (pH 10.0, 2.0 M) and acetate-sodium acetate buffer solution (pH 4, 100 mM), respectively. After mixing thoroughly, incubate the sample at room temperature. The remaining enzyme activity was measured at regular intervals, and the relative activity at time 0 before incubation was defined as 100%. The experimental results are shown in Table 5.

[0155] Table 5 pH stability of amine dehydrogenases

[0156]

[0157] As shown in Table 5, the amine dehydrogenase of the present invention has good stability under both acidic and alkaline conditions.

[0158] Example 6: Storage stability of the amine dehydrogenase of the present invention

[0159] Add 1.0 mL of purified AmDH to a 10 mL EP tube, followed by a certain volume of PBS buffer (50 mM, pH 7.5) to dilute the protein concentration to 1.0 mg / mL. After mixing thoroughly, store the sample at 4 °C. The remaining enzyme activity was measured at regular intervals, with the relative activity at time 0 before storage defined as 100%. The experimental results are shown in Table 6.

[0160] Table 6 Storage stability of amine dehydrogenases

[0161]

[0162] As shown in Table 6, the amine dehydrogenase of the present invention is resistant to storage.

[0163] Example 7: Reaction Condition Optimization Experiment

[0164] See the synthetic route. Figure 4 .

[0165] Figure 4 The present invention provides the synthetic route for 3-hydroxy-D-tyrosine (D-DOPA).

[0166] Initial reaction conditions: 3,4-dihydroxyphenylpyruvic acid (0.5 mmol), coenzyme NAD... + Add 5 mL of 2M NH4Cl / NH4OH solution (pH 10.0), 10 U of amine dehydrogenase mutant 13 (Sa-AmDH-M13), 10 U of GDH (glucose dehydrogenase), 0.6 mmol of glucose, and 50 μL of DMSO (dimethyl sulfoxide) to a 10 mL glass tube. Stir the reaction mixture at 30 °C for 24 h.

[0167] After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 5 mL), and the organic layer was collected. The organic layer was then dried with Na₂SO₄ and separated by silica gel column chromatography (elution with ethyl acetate:methanol at a ratio of 99:1 (v / v)-100 mL; followed by elution with ethyl acetate:methanol at a ratio of 90:10 (v / v)-100 mL). The product-rich eluent was collected, placed in a pistol flask, and rotary evaporated at 45 °C. The yield was calculated after weighing.

[0168] The configuration and ee value of the asymmetric reduction reaction product were determined by gas chromatography. The specific procedure was as follows: 1 μl of the collected product was redissolved in 1 mL of ethyl acetate, 10 μL of acetic anhydride was added, and the mixture was vortexed for 10 s. 1 μl of the vortex product was then analyzed. The chromatographic conditions were: Agilent J&W CP-Chiralsil-DEXCB column (25 m × 0.32 mm × 0.25 μm), column temperature program: initial temperature 100 °C, temperature ramped to 200 °C at a rate of 5 °C / min, and held at 200 °C for 5 min. The results showed that the synthesized product was 3-hydroxy-D-tyrosine. The yield and ee value under different experimental conditions are shown in Table 7.

[0169] Table 7 Optimization of Reaction Conditions

[0170]

[0171] The coenzyme concentrations in the table are molar concentrations relative to the substrate 3,4-dihydroxyphenylpyruvic acid.

[0172] As shown in Table 7, the yields of sequences 24, 25, 28, 29 and 30 all reached 99%. The optimal reaction conditions were those with the least amount of enzyme and the shortest reaction time (i.e., sequence 24 was the best).

[0173] Example 8: Asymmetric Synthesis of Optically Pure 3-Hydroxy-D-Tyrosine

[0174] 3,4-Dihydroxyphenylpyruvic acid (0.5 mmol) and coenzyme NAD+ were added. + Add 5 mL of 2M NH4Cl / NH4OH solution (pH 9.5), 15 U of amine dehydrogenase mutant 13 (Sa-AmDH-M13), 15 U of GDH (glucose dehydrogenase), 0.6 mmol of glucose, and 50 μL of DMSO (dimethyl sulfoxide) to a 10 mL glass tube. Stir the reaction mixture at 35 °C for 24 h.

[0175] After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 5 mL), and the organic layer was collected. The organic layer was then dried with Na₂SO₄ and separated by silica gel column chromatography (elution with ethyl acetate:methanol at a ratio of 99:1 (v / v)-100 mL; followed by elution with ethyl acetate:methanol at a ratio of 90:10 (v / v)-100 mL). The product-rich eluent was collected, placed in a pistol flask, and rotary evaporated at 45 °C. The yield was calculated after weighing.

[0176] The configuration and ee value of the asymmetric reduction reaction product were determined by gas chromatography. The specific procedure was as follows: 1 μl of the collected product was redissolved in 1 mL of ethyl acetate, 10 μL of acetic anhydride was added, and the mixture was vortexed for 10 s. Then, 1 μl was taken for analysis. The chromatographic conditions were: 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, and held at 200 °C for 5 min. The results showed that the synthesized product was 3-hydroxy-D-tyrosine, with a yield of 99% and an ee value of 99%. The results are shown in the table below. Figure 5 See Tables 8 and 9.

[0177] Figure 5 The above are gas chromatograms of 3-hydroxy-D-tyrosine according to the present invention. Figure A shows the gas chromatogram of racemic 3-hydroxy-tyrosine, and Figure B shows the gas chromatogram of optically pure 3-hydroxy-D-tyrosine (D-DOPA).

[0178] Table 8. Gas chromatographic analysis data of racemic 3-hydroxy-tyrosine

[0179] Peak time / min Peak area Peak Peak area percentage (%) S 14.889 2928925 525373 49.888 R 15.150 2942077 596560 50.112

[0180] Table 93 Gas Chromatographic Analysis Data of 3-Hydroxy-D-Tyrosine (D-DOPA)

[0181] Peak time / min Peak area Peak Peak area percentage (%) R 15.134 2954750 605344 >99％

[0182] Example 9: Asymmetric Synthesis of Substrates

[0183] Substrate 1s-15s (0.5mmol), coenzyme NAD + Add 0.0025 mmol of 2M NH4Cl / NH4OH solution (pH 9.5), 15 U of amine dehydrogenase mutant 13, 15 U of GDH (glucose dehydrogenase), 0.6 mmol of glucose, and 50 μL of DMSO to a 10 mL glass tube. Stir the reaction mixture at 35 °C for 24 h.

[0184] After the reaction was complete, the mixture was extracted with ethyl acetate (3 × 5 mL), and the organic layer was collected. The organic layer was then dried with Na₂SO₄ and separated by silica gel column chromatography (elution with ethyl acetate:methanol at a ratio of 99:1 (v / v)-100 mL; followed by elution with ethyl acetate:methanol at a ratio of 90:10 (v / v)-100 mL). The product-rich eluent was collected, placed in a pistol flask, and rotary evaporated at 45 °C. The yield was calculated after weighing.

[0185] The configuration and ee value of the asymmetric reduction reaction products were determined by gas chromatography. The specific procedure was as follows: 1 μL of the collected product was redissolved in 1 mL of ethyl acetate, 10 μL of acetic anhydride was added, and the mixture was vortexed for 10 s. 1 μL of the vortex product was then analyzed. The chromatographic conditions were: 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, and held at 200 °C for 5 min. The yields and ee values ​​of different substrates and their corresponding products are shown in Table 10. All products listed below are R-configuration compounds.

[0186] Table 10 Asymmetric Synthesis of Different Substrates

[0187]

[0188]

[0189] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An amine dehydrogenase, characterized in that: It was obtained by mutating the amino acid dehydrogenase shown in SEQ ID NO.1, wherein the mutation is as follows: the 69th amino acid residue of SEQ ID NO.1 is mutated from K to S, the 262nd amino acid residue is mutated from N to L; the 115th amino acid residue is mutated from E to V; and the 293rd amino acid residue is mutated from V to G.

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 obtained by the following mutations to SEQ ID NO.2: nucleotides 205-207 are mutated from AAG to AGC, nucleotides 343-345 are mutated from GAG to GTG, nucleotides 784-786 are mutated from AAT to TTA, and nucleotides 877-879 are mutated from GTT to GGG.

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

6. The expression vector according to claim 5, characterized in that: The expression vector is pET28a.

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

8. The cell according to claim 7, characterized in that: The cells are bacteria.

9. The cell according to claim 8, characterized in that: The cells are either Escherichia coli or fungi.

10. 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 or 6 into cells, culturing the cells, collecting bacterial cells, and obtaining the amine dehydrogenase; or The preparation method includes directly culturing the cells according to any one of claims 7-9, collecting the bacterial cells, and obtaining the amine dehydrogenase.

11. The method for preparing amine dehydrogenase according to claim 10, characterized in that: The cells are bacteria.

12. The method for preparing amine dehydrogenase according to claim 11, characterized in that: The cells are either Escherichia coli or fungi.

13. The method for preparing amine dehydrogenase according to any one of claims 10-12, characterized in that: The preparation method further includes the following steps: breaking the bacterial cells, centrifuging to collect the supernatant, and obtaining crude amine dehydrogenase solution.

14. The use of the amine dehydrogenase of claim 1 as a catalyst in the preparation of compounds of formula II with configuration R, characterized in that: Using the compound shown in Formula I as a substrate and the deamination hydrogenase described in claim 1 as a catalyst, the compound shown in Formula II with the R configuration was prepared. Equation I Formula II; Wherein, R1 represents any one of H, hydroxyl, fluorine, chloro, bromide, alkyl, methoxy, or nitro; R2 represents any one of H, hydroxyl, fluorine, chloro, bromine, methyl, alkyl, methoxy, or nitro; R1 and R2 may be the same or different.

15. The application according to claim 14, characterized in that: The alkyl group has 1-4 carbon atoms.

16. The application according to claim 14 or 15, characterized in that: Both R1 and R2 are hydroxyl groups.

17. A method for catalytic synthesis of a compound of formula II with configuration R, characterized in that: Using the compound shown in Formula I as a substrate and the deamination hydrogenase described in claim 1 as a catalyst, the compound shown in Formula II with the R configuration was prepared. Wherein, R1 represents any one of H, hydroxyl, fluorine, chloro, bromide, alkyl, methoxy, or nitro; R2 represents any one of H, hydroxyl, fluorine, chloro, bromine, methyl, alkyl, methoxy, or nitro; R1 and R2 may be the same or different.

18. A method for catalytic synthesis of a compound of formula II with configuration R according to claim 17, characterized in that: The alkyl group has 1-4 carbon atoms.

19. A method for catalytic synthesis of a compound of formula II with configuration R according to claim 17 or 18, characterized in that: Both R1 and R2 are hydroxyl groups.

20. A method for catalytic synthesis of a compound of formula II with configuration R according to claim 17, characterized in that: The compound shown in Formula I, coenzyme NAD + Glucose, the amine dehydrogenase of claim 1, glucose dehydrogenase, organic solvent dimethyl sulfoxide and inorganic solvent are mixed evenly and reacted at 25-45℃ for 12h-36h to obtain the compound of formula II with R configuration; the inorganic solvent is NH4Cl / NH4OH solution.

21. A method for catalytic synthesis of a compound of formula II with configuration R according to claim 20, characterized in that: The inorganic solvent has a pH value of 8-10.

22. The method for catalytic synthesis of the compound of formula II with configuration R according to claim 21, characterized in that: The inorganic solvent has a pH value of 9.

5.

23. The method for catalytic synthesis of the R-configuration compound of formula II according to claim 20, characterized in that: The coenzyme NAD + The concentration is 0.5-2.0 mol%.

24. The method for catalytic synthesis of the R-configuration compound of formula II according to claim 23, characterized in that: The coenzyme NAD + The concentration is 0.5 mol%.

25. A method for catalytic synthesis of a compound of formula II with configuration R according to claim 20, characterized in that: The amount of amine dehydrogenase added is 1-4 U / ml, and the amount of glucose dehydrogenase added is 1-4 U / ml.

26. A method for catalytic synthesis of a compound of formula II with configuration R according to claim 25, characterized in that: The amount of amine dehydrogenase added is 3 U / ml, and the amount of glucose dehydrogenase added is 3 U / ml.

27. A method for catalytic synthesis of a compound of formula II with configuration R according to claim 20, characterized in that: React at 35℃ for 24 hours.

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