Amino acid dehydrogenase and application thereof in preparation of aminobutanol

Abstract

The invention discloses amino acid dehydrogenase and application thereof in preparation of aminobutanol. The amino acid dehydrogenase is selected from the following components: (1) an amino acid sequence as shown in SEQ ID NO: 1; (2) compared with an amino acid sequence with at least 90% sequence identity as shown in SEQ ID NO: 1, the amino acid sequence comprises amino acid residues at the P148 and / or N293 sites which are different from the amino acid sequence with at least 90% sequence identity as shown in SEQ ID NO: 1; wherein sites 148 and 293 refer to amino acid residue numbers in SEQ ID NO: 1. According to the amino acid dehydrogenase disclosed by the invention, 4-hydroxy-2-butanone is used as a substrate for catalyzing to generate a target product (R)-3-aminobutanol, the substrate has a relatively high conversion rate, and the target product has relatively high stereoselectivity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme catalysis, and particularly relates to an amino acid dehydrogenase and application thereof in preparation of aminobutanol. BACKGROUND

[0002] The English name of (R)-3-aminobutanol is (R)-3-amino-1-butanol, the molecular formula is C4H 11 NO, the molecular weight is 89.14, and the structural formula is shown as formula I:

[0003]

[0004] (R)-3-aminobutanol is an important intermediate of anti-AIDS drugs dolutegravir and penicillin antibiotics. The synthesis methods of (R)-3-aminobutanol mainly include chemical synthesis and biological enzyme methods.

[0005] The chemical synthesis method mostly involves chiral induction or chiral resolution, and has harsh reaction conditions, poor selectivity, many by-products, and complex purification and separation, and is high in production cost. For example, CN 113336655A discloses that (R)-3-aminobutanol is prepared by reduction with (R)-3-aminobutyric acid as a raw material and zinc borohydride inert solvent as a reducing agent, but the yield is low, and subsequent steps such as quenching, filtration, concentration and rectification are required.

[0006] The biological enzyme method has the characteristics of mild reaction conditions, high efficiency, strong stereoselectivity and environmental friendliness, and is easy to realize industrial production. Amino acid dehydrogenase can react to generate (R)-3-aminobutyric acid with 4-hydroxy-2-butanone as a substrate in the presence of an amino donor, so it has great research value to develop an amino acid dehydrogenase with high efficiency, stereoselectivity and better stability. SUMMARY

[0007] The application provides an amino acid dehydrogenase and application thereof in preparation of aminobutanol, and a route for preparing (R)-3-aminobutanol with 4-hydroxy-2-butanone as a substrate is shown in the following formula:

[0008]

[0009] Wherein, AmDH is amino acid dehydrogenase, and FDH is formate dehydrogenase.

[0010] The application solves the above technical problems through the following technical solutions.

[0011] The first aspect of the present application provides an amino acid dehydrogenase selected from the group consisting of:

[0012] (1) the amino acid sequence shown as SEQ ID NO: 1;

[0013] (2) an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown as SEQ ID NO: 1, comprising amino acid residue difference at position P148 and / or N293; wherein the positions of 148 and 293 refer to the amino acid residue numbering in SEQ ID NO: 1.

[0014] In some embodiments, the amino acid dehydrogenase has a conversion rate of 4-hydroxy-2-butanone no less than 3%, 5%, 10%, 15%, 20% or 30% when preparing (R)-3-aminobutanol with 4-hydroxy-2-butanone as substrate.

[0015] In some embodiments, the amino acid dehydrogenase has an amino acid residue difference at position P148 is P148A or P148L; and / or

[0016] the amino acid residue difference at position N293 is N293V, N293A or N293L; wherein the positions of 148 and 293 refer to the amino acid residue numbering in SEQ ID NO: 1.

[0017] In some specific embodiments, the amino acid dehydrogenase comprises one of the following amino acid residue differences compared with the amino acid sequence shown as SEQ ID NO: 1:

[0018] (1) P148A;

[0019] (2) P148L;

[0020] (3) N293V;

[0021] (4) N293A;

[0022] (5) N293L;

[0023] (6) P148L and N293V;

[0024] (7) P148L and N293L;

[0025] (8) P148A and N293A.

[0026] In some embodiments, the amino acid dehydrogenase is selected from the group consisting of:

[0027] (1) the amino acid sequence shown as SEQ ID NO: 3;

[0028] (2) deletion of S137 / P138 / E139 / F140 / G141 / S142 compared to the amino acid sequence as set forth in SEQ ID NO: 1, and comprising the following amino acid residue differences: P148A and N293A; wherein the positions 148 and 293 refer to the amino acid residue numbering in SEQ ID NO: 1.

[0029] In some embodiments, the amino acid dehydrogenase further comprises an amino acid residue difference at one or more of the following positions: G64, Y67, E121, G144, V285, A288, A295, C134, N69, and V114 compared to the amino acid sequence as set forth in SEQ ID NO: 1; wherein the positions 64, 67, 121, 144, 285, 288, 295, 134, 69, and 114 refer to the amino acid residue numbering in SEQ ID NO: 1.

[0030] In some embodiments, the amino acid difference at the G64 position is a substitution of G to a polar amino acid; preferably an acidic amino acid or an amide thereof, or a basic amino acid.

[0031] In some specific embodiments, the amino acid residue difference at the G64 position of the amino acid dehydrogenase is G64N, G64H, or G64D; and / or

[0032] the amino acid residue difference at the Y67 position of the amino acid dehydrogenase is Y67E, Y67K, or Y67F; and / or

[0033] the amino acid residue difference at the E121 position of the amino acid dehydrogenase is E121P; and / or

[0034] the amino acid residue difference at the G144 position of the amino acid dehydrogenase is G144I, G144H, G144V, G144C, or G144S; and / or

[0035] the amino acid residue difference at the V285 position of the amino acid dehydrogenase is V285A, V285S, V285I or V285Q; and / or

[0036] the amino acid residue difference at the A288 position of the amino acid dehydrogenase is A288G, A288S, A288P, or A288C; and / or

[0037] the amino acid residue difference at the A295 position of the amino acid dehydrogenase is A295M, A295W, A295G, A295E, A295F, A295T, A295Q, A295H, or A295C; and / or

[0038] the amino acid dehydrogenase differs at the amino acid residue at position C134 to C134Q or C134L; and / or

[0039] the amino acid dehydrogenase differs at the amino acid residue at position N69 to N69E, N69D, N69G, or N69I; and / or

[0040] the amino acid dehydrogenase differs at the amino acid residue at position V114 to V114C, V114L, or V114P; wherein the positions 64, 67, 121, 144, 285, 288, 295, 134, 69, and 114 refer to the numbering of the amino acid residues in SEQ ID NO: 1.

[0041] In some further particular embodiments, the amino acid dehydrogenase lacks S137 / P138 / E139 / F140 / G141 / S142 as compared to the amino acid sequence set forth in SEQ ID NO: 1 and comprises one of the following combinations of amino acid residue differences:

[0042] (1) P148A and N293A;

[0043] (2) P148A, N293A, and G64N;

[0044] (3) P148A, N293A, G64H, and Y67E;

[0045] (4) P148A, N293A, G64D, and Y67K;

[0046] (5) P148A, N293A, G64H, and Y67F;

[0047] (6) P148A, N293A, G64H, Y67E, and G144I;

[0048] (7) P148A, N293A, G64H, Y67E, and G144H;

[0049] (8) P148A, N293A, G64H, Y67E, and G144V;

[0050] (9) P148A, N293A, G64H, Y67E, and G144C;

[0051] (10) P148A, N293A, G64H, Y67E, and G144S;

[0052] (11) P148A, N293A, G64H, Y67E, G144S, and E121P;

[0053] (12) P148A, N293A, G64H, Y67E, G144S, and V285A;

[0054] (13) P148A, N293A, G64H, Y67E, G144S, and V285S;

[0055] (14) P148A, N293A, G64H, Y67E, G144S, and V285I;

[0056] (15) P148A, N293A, G64H, Y67E, G144S, and V285Q;

[0057] (16) P148A, N293A, G64H, Y67E, G144S, and A288G;

[0058] (17) P148A, N293A, G64H, Y67E, G144S, and A288S;

[0059] (18) P148A, N293A, G64H, Y67E, G144S, and A288P;

[0060] (19) P148A, N293A, G64H, Y67E, G144S, and A288C;

[0061] (20) P148A, N293A, G64H, Y67E, G144S, and A295M;

[0062] (21) P148A, N293A, G64H, Y67E, G144S, and A295W;

[0063] (22) P148A, N293A, G64H, Y67E, G144S, and A295G;

[0064] (23) P148A, N293A, G64H, Y67E, G144S, and A295E;

[0065] (24) P148A, N293A, G64H, Y67E, G144S, and A295F;

[0066] (25) P148A, N293A, G64H, Y67E, G144S, and A295T;

[0067] (26) P148A, N293A, G64H, Y67E, G144S, and A295Q;

[0068] (27) P148A, N293A, G64H, Y67E, G144S, and A295H;

[0069] (28) P148A, N293A, G64H, Y67E, G144S and A295C;

[0070] (29) P148A, N293A, G64H, Y67E, G144S and C134Q;

[0071] (30) P148A, N293A, G64H, Y67E, G144S and C134L;

[0072] (31) P148A, N293A, G64H, Y67E, G144S and N69E;

[0073] (32) P148A, N293A, G64H, Y67E, G144S and N69D;

[0074] (33) P148A, N293A, G64H, Y67E, G144S and N69G;

[0075] (34) P148A, N293A, G64H, Y67E, G144S and N69I;

[0076] (35) P148A, N293A, G64H, Y67E, G144S and V114C;

[0077] (36) P148A, N293A, G64H, Y67E, G144S and V114L;

[0078] (37) P148A, N293A, G64H, Y67E, G144S and V114P; wherein the positions of 64, 67, 121, 144, 285, 288, 295, 134, 69 and 114 refer to the amino acid residue numbers in SEQ ID NO: 1.

[0079] The second aspect of the present application provides an isolated nucleic acid encoding the amino acid dehydrogenase according to the first aspect of the present application; the nucleotide sequence of the nucleic acid is for example as set forth in SEQ ID NO: 2 or 4.

[0080] The third aspect of the present application provides a recombinant expression vector comprising the nucleic acid according to the second aspect of the present application; the backbone of the recombinant expression vector is for example pET28a.

[0081] The fourth aspect of the present application provides a transformant comprising the nucleic acid according to the second aspect of the present application or the recombinant expression vector according to the third aspect of the present application.

[0082] In some embodiments, the host cell used in the construction of the transformant is Escherichia coli, for example, Escherichia coli BL21 (DE3).

[0083] The fifth aspect of the present application provides a method for preparing an amino acid dehydrogenase, the method comprising culturing the transformant according to the fourth aspect of the present application to obtain a fermentation product.

[0084] In some embodiments, the culture medium used in the culturing is LB liquid medium.

[0085] In some embodiments, the conditions of the culturing are that the culturing is induced overnight at a temperature of 37±1°C, and the inducer is further preferably IPTG.

[0086] In some embodiments, the method further comprises the step of purifying the fermentation product to obtain the amino acid dehydrogenase.

[0087] The sixth aspect of the present application provides an enzyme composition, the enzyme composition comprising the amino acid dehydrogenase according to the first aspect of the present application and a coenzyme regenerating enzyme.

[0088] In some embodiments, the coenzyme regenerating enzyme is formate dehydrogenase.

[0089] In some specific embodiments, the amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO: 5.

[0090] The seventh aspect of the present application provides the use of the amino acid dehydrogenase according to the first aspect of the present application, the nucleic acid according to the second aspect of the present application, the recombinant expression vector according to the third aspect of the present application, the transformant according to the fourth aspect of the present application, or the enzyme composition according to the sixth aspect of the present application in the preparation of (R)-3-aminobutanol.

[0091] In some specific embodiments, the (R)-3-aminobutanol is prepared using 4-hydroxy-2-butanone as the substrate.

[0092] The eighth aspect of the present application provides a method for preparing (R)-3-aminobutanol, the method comprising: contacting and reacting at least the amino acid dehydrogenase according to the first aspect of the present application with a substrate and an amino donor to obtain (R)-3-aminobutanol.

[0093] In some embodiments, the substrate is 4-hydroxy-2-butanone.

[0094] In some embodiments, the amino donor is ammonium formate, ammonium chloride, isopropylamine, or D-alanine.

[0095] In some embodiments, the reaction further comprises the use of a coenzyme, which includes an oxidized coenzyme and a reduced coenzyme.

[0096] In some embodiments, the reaction further comprises a step of regenerating the oxidized coenzyme into the reduced coenzyme.

[0097] In the present application, the term “coenzyme” refers to a small molecule organic compound that can bind to an enzyme and participate in catalyzing a reaction. The term “regenerating the oxidized coenzyme into the reduced coenzyme” refers to generating the reduced coenzyme from the oxidized coenzyme in the presence of a hydrogen donor.

[0098] In some embodiments, the reduced coenzyme is any one or more of NADH and NADPH, and the oxidized coenzyme is any one or more of NAD + and NADP + .

[0099] In some specific embodiments, the oxidized coenzyme is regenerated into the reduced coenzyme using any one of NAD + , NADP + , NAD + and NADP + , a hydrogen donor and a coenzyme regeneration enzyme; the hydrogen donor and the coenzyme regeneration enzyme are selected from one or more of:

[0100] (1) glucose and glucose dehydrogenase;

[0101] (2) formic acid / formate and formate dehydrogenase; the amino acid sequence of the formate dehydrogenase is preferably as shown in SEQ ID NO: 5;

[0102] (3) alcohol and alcohol dehydrogenase.

[0103] In some embodiments, the coenzyme regeneration enzyme or the amino acid dehydrogenase is used in the form of a liquid enzyme, a solid enzyme powder, an immobilized enzyme, a wet bacterial cell or a bacterial powder.

[0104] In some embodiments, the 4-hydroxy-2-butanone is added in the reaction at a concentration of 1-100 mg / mL; preferably 10-80 mg / mL.

[0105] In some embodiments, the hydrogen donor is added in the reaction at a molar ratio of 1-5:1, preferably 1-2:1, relative to the 4-hydroxy-2-butanone.

[0106] In some embodiments, the NAD + and / or NADP + is added in the reaction at a molar ratio of 1:1-500, preferably 1:300-400, relative to the 4-hydroxy-2-butanone.

[0107] In some embodiments, the pH value of the reaction is 8.0-10, preferably 8.0-9.0.

[0108] In some embodiments, the temperature of the reaction is 30-50℃, preferably 30-38℃.

[0109] In some embodiments, the time of the reaction is 10-25h, preferably 15-20h.

[0110] In some embodiments, the added amount of the amino acid dehydrogenase is 1:0.1-10, preferably 1:0.5-2, of the mass of the wet bacteria of the amino acid dehydrogenase to the mass of the substrate in the reaction system in terms of the mass of the corresponding wet bacteria.

[0111] In some embodiments, the added amount of the formate dehydrogenase is 1:0.1-10, preferably 1:0.5-2, of the mass of the wet bacteria of the formate dehydrogenase to the mass of the substrate in the reaction system in terms of the mass of the corresponding wet bacteria.

[0112] The ninth aspect of the present application provides a reaction end product system, which comprises:

[0113] (R)-3-aminobutanol;

[0114] 4-hydroxy-2-butanone; and

[0115] The amino acid dehydrogenase as described in the first aspect of the present application.

[0116] In some embodiments, the reaction end product system further comprises a coenzyme regenerating enzyme, which is preferably a formate dehydrogenase.

[0117] In some specific embodiments, the amino acid sequence of the formate dehydrogenase is as shown in SEQ ID NO: 5.

[0118] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined in any manner, thereby obtaining various preferred examples of the present application.

[0119] The reagents and raw materials used in the present application are commercially available.

[0120] The positive progress effect of the present application is that:

[0121] The amino acid dehydrogenase of the present application catalyzes the generation of the target product (R)-3-aminobutanol from the substrate 4-hydroxy-2-butanone, and the substrate has a high conversion rate, and the target product has high stereoselectivity.

[0122] The whole reaction process of the application is simple in operation, and the reaction condition is mild, without high temperature and high pressure, strong acid and strong base, large amount of organic solvent and precious chemical catalyst, only needs to be reacted at normal temperature and pressure, effectively reduces the production cost, and is environment-friendly, in line with the green and safe production concept. DETAILED DESCRIPTION

[0123] The application will be further described below by way of examples, but the application is not limited to the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to the conventional methods and conditions, or according to the product instructions. The experimental methods in the application are conventional methods unless otherwise specified, and the gene cloning operation can refer to J. Sambrook et al. Molecular Cloning Experiment Guide. The gene synthesis, primer synthesis and gene sequencing in the examples are completed by Shanghai Shengong Bioengineering Co., Ltd.

[0124] 1, part of the reagent information used in the application is shown in Table 1.

[0125] Table 1 reagent information table

[0126] Reagent name Manufacturer Catalogue number Taq DNA polymerase Shanghai Biotech B600001-0200 pET28a Novagen 69864 E. coli BL21 (DE3) Novagen 69450 BugBuster lysis buffer Merck 70584-4 T4 DNA ligase Thermo Fisher Scientific EL0014 Marfey derivatization reagent Bide Pharmatech BD130031 4-Hydroxy-2-butanone standard Adamas-beta 68120H (R)-3-Aminobutanol standard aladdin A123608 (S)-3-Aminobutanol standard aladdin S176942

[0127] 2, part of the reagent preparation method used in the application is as follows:

[0128] (1) Marfey derivative solution: weigh 75 mg of (R)-2-((5-fluoro-2.4-dinitrophenyl) amino) propionamide (Marfey derivative reagent) in a 25 ml volumetric flask, dissolve and dilute to the mark with acetonitrile.

[0129] (2) TB liquid medium: 10 g / L of proteose peptone, 18 g / L of yeast powder, 4 mL / mL of glycerol, 2.31 g / L of KH2PO4 (anhydrous), 16.43 g / L of K2HPO4 (trihydrate), dissolved with RO water, and then constant volume, sterilized at 121 ℃ for 20 min, and used.

[0130] (3) LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, dissolved with RO water, and then constant volume, sterilized at 121 ℃ for 20 min, and used.

[0131] (4) LB agar plate: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, 18 g / L of agar, pH 7.2 (adjusted with 1 mol / L NaOH).

[0132] 3, the substrate conversion rate is determined by HPLC method, specifically as follows:

[0133] Chromatographic column: Eclipse Plus C18 (1.8 μm, 50 x 2.1 mm); mobile phase A: 0.1% TFA in water (solvent: water), mobile phase B: 0.1% (v / v) TFA in acetonitrile (solvent: acetonitrile); detection wavelength: 360 nm; flow rate: 0.5 mL / min; injection volume of sample solution: 1.0 μL; column temperature: 35 °C; gradient elution according to the procedure of Table 2:

[0134] Table 2 Gradient elution procedure for HPLC

[0135] Time (min) Mobile phase A % Mobile phase B % 0.00 70 30 2.00 70 30 2.50 10 90 3.00 10 90 3.10 70 30 5.00 70 30

[0136] In the obtained HPLC chromatogram, the retention time of the product (3-aminobutanol) is:

[0137] 2.501 min.

[0138] The content of 3-aminobutanol is calculated by external standard method. The calculation formula of the content of 3-aminobutanol is:

[0139] Note: A 供试品 is the peak area (mAU) of the test sample; A 对照品 is the peak area (mAU) of the control sample; C 对照品 is the concentration (mg / mL) of the control sample; X 对照品 is the content (%) of the control sample; N is the dilution multiple of the test sample solution. The substrate conversion rate is calculated from the content of 3-aminobutanol.

[0140] Test sample solution: Take the reaction solution into a centrifuge tube, adjust the acid to inactivate the enzyme, centrifuge, take the supernatant, adjust the pH of the supernatant to about 6 to 7 with alkali, dilute with water to a certain multiple, take 1 mL of the solution, add 1 mL of sodium bicarbonate solution, 1 mL of 40% ethanol solution and 1 mL of derivatization solution into a centrifuge tube, shake well, wrap with tin foil paper to avoid light, then place in a 60 °C oven for 1 h for derivatization, to obtain the test sample solution. Among them, the derivatization solution is: 1 mL of 2,4-dinitrofluorobenzene (DNFB) is diluted to 100 mL with acetonitrile, and shaken well to prepare.

[0141] 4. Chiral analysis and concentration analysis of the product are carried out by pre-column derivatization high performance liquid chromatography, as follows:

[0142] (1) Derivatization reaction: add the reaction solution obtained after the reaction into Marfey derivatization solution and sodium bicarbonate solution, derivatize in a 50 °C oven for 1 hour, then cool to room temperature, add hydrochloric acid solution, dilute and filter to obtain the sample solution.

[0143] (2) Chromatographic detection: chromatographic column: Diamonsil C18, (5 μm, 250 x 4.6 mm); mobile phase: 0.1% formic acid aqueous solution: 0.1% formic acid acetonitrile solution = 70:30; detection wavelength: 340 nm; flow rate: 0.8 ml / min; injection volume: 10 μl; column temperature: 40 °C; running time: 35 min.

[0144] The retention time of (R)-3-aminobutanol was 16.493 min, and the retention time of (S)-3-aminobutanol was 24.836 min.

[0145] Obtaining and preparation of amino acid dehydrogenase E2 of Example 1

[0146] 1.1 An amino acid dehydrogenase E1 with accession number WP_003249296.1 was obtained from Parageobacillus thermoglucosidasius. The amino acid dehydrogenase E1 was subjected to multiple rounds of random mutation screening to obtain an amino acid dehydrogenase E2 with mutation sites K68S / E114V / T134C / P146V / H187D / N261L / I294C / R350A, and the amino acid sequence thereof is shown as SEQ ID NO: 1, and the nucleotide sequence thereof is shown as SEQ ID NO: 2.

[0147] 1.2 Transformation of the amino acid dehydrogenase E2 gene

[0148] The gene fragment of the amino acid dehydrogenase E2 was synthesized and cloned into the Ndel / Hindlll enzyme cutting sites between the expression vector pET28a (Kan resistance) to obtain a recombinant plasmid pET28a-E2. The recombinant plasmid pET28a-E2 was transformed into E. coli BL21 (DE3) competent cells, and the transformed bacterial liquid was spread on LB agar plates containing 50 μg / mL kanamycin (Kan) and cultured at 37 °C overnight.

[0149] 1.3 Expression of the amino acid dehydrogenase E2 gene

[0150] A single colony on the LB agar plate was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37 °C for 4 h, and then inoculated into TB liquid medium containing 50 μg / mL kanamycin at a 1% (v / v) inoculation amount, and cultured at 37 °C, 250 rpm until the bacterial concentration OD 600 was 0.6-0.8. After induction at 25 °C, 250 rpm overnight with an inducer IPTG at a final concentration of 0.1 mM, centrifugation at 4000 rpm for 20 min, the supernatant was discarded, and the precipitate was collected to obtain wet bacteria expressing the amino acid dehydrogenase E2.

[0151] The wet bacterial cells were resuspended with 50 mM PBS buffer at pH 8.0 at a ratio of 1 g wet bacterial cells: 10 mL PBS buffer (i.e. homogenization ratio), and then homogenized with a homogenizer. After centrifugation, the precipitate was discarded, and a crude enzyme solution of amino acid dehydrogenase E2 was obtained, which was stored at -20 °C for later use.

[0152] Example 2 Construction and screening of amino acid dehydrogenase E2 mutant library

[0153] The amino acid dehydrogenase E2 was simulated and analyzed using protein three-dimensional structure analysis software and molecular docking software, and was modified based on its protein structure, molecular conformation, docking results with substrates, characteristics of the enzyme-substrate binding pocket, stereoselectivity of the enzyme in recognizing substrates, and catalytic mechanism of the enzyme, etc. A series of mutants with improved stereoselectivity and conversion rate were obtained.

[0154] 2.1 Construction of E2 mutant library

[0155] (1) The pET28a-E2 plasmid in Example 1 was used as a template to design error-prone PCR primers (forward primer P-F: 5'-cagcggcctggtgccgcgcggcagccat-3' (SEQ ID NO: 6), reverse primer P-R: 5'-gtggtggtgctcgagtgcggccgcaagctta-3' (SEQ ID NO: 7), which were used to amplify the E2 gene. Random mutagenesis. PCR amplification reaction The PCR amplification reaction system (total volume 25 μL) is shown in Table 3, and the PCR amplification process The PCR amplification program is shown in Table 4.

[0156] (2) The amplified product and the pET28a vector were recombined using a seamless cloning kit, and the recombined plasmid was transformed into E. coli BL21 (DE3) competent cells. The transformed bacterial solution was spread on an LB agar plate containing 50 μg / mL kanamycin, and incubated at 37 °C overnight until single colonies of the recombined E. coli were formed on the LB agar plate, thereby obtaining an E2 mutant enzyme expression library.

[0157] Table 3 PCR amplification reaction system

[0158] Reagent name Reagent addition volume Template (pET28a-E3 recombinant plasmid) 1 μL Forward primer F 1 μL Reverse primer R 1 μL KOD One TM PCR Master Mix 12.5 μL ddH2O 9.5 μL

[0159] Table 4 PCR amplification program

[0160]

[0161] 2.2 Screening of E2 mutant library

[0162] (1) Preparation of crude enzyme solution

[0163] Reference Example 1.3 Preparation of the crude enzyme solution of amino acid dehydrogenase E2 mutant and formate dehydrogenase E3, wherein the amino acid sequence of formate dehydrogenase E3 is shown as SEQ ID NO: 5.

[0164] (2) Construction and screening of enzyme catalytic reaction system

[0165] An enzyme catalytic system for preparing (R)-3-aminobutanol with 4-hydroxy-2-butanone as substrate was constructed in order to screen the amino acid dehydrogenase in the gene mutation library. The enzyme catalytic system is shown in Table 5. The construction method of the enzyme catalytic system is as follows: 250 mg of 4-hydroxy-2-butanone (2.84 mmol, MV: 88.11) was added into a reaction container, followed by the addition of the crude enzyme solution of amino acid dehydrogenase, 315.3 mg of ammonium formate (5 mmol, MV: 63.06), NADH (0.5 mmol, MV: 0.25), and 0.5 mL of 1 mol / L Tris-HCl buffer (pH 9.0). The total volume of the enzyme catalytic system was 5 mL. The enzyme catalytic system was placed at 45 °C for 18 h. After the reaction was completed, the reaction solution was taken to determine the conversion rate of the substrate and the ee value of the product. + The crude enzyme solution of formate dehydrogenase E3 was prepared according to the method of Reference Example 1.3. The pH value was adjusted to 9.0 with ammonia water, and the volume was made up to 5 mL with water. The reaction conditions were as follows: the enzyme catalytic system was placed at 45 °C for 18 h. After the reaction was completed, the reaction solution was taken to determine the conversion rate of the substrate and the ee value of the product. Eight amino acid dehydrogenases with improved conversion rate and ee value were screened from the gene mutation library, which were numbered as E2-1 to E2-8, and the conversion rate and ee value are shown in Table 7.

[0166] Table 5 Enzyme catalytic reaction system (total volume 5 mL)

[0167] Component Addition amount Final concentration 4-Hydroxy-2-butanone (substrate) 250 mg (2.84 mmol) 50 mg / mL Crude enzyme solution of amino acid dehydrogenase 1.25 ml of homogeneous enzyme solution (equivalent to 125 mg of wet bacteria) 25 mg / mL Ammonium formate (hydrogen donor) 315.3 mg 1M 50 mM NAD + (oxidized coenzyme) 100 μL 1 mM Crude enzyme solution of formate dehydrogenase E3 (coenzyme regeneration enzyme) 2.5 ml of homogeneous enzyme solution (equivalent to 250 mg of wet bacteria) 50 mg / mL Ammonia solution (pH adjuster) Adjust the pH to 9.0 / ddH2O Dilute to 5 mL /

[0168] Example 3 Truncation of amino acid dehydrogenase E2

[0169] According to the results of three-dimensional structure analysis and molecular docking simulation analysis of amino acid dehydrogenase E2, the amino acid residues from the 137th to the 142nd site of amino acid dehydrogenase E2, i.e. S137- / P138- / E139- / F140- / G141- / S142, were truncated, which was numbered as amino acid dehydrogenase E2-9, and the amino acid sequence thereof is shown as SEQ ID NO: 3, and the nucleotide sequence thereof is shown as SEQ ID NO: 4.

[0170] The conversion rate of the substrate and the ee value of the product of amino acid dehydrogenase E2-9 were determined according to Reference Example 2.2, and the conversion rate of the substrate was 5.46%, and the ee value of the product was 98.95%, which was 66% higher than that of E2, and the ee value was comparable to that of E2 (as shown in Table 7).

[0171] Example 4 Construction and screening of amino acid dehydrogenase E2-9 mutation library

[0172] 4.1 Construction of amino acid dehydrogenase E2-9 mutation library

[0173] According to the screening results of the E2 mutant library, three-dimensional structure analysis and molecular docking simulation analysis, some sites of the amino acid dehydrogenase E2 were mutated based on E2-9 to construct the mutant library of the amino acid dehydrogenase E2-9. The primers are shown in Table 6 below, and the specific steps are referred to Example 2.1.

[0174] Table 6 Mutant primers of E2-9

[0175]

[0176] In Table 6, N represents any one of A, G, C, T, and K represents G or T.

[0177] 4.2 Screening of the mutant library of the amino acid dehydrogenase E2-9

[0178] The screening of the mutant library of the amino acid dehydrogenase E2-9 is referred to Example 2.2, and the results are shown in Table 7 below. S137- / P138- / E139- / F140- / G141- / S142- represents that the amino acids SPEFGS from positions 137 to 142 are truncated.

[0179] Table 7 Conversion rate and ee value under different amino acid dehydrogenases

[0180]

[0181]

[0182]

[0183]

[0184] Results and analysis: The conversion rates of the above mutants have been greatly improved compared with the control. The conversion rate of E2-35 is the highest, which is 37.10%, and the ee value is 99.48%; the second is E2-32, the conversion rate is 36.40%, and the ee value is 99.57%; the third is E2-29, the conversion rate is 35.5%, and the ee value is 99.48%.

[0185] Example 5 Preparation of (R)-3-aminobutanol

[0186] The mutant with a higher conversion rate in Example 4 was selected to construct a 100 mL enzyme catalytic system for preparing (R)-3-aminobutanol with 4-hydroxy-2-butanone as the substrate. The enzyme catalytic system is shown in Table 8. The specific steps are referred to Example 2.2. The steps are as follows: take a 250 mL jacketed bottle, add 85 mL of water and 5.36 g of ammonium formate (85.0 mmoll), after the solution was cleared, ammonia was added dropwise to adjust the pH of the solution to 8.5; then 5g of 4-hydroxy-2-butanone (56.7mmol) was added and mixed, and then 5g of formate dehydrogenase E3 wet bacteria and 5g of amino acid dehydrogenase mutant wet bacteria were added and mixed, and then 0.1g of NAD+ (0.15mmol) was added; the pH of the system was controlled by ammonia, the reaction was carried out in a closed system, and the enzyme catalytic system was placed in a 35℃ environment for 18h. After the reaction was completed, the reaction solution was taken, and the conversion rate of the substrate and the ee value of the product were determined (as shown in Table 9).

[0187] Table 8: Enzyme catalytic reaction system (total volume 100mL)

[0188] Component Addition amount Final concentration 4-Hydroxy-2-butanone (substrate) 5g 50 mg / mL Amino acid dehydrogenase wet bacteria 5g 50 mg / mL Ammonium formate (hydrogen donor) (MV: 63.06) 5.36 g (0.085 mol) 0.85M NAD + (oxidized form of coenzyme) (MV: 663.4) 0.1g 1 g / L Formate dehydrogenase E3 wet bacteria (coenzyme regeneration enzyme) 5.0g 50 mg / mL Ammonia solution (pH adjuster) Stabilize the pH to 8.5 / ddH2O Dilute to 100 mL /

[0189] Table 9: Conversion rate and ee value under different amino acid dehydrogenases

[0190]

[0191]

[0192] Among them, the conversion rate of E2-35 is the highest, which is 70%, and the ee value is 99.80%; followed by E2-32, the conversion rate is 68%, and the ee value is 99.42%; and then E2-11, the conversion rate is 67%, and the ee value is 99.35%.

[0193] 32, the conversion rate is 68%, and the ee value is 99.42%; and then E2-11, the conversion rate is 67%, and the ee value is 99.35%.

[0194] The sequence used in the application is as follows:

[0195] 1, E2 amino acid sequence (SEQ ID NO: 1)

[0196] MELFKYMEQYDYEQLLFCQDKESGLKAIIAIHDTTLGPALGGTRMWMYESEEAAIEDALRLARGMTYSNAAAGLNLGGGKAVIIGDPRKDKNEAMFRAFGRFIQGLNGRYITAVDVGTTVEDMDIIYQETDYVCGISPEFGSSGNVSPATAYGVYRGMKAAVKEAFGSDSLEGKVVAVQGVGSVAYDLCRHLHEEGAKLIVTDINKEAVQRAIEEFGAKAVDPNDIYGVDCDVFAPCALGGIINDETIPQLKAKVIAGSALNQLKEPRHGDIIHELGIVYAPDYVINAGGVINCADELHGYNRERAMKKIEQIYDNIKKIFVIAKRDGIPAYQAADRLAEERIETLRKSASQFLQNGHHILSRRRGR

[0197] 2. E2 nucleotide sequence (SEQ ID NO: 2)

[0198] ATGGAGCTGTTTAAGTACATGGAACAGTATGATTACGAGCAGCTGCTGTTTTGTCAGGATAAAGAAAGTGGTCTGAAGGCCATTATTGCAATTCATGATACCACCCTGGGTCCGGCCCTGGGTGGTACCCGTATGTGGATGTATGAAAGTGAAGAAGCAGCAATTGAGGATGCCCTGCGTCTGGCCCGTGGTATGACATATAGCAATGCAGCCGCAGGCCTGAATCTGGGTGGTGGTAAAGCCGTGATTATTGGCGATCCGCGCAAAGATAAAAATGAAGCAATGTTTCGCGCCTTTGGCCGTTTTATTCAGGGCCTGAATGGTCGTTATATTACCGCCGTTGATGTGGGTACCACCGTGGAAGATATGGATATTATCTATCAGGAGACCGATTACGTGTGTGGTATTAGTCCGGAATTTGGTAGCAGCGGTAATGTTAGTCCGGCAACCGCATATGGTGTTTATCGTGGCATGAAAGCCGCAGTTAAAGAAGCATTTGGTAGCGATAGCCTGGAAGGTAAAGTTGTGGCCGTGCAGGGTGTGGGCAGTGTTGCATATGATCTGTGTCGCCATCTGCATGAAGAAGGTGCCAAACTGATTGTTACCGATATTAATAAGGAGGCCGTTCAGCGCGCAATTGAAGAATTTGGTGCAAAAGCCGTTGATCCGAATGATATCTATGGCGTTGATTGCGATGTTTTTGCACCGTGCGCACTGGGCGGCATTATTAATGATGAAACCATTCCGCAGCTGAAAGCAAAAGTTATTGCAGGTAGTGCACTGAATCAGCTGAAAGAACCGCGCCATGGCGATATTATTCATGAACTGGGCATTGTTTACGCACCGGATTATGTGATTAACGCCGGTGGTGTGATTAATTGTGCAGATGAACTGCATGGCTATAATCGTGAACGCGCAATGAAAAAGATTGAACAGATCTATGACAACATCAAGAAGATCTTCGTGATCGCCAAACGTGATGGCATTCCGGCCTATCAGGCAGCAGATCGTCTGGCTGAAGAACGTATTGAAACCCTGCGTAAAAGCGCCAGTCAGTTTCTGCAGAATGGCCATCATATTCTGAGTCGTCGTCGCGGCCGC

[0199] 3. E2-9 amino acid sequence (SEQ ID NO: 3)

[0200] MELFKYMEQYDYEQLLFCQDKESGLKAIIAIHDTTLGPALGGTRMWMYESEEAAIEDALRLARGMTYSNAAAGLNLGGGKAVIIGDPRKDKNEAMFRAFGRFIQGLNGRYITAVDVGTTVEDMDIIYQETDYVCGISGNVSPATAYGVYRGMKAAVKEAFGSDSLEGKVVAVQGVGSVAYDLCRHLHEEGAKLIVTDINKEAVQRAIEEFGAKAVDPNDIYGVDCDVFAPCALGGIINDETIPQLKAKVIAGSALNQLKEPRHGDIIHELGIVYAPDYVINAGGVINCADELHGYNRERAMKKIEQIYDNIKKIFVIAKRDGIPAYQAADRLAEERIETLRKSASQF LQNGHHILSRRRGR

[0201] 4. E2-9 nucleotide sequence (SEQ ID NO: 4)

[0202]

[0203] 5. Formate dehydrogenase E3 amino acid sequence (SEQ ID NO: 5)

[0204] MPLGTAKVLCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAI DFTPGQLLGSVSGELGLRKYL ESNGHTLVVTSDKDGPDSVFERELVDADV VISQPFWPAYLTPERIAKAKNLKLALTAGVGSDHVDLQAAIDRNVTVAEVTGANSISVAEHVVMMILALVRNYLPSHEWARKGGWNIADCVAHAYDLEAMHVGTVGAGRIGLAVLRRLAPFDVHLHYTDRHRLPEAVEKELNLTWHATREDMYPVCDVVTLNAPLHPETEHMINDETLKLFKRGAYIVNTARGKLCDRDAVARALESGRLAGYAGDVWFPQPAPKDHPWRTMPYNGMTPHISGTTLTAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAV

[0205] Although the specific embodiments of the present application are described above, it should be understood by those skilled in the art that these are only illustrative, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present application. Therefore, the scope of protection of the present application is defined by the appended claims.

Claims

1. An amino acid dehydrogenase, characterized in that, The amino acid dehydrogenase is selected from the following: (1) The amino acid sequence is shown in SEQ ID NO:1; (2) The amino acid residues contained in the P148 and / or N293 sites differ from the amino acid sequence having at least 90% sequence identity as shown in SEQ ID NO:1; wherein the sites 148 and 293 refer to the amino acid residue numbers in SEQ ID NO:

1. Preferably, when the amino acid dehydrogenase prepares (R)-3-aminobutanol using 4-hydroxy-2-butanone as a substrate, the conversion rate of 4-hydroxy-2-butanone is not less than 3%, 5%, 10%, 15%, 20%, or 30%.

2. The amino acid dehydrogenase according to claim 1, characterized in that, The amino acid dehydrogenase differs from other amino acid dehydrogenases at the P148 site by either P148A or P148L; and / or The amino acid dehydrogenases differ in amino acid residues at the N293 position as N293V, N293A, or N293L; Preferably, the amino acid dehydrogenase contains one of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO:1: (1) P148A; (2)P148L; (3) N293V; (4) N293A; (5) N293L; (6) P148L and N293V; (7) P148L and N293L; (8) P148A and N293A.

3. The amino acid dehydrogenase according to claim 1, characterized in that, The amino acid dehydrogenase is selected from: (1) The amino acid sequence is shown in SEQ ID NO:3; (2) Compared with the amino acid sequence shown in SEQ ID NO:1, S137 / P138 / E139 / F140 / G141 / S142 are missing, and the following amino acid residues are different: P148A and N293A; wherein the positions of 148 and 293 refer to the amino acid residue numbers in SEQ ID NO:1; Preferably, the amino acid dehydrogenase, compared with the amino acid sequence shown in SEQ ID NO:1, further includes amino acid residue differences at one or more sites among G64, Y67, E121, G144, V285, A288, A295, C134, N69, and V114; wherein the sites of 64, 67, 121, 144, 285, 288, 295, 134, 69, and 114 refer to the amino acid residue numbers in SEQ ID NO:1; More preferably, the amino acid difference at the G64 site is to replace G with a polar amino acid; for example, an acidic amino acid or its amide, or a basic amino acid; More preferably, the amino acid dehydrogenase differs in the amino acid residues at the G64 position by G64N, G64H, or G64D; and / or The amino acid dehydrogenase differs in amino acid residues at the Y67 position by Y67E, Y67K, or Y67F; and / or The amino acid dehydrogenase differs from the other amino acid at the E121 site by E121P; and / or The amino acid dehydrogenase differs in the following amino acid residues at the G144 position: G144I, G144H, G144V, G144C, or G144S; and / or The amino acid dehydrogenase differs in amino acid residues at position V285 as V285A, V285S, V285I, or V285Q; and / or The amino acid dehydrogenase differs from other amino acid dehydrogenases at position A288 by A288G, A288S, A288P, or A288C; and / or The amino acid dehydrogenase differs in the following amino acid residues at position A295: A295M, A295W, A295G, A295E, A295F, A295T, A295Q, A295H, or A295C; and / or The amino acid dehydrogenase differs from the other amino acid at the C134 site by either C134Q or C134L; and / or The amino acid dehydrogenase differs in the amino acid residues at the N69 position as N69E, N69D, N69G, or N69I; and / or The amino acid dehydrogenases differ in the amino acid residues at the V114 site as V114C, V114L, or V114P.

4. The amino acid dehydrogenase according to claim 3, characterized in that, The amino acid dehydrogenase is missing S137 / P138 / E139 / F140 / G141 / S142 compared to the amino acid sequence shown in SEQ ID NO:1, and contains one of the following combinations of amino acid residue differences: (1) P148A and N293A; (2) P148A, N293A and G64N; (3) P148A, N293A, G64H and Y67E; (4) P148A, N293A, G64D and Y67K; (5) P148A, N293A, G64H and Y67F; (6) P148A, N293A, G64H, Y67E and G144I; (7) P148A, N293A, G64H, Y67E and G144H; (8) P148A, N293A, G64H, Y67E and G144V; (9) P148A, N293A, G64H, Y67E and G144C; (10) P148A, N293A, G64H, Y67E and G144S; (11) P148A, N293A, G64H, Y67E, G144S and E121P; (12) P148A, N293A, G64H, Y67E, G144S and V285A; (13) P148A, N293A, G64H, Y67E, G144S and V285S; (14) P148A, N293A, G64H, Y67E, G144S and V285I; (15) P148A, N293A, G64H, Y67E, G144S and V285Q; (16) P148A, N293A, G64H, Y67E, G144S and A288G; (17) P148A, N293A, G64H, Y67E, G144S and A288S; (18) P148A, N293A, G64H, Y67E, G144S and A288P; (19) P148A, N293A, G64H, Y67E, G144S and A288C; (20) P148A, N293A, G64H, Y67E, G144S and A295M; (21) P148A, N293A, G64H, Y67E, G144S and A295W; (22) P148A, N293A, G64H, Y67E, G144S and A295G; (23) P148A, N293A, G64H, Y67E, G144S and A295E; (24) P148A, N293A, G64H, Y67E, G144S and A295F; (25) P148A, N293A, G64H, Y67E, G144S and A295T; (26) P148A, N293A, G64H, Y67E, G144S and A295Q; (27) P148A, N293A, G64H, Y67E, G144S and A295H; (28) P148A, N293A, G64H, Y67E, G144S and A295C; (29) P148A, N293A, G64H, Y67E, G144S and C134Q; (30) P148A, N293A, G64H, Y67E, G144S and C134L; (31) P148A, N293A, G64H, Y67E, G144S and N69E; (32) P148A, N293A, G64H, Y67E, G144S and N69D; (33) P148A, N293A, G64H, Y67E, G144S and N69G; (34) P148A, N293A, G64H, Y67E, G144S and N69I; (35) P148A, N293A, G64H, Y67E, G144S and V114C; (36) P148A, N293A, G64H, Y67E, G144S and V114L; (37) P148A, N293A, G64H, Y67E, G144S and V114P.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes an amino acid dehydrogenase as described in any one of claims 1-4; the nucleotide sequence of the nucleic acid is, for example, as shown in SEQ ID NO:2 or 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 5; the backbone of the recombinant expression vector is, for example, pET28a.

7. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 5 or the recombinant expression vector as described in claim 6; Preferably, the host cell used in the construction of the transformant is Escherichia coli, such as Escherichia coli BL21(DE3).

8. A method for preparing amino acid dehydrogenase, characterized in that, The method includes culturing the transformant as described in claim 7 to obtain a fermentation product; Preferably, the culture medium used for the culture is LB liquid medium, and / or the culture conditions are: overnight induction culture at a temperature of 37±1℃, and the inducer is preferably IPTG; More preferably, the method further includes the step of purifying the fermentation product to obtain the amino acid dehydrogenase.

9. An enzyme composition, characterized in that, The enzyme combination includes the amino acid dehydrogenase and coenzyme regeneration enzyme as described in any one of claims 1-4; Preferably, the coenzyme regenerating enzyme is formate dehydrogenase; the amino acid sequence of the formate dehydrogenase is preferably as shown in SEQ ID NO:

5.

10. The use of the amino acid dehydrogenase according to any one of claims 1-4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the transformant according to claim 7, or the enzyme composition according to claim 9 in the preparation of (R)-3-aminobutanol; Preferably, 4-hydroxy-2-butanone is used as a substrate when preparing (R)-3-aminobutanol.

11. A method for preparing (R)-3-aminobutanol, characterized in that, The method includes: contacting and reacting the amino acid dehydrogenase as described in any one of claims 1-4 with a substrate and an amino donor to obtain (R)-3-aminobutanol; Preferably, the substrate is 4-hydroxy-2-butanone; and / or, the amino donor is ammonium formate, ammonium chloride, isopropylamine, or D-alanine; and / or, the reaction further includes the use of a coenzyme, the coenzyme including oxidized coenzyme and reduced coenzyme. More preferably, the reaction further includes the step of regenerating the oxidized coenzyme into the reduced coenzyme.

12. The method as described in claim 11, characterized in that, The reduced coenzyme is any one or more of NADH and NADPH, and the oxidized coenzyme is NAD. + and NADP + Any one or more of the following; Preferably, the oxidized coenzyme is regenerated into a reduced coenzyme using NAD+. + NADP + NAD + and NADP + Any one of the following, a hydrogen donor and a coenzyme regenerating enzyme; wherein the hydrogen donor and the coenzyme regenerating enzyme are selected from one or more of the following: (1) Glucose and glucose dehydrogenase; (2) Formic acid / formate salt and formic acid dehydrogenase; the amino acid sequence of the formic acid dehydrogenase is preferably as shown in SEQ ID NO:5; (3) Alcohols and alcohol dehydrogenases.

13. The method as described in claim 12, characterized in that, The concentration of 4-hydroxy-2-butanone added in the reaction is 1-100 mg / mL; preferably 10-80 mg / mL. And / or, the molar ratio of the hydrogen donor to 4-hydroxy-2-butanone is 1-5:1, preferably 1-2:1; And / or, the NAD + and / or NADP + The molar ratio of 4-hydroxy-2-butanone to 4-hydroxy-2-butanone is 1:1-500, preferably 1:300-400; And / or, the pH value of the reaction is 8.0-10, preferably 8.0-9.0; And / or, the reaction temperature is 30-50°C, preferably 30-38°C; And / or, the reaction time is 10-25 h, preferably 15-20 h.

14. The method as described in claim 13, characterized in that, The method includes one or more of the following conditions: (1) The amount of amino acid dehydrogenase added is based on the mass of the corresponding wet cells. In the reaction system, the mass ratio of the wet cells of the amino acid dehydrogenase to the added substrate can be 1:0.1-10, preferably 1:0.5-2. (2) The amount of formate dehydrogenase added is based on the mass of the corresponding wet cells. In the reaction system, the mass ratio of the wet cells of formate dehydrogenase to the added substrate is 1:0.1-10, preferably 1:0.5-2.

15. A reaction end product system, characterized in that, The reaction end product system includes: (R)-3-aminobutanol; 4-Hydroxy-2-butanone; and The amino acid dehydrogenase as described in any one of claims 1-4; The reaction end product system may optionally further include a coenzyme regenerating enzyme, wherein the coenzyme regenerating enzyme is preferably formate dehydrogenase; the amino acid sequence of the formate dehydrogenase is more preferably as shown in SEQ ID NO:5.

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