Amine dehydrogenase mutants, engineered bacteria, and their applications in synthesizing (R)-3-aminobutanol

Abstract

The present invention discloses an amine dehydrogenase mutant, an engineered bacterium, and its use in the synthesis of (R)-3-aminobutanol. The mutant is obtained by single or multiple mutations at amino acids 100 and 206 of the amino acid sequence shown in SEQ ID No. 1. The enzyme activity of the amine dehydrogenase mutant provided by the present invention is significantly improved from 55.3 U / g before the mutation to 93.9 U / g, and the thermal stability is also significantly improved, with the half-life increasing from 9.0 hours before the mutation to 33.6 hours, effectively addressing the low activity and low thermal stability of existing amine dehydrogenases.

Description

(1) Technical field

[0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and relates to an amine dehydrogenase mutant with improved enzyme activity and thermal stability, an engineered bacterium, and application of the mutant in synthesizing (R)-3-aminobutanol. (2) Background technology

[0002] (R)-3-Aminobutanol is an important pharmaceutical intermediate with significant applications. It is a key intermediate in the synthesis of the chiral six-membered ring of the anti-AIDS drug Dolutegravir. It can also be derived into β-lactams and then into penicillin antibiotics, and has broad application value in the pharmaceutical field.

[0003] Currently, the synthesis of (R)-3-aminobutanol is primarily achieved through chemical methods. These methods primarily include chiral raw material synthesis, chemical induction, chiral resolution, and preparative chromatography. These methods suffer from complex procedures, severe pollution, harsh reaction conditions, and low regio / enantioselectivity.

[0004] Compared with chemical methods, bioenzymatic catalytic synthesis has the advantages of stereoselectivity, high catalytic efficiency, mild catalytic conditions and high atom utilization. It has been widely used in the industrial production of chiral chemicals such as pharmaceutical intermediates and fine chemicals. In recent years, transaminases (Catalysts.2018;8(7):254), lyases (Angewandte Chemie (Internationled.).2004;43:788-824), imine reductases (Organic processes reswarch & development.2019;23:1262-1268), and amine dehydrogenases (Angewandte Chemie (Internationaled.).2012;51:2969-3972) have been developed for the synthesis of chiral amines. In the reaction process of transaminases, different configurations of enzymes require different configurations of ammonia donors, resulting in a limited range of donor selection. In addition, the limitations of enzyme affinity, coenzyme binding, and the generation of reaction by-products have seriously restricted the application of transaminases. Lyases are also widely used in the preparation of chiral amines, such as in the synthesis of L-aspartic acid and alanine. However, lyases cannot mediate C-N bond formation in certain reactions, such as the synthesis of L-phenylalanine (Angewandte Chemie (Internationled.). 2004; 43: 788-824). Furthermore, the optimal pH of lyases differs significantly from that of cascade enzymes, severely limiting their widespread application. Imine reductases have low activity, an unclear catalytic mechanism, and are difficult to design semi-rationally. Their limited substrate range makes them difficult to apply in industrial production (Angewandte Chemie (Internationaled.). 2012; 51: 2969-3972).

[0005] Compared to other biocatalytic methods, (R)-selective amine dehydrogenase (ADE) can synthesize (R)-3-aminobutanol in a one-pot reaction using 4-hydroxy-2-butanone as a substrate and inorganic ammonia (such as ammonium chloride) as an amino donor. Coupled with a glucose dehydrogenase (GDH) cofactor regeneration system for hydrogen supply, it produces only H2O and gluconic acid as byproducts. These products are easily separable, meeting the requirements of green and sustainable production. It shows promising application prospects in the synthesis of various chiral amines and is expected to be applied in industrial production. However, current ADEs generally have low enzymatic activity and poor thermal stability, which cannot meet the requirements of practical applications.

[0006] Therefore, it is of great significance to develop highly active and thermally stable amine dehydrogenases to meet the biosynthesis of (R)-3-aminobutanol and other chiral amino alcohol compounds. (3) Summary of the invention

[0007] The present invention aims to provide an amine dehydrogenase mutant with improved enzyme activity and thermal stability, an engineered bacterium, and use thereof in the synthesis of (R)-3-aminobutanol. The present invention obtains an amine dehydrogenase mutant with synergistically improved enzyme activity and thermal stability through molecular modification, thereby solving the problem of low amine dehydrogenase activity and thermal stability.

[0008] The technical solution adopted in the present invention is:

[0009] The present invention provides an amine dehydrogenase mutant with improved enzyme activity and thermal stability. The mutant is obtained by subjecting the amino acids at positions 100 and 206 of the amino acid sequence shown in SEQ ID No. 1 (the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2) to single or multiple mutations.

[0010] Furthermore, it is preferred that the amine dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID No. 1 is mutated into one of the following: (1) the phenylalanine at position 100 is mutated into alanine, which is denoted as GKGBAmDH F100A The amino acid sequence is shown in SEQ ID No. 3, and the nucleotide sequence of the coding gene is shown in SEQ ID No. 4; (2) the asparagine at position 206 is mutated to alanine, denoted as GKGBAmDH N206A , the amino acid sequence is shown in SEQ ID No.5, and the nucleotide sequence of the coding gene is shown in SEQ ID No.6; (3) the phenylalanine at position 100 is mutated to alanine, and the asparagine at position 206 is mutated to alanine, denoted as GKGBAmDH F100A / N206A The amino acid sequence is shown in SEQ ID No.7, and the nucleotide sequence of the encoding gene is shown in SEQ ID No.8.

[0011] The last three codons tga in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 8 of the present invention are terminators and do not participate in the editing of the amino acid sequence.

[0012] The present invention also provides a coding gene for the amine dehydrogenase mutant, a recombinant expression vector containing the coding gene, and a recombinant genetic engineering bacterium prepared by transforming the recombinant expression vector.

[0013] The recombinant expression vector can be constructed by ligating a nucleic acid encoding the mutant amine dehydrogenase gene of the present invention to various suitable vectors using conventional methods in the art. The vector can be any conventional vector in the art, such as commercially available plasmids, cosmids, phage, or viral vectors, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the mutant amine dehydrogenase. The mutant amine dehydrogenase gene can be operably linked downstream of appropriate regulatory sequences in the vector to achieve constitutive or inducible expression of the mutant amine dehydrogenase. The vector is preferably a plasmid, more preferably plasmid pET-28a. The host cell can be any conventional host cell in the art, as long as the recombinant expression vector can stably replicate and effectively express the target protein after induction with an inducer. Escherichia coli is the preferred host cell in the present invention, and E. coli BL21 (DE3) is more preferably used for efficient expression of the mutant amine dehydrogenase of the present invention. The recombinant expression transformant can be cultured using conventional methods and conditions in the art.

[0014] The present invention also provides an application of the amine dehydrogenase mutant in catalyzing the synthesis of (R)-3-aminobutanol from 4-hydroxy-2-butanone. The application method comprises the following steps: using wet bacteria obtained by fermentation culture of a recombinant genetically engineered bacterium containing a gene encoding the amine dehydrogenase mutant as a catalyst, using glucose dehydrogenase (GDH) as a coenzyme, using 4-hydroxy-2-butanone as a substrate, using glucose as a cosubstrate, and using NAD as a catalyst. + As a cofactor, a reaction system is formed with a buffer solution of pH 8.5 to 11.0 (preferably a 0.1 M NH4Cl / NH3·H2O buffer solution of pH 10.0) as a reaction medium, and the reaction is carried out at a temperature of 20 to 45° C. and a rotation speed of 1000 to 1500 rpm (preferably 40° C. and 1100 rpm). After the reaction is completed, the reaction solution is separated and purified to obtain (R)-3-aminobutanol.

[0015] Furthermore, in the reaction system, the catalyst dosage is 10-100 g / L (preferably 50 g-100 g / L, most preferably 50 g / L) based on the weight of the wet cells, the final substrate concentration is 50-300 mM (preferably 50-200 mM, more preferably 50 mM), the final cofactor concentration is 1-3 mM (preferably 3 mM), the final glucose concentration is 100-300 mM (preferably 200 mM), and the final glucose dehydrogenase concentration is 1-5 g / L (preferably 2 g / L). The glucose dehydrogenase is added in the form of lyophilized enzyme powder, with a specific enzyme activity of 56.12 U / mg.

[0016] Furthermore, the reaction medium is a 0.1M NH4Cl / NH3·H2O buffer solution with a pH of 10.0.

[0017] Furthermore, the catalyst is prepared according to the following method: a recombinant genetically engineered bacterium containing a gene encoding a mutant of amine dehydrogenase is inoculated into an LB solid culture medium containing 50 μg / mL of kanamycin, and cultured in a shaker at 37° C. and 180 rpm. When the OD600 of the culture solution reaches 0.6 to 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) is added as an inducer at a final concentration of 0.1 to 0.5 mmol / L. After induction at 28° C. for 12 hours, the culture solution is centrifuged and the precipitate is collected to obtain the catalyst.

[0018] LB medium composition: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, solvent is water, pH natural. LB solid medium is prepared by adding 20g / L agar powder to the above LB liquid medium.

[0019] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0020] (1) The enzyme activity of the amine dehydrogenase mutant provided by the present invention has been significantly improved, from 55.3 U / g before mutation to 93.9 U / g, and the thermal stability has also been significantly improved, with the half-life increased from 9.0 h before mutation to 33.6 h, effectively solving the defects of the existing amine dehydrogenase with low activity and low thermal stability.

[0021] (2) The amine dehydrogenase mutant of the present invention is used as a catalyst to catalyze the synthesis of (R)-3-aminobutanol from 4-hydroxy-2-butanone using a "one-pot" reaction. The reaction begins after the substrate and enzyme are added, and the final product, (R)-3-aminobutanol, is directly obtained, resulting in low industrial cost. Furthermore, the initial raw material is 4-hydroxy-2-butanone, and the amino donor is inorganic ammonia, which is inexpensive. The catalyst can be prepared in large quantities by constructing genetically engineered Escherichia coli bacteria and then fermenting them, making it relatively easy and inexpensive.

[0022] (3) Compared with the parent, the amine dehydrogenase mutant provided by the present invention has better catalytic activity. Under the optimal system, the product yield of 200 mM substrate reached 89.04% after 24 hours of reaction, and the ee value reached 99.9%, providing a basis for the efficient preparation of (R)-3-aminobutanol. (IV) Description of the accompanying drawings

[0023] Figure 1 , Schematic diagram of the reaction process of 4-hydroxy-2-butanone catalyzed by amine dehydrogenase.

[0024] Figure 2 , relative enzyme activity curve of amine dehydrogenase mutants at 40℃.

[0025] Figure 3, high performance liquid chromatogram; a represents the reaction solution of reaction system 3 of Example 3; b represents 10 mM (S)-3-aminobutanol standard solution. (V) Specific implementation methods

[0026] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0027] In the examples of the present invention, the term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, "F100A" means that the phenylalanine F at position 69 is mutated to serine alanine A, and so on.

[0028] LB medium composition: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, solvent is water, pH natural. LB solid medium is prepared by adding 20g / L agar powder to the above LB liquid medium.

[0029] The specific enzyme activity of the glucose dehydrogenase (GDH) lyophilized powder in the embodiment is 56.12 U / mg, where the enzyme activity is defined as the amount of lyophilized enzyme powder required to convert 1 mmol of NADH per unit time, i.e., 1 minute.

[0030] Example 1: Screening of amine dehydrogenase mutants and preparation of recombinant expression transformants

[0031] 1. Construction of original amine dehydrogenase expression vector and engineered bacteria

[0032] The GKGBAmDH gene from Geobacillus thermophilus (the encoded amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence is shown in No. 2) was inserted into the multiple cloning site of plasmid pET-28a to construct a recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells, spread on LB plates containing 50 μg / mL kanamycin resistance, and placed in a 37°C incubator for static culture for approximately 12 hours to obtain E. coli BL21 (DE3) / pET-28a-GKGBAmDH recombinant bacteria.

[0033] 2. Screening of mutation sites of amine dehydrogenase mutants

[0034] The structure of GKGBAmDH protein (amino acid sequence shown in SEQ ID No. 1) was modeled using the AlphaFold2 (AlphaFold2 TIB Server (biodesign.ac.cn)) website, and predictions were made using the HotSpot wizard. Saturation mutations were performed on sites 23, 92, 93, 100, 102, 206, 223, 224, 277, and 270 in the predicted structural and functional hotspots.

[0035] SEQ ID No.1

[0036] MMELFQYMEKYDYEQ VL ​​FCQD KE SGLKAIIVIHDT TL GPALGGTRMWMYNSEEEALED ALRLARGMTYSNAAAG LN LGGGKTVIIGDPRKDKNEAMF RAFGRF IQ GLNGRYIT AE DVGTTVADMDIIYQETDYVNG ISPEFGSSGNVSPATA YG VYRGMKAAAKEAFG SD SLEGKVVAVQGVGNVAYHLCR HLH EEGAKLIVTDINKEAVARAVE EFGAKAVDPNDIYGVE CD IFAPCALGGIINDQT IP QLKAKVIAGSALNQLKEPRH GDM IHEMGIVYAPDYVINAGGVIN VADELYGYN RE RAMKK IE QIYDNIEKVFAIAKRD NIP TYVAADRMAEERIETMRKA RS QFLQNGHHILSRRRARHHHHH HLEHHHHHH.

[0037] 3. Construction of saturation mutation library

[0038] Using the recombinant expression plasmid pET-28a-GKGBAmDH obtained in step 1 as a DNA template, saturation mutagenesis primers (Table 1) were designed and a mutation library was obtained by PCR amplification.

[0039] Table 1 Saturation mutagenesis primers

[0040]

[0041] The bold font “NNK” in the table represents that the inter-codon contains 32 codon combinations, covering all 20 amino acid residues, N = A / C / G / T, K = G / T.

[0042] PCR extension system (total volume 50 μL): 25 μL 2× Phanta Max buffer, 1 μL dNTP Mix (10 mM each), 0.5 μL template, 1 μL each of a pair of mutant primers (10 μM), 0.5 μL Phanta Max Super-Fidelity DNA Polmerase, and add sterile distilled water to 50 μL.

[0043] PCR reaction procedure: (1) denaturation at 95°C for 5 min, (2) denaturation at 95°C for 30 s, (3) annealing at 59°C for 30 s, (4) extension at 72°C for 4 min 30 s, steps (2) to (4) were performed for a total of 30 cycles, (5) extension at 72°C for 10 min, and (6) insulation at 16°C.

[0044] Store PCR products at 4°C. After verification by agarose gel electrophoresis, digest with the restriction endonuclease DpnI at 37°C for 2-3 hours. Transform the digested product into E. coli BL21(DE3) competent cells and plate onto LB plates containing 50 μg / mL kanamycin. Incubate in a 37°C incubator for approximately 12 hours to obtain the mutant library.

[0045] 4. Screening of single mutants

[0046] (1) Cultivation of positive clones

[0047] A single clone was picked from the mutation library and inoculated into LB liquid culture medium. The culture was incubated in an incubator at 37°C and 180 rpm for 12 hours. The inoculum was transferred to new LB liquid culture medium at a volume concentration of 1%. After incubation at 37°C and 180 rpm for another 2 hours, 0.1 mM isopropylthiogalactoside (IPTG) was added and the culture was continued at 28°C and 180 rpm for 12 hours. The wet cells were collected by centrifugation to obtain the wet cells of amine dehydrogenase, which were used in the study of catalytic synthesis of (R)-3-aminobutanol.

[0048] (2) Synthesis of (R)-3-aminobutanol

[0049] The final concentration of 1mL reaction system is: 4-hydroxy-2-butanone substrate 50mM, 200mM glucose, 2.0mM NAD +, 2 mg / mL GDH lyophilized powder and 100 g / L amine dehydrogenase wet cells in 1 M ammonium chloride buffer (pH 10.0). After incubation at 40°C, 1100 rpm for 24 hours, the reaction solution was boiled for 5-10 minutes and centrifuged. The supernatant was derivatized with FDAA, and the peak area of ​​the product (R)-3-aminobutanol was measured by HPLC. The product concentration and specific enzyme activity were calculated based on a standard curve prepared under the same conditions using a standard sample. The results are shown in Table 2, and recombinant strains with enhanced activity were screened.

[0050] The derivatization reaction was as follows: 400 μL of 6 mM FDAA (solvent: DMSO) was added to 100 μL of the supernatant after centrifugation, and then 25 μL of 1 M NaHCO3 solution was added thereto. After mixing, the mixture was reacted in a metal bath at 40°C and 300 rpm for 1 hour. After the reaction was completed, 50 μL of 2 M HCl was immediately added to terminate the reaction.

[0051] Yield = (product concentration / initial substrate concentration) × 100%

[0052] The enzyme activity is defined as: under the above reaction conditions, the amount of enzyme required to catalyze the conversion of 1 μmol of substrate 4-hydroxy-2-butanone into (R)-3-aminobutanol per hour is one enzyme activity unit, represented by U.

[0053] Specific enzyme activity: the number of enzyme activity units per unit weight of protein, generally expressed as U / g protein.

[0054] Liquid chromatography detection conditions: Agilent 1260 Infinity II, C18 column (Unitary, 5 μm × 250 mm × 4.6 mm), detection wavelength 220 nm, flow rate 1.0 mL / min, injection volume 5 μL, column temperature 40°C.

[0055] Table 2 Enzyme activity of single mutants

[0056]

[0057] Sequencing analysis revealed mutants E23D, F100A, R102Q, N206A, P224T, and D223R with activity units, exhibiting enzyme activities of 62.3, 79.0, 67.9, 63.4, 69.1, and 66.1 U / g for 4-hydroxy-2-butanone, respectively. Individual mutations at positions 92, 93, 277, and 270 did not yield mutants with activity higher than that of the parent. The single mutant F100A exhibited the highest activity and was designated GKGBAmDH. F100A The amino acid sequence is shown in SEQ ID No.3, and the nucleotide sequence of the coding gene is shown in SEQ ID No.4. F100ARecombinant genetic engineering bacteria E. coli BL21 (DE3)-pET28a-GKGBAmDH F100A The following reactions were performed for the dominant strain.

[0058] 5. Double mutation

[0059] The single-site mutations that enhance activity were combined to mutate the phenylalanine at position 100 obtained by saturation mutation to alanine in the plasmid pET28a-GKGBAmDH. F100A As a template, the primers for amino acid mutation at position 206 in Table 1 were used to mutate the asparagine at position 206 of the amino acid sequence to alanine by whole-plasmid PCR. The DNA template was then removed by degradation with DpnI enzyme, and the mutant plasmid was transformed into E. coli BL21 (DE3) to obtain the recombinant genetically engineered bacteria E. coli BL21 (DE3)-pET28a-GKGBAmDH. F100A / N206A The corresponding amine dehydrogenase mutant is a double mutant in which phenylalanine at position 100 is mutated to alanine and asparagine at position 206 is mutated to alanine, and is denoted as GKGBAmDH. F100A / N206A (The amino acid sequence is shown in SEQ ID No. 7, and the nucleotide sequence is shown in SEQ ID No. 8.) Using the single mutant enzyme activity assay method and conditions, the mutant wet cells had a specific enzyme activity of 93.9 U / g in catalyzing the synthesis of (R)-3-aminobutanol from 4-hydroxy-2-butanone.

[0060] In the same way, GKGBAmDH was obtained. F100A / N206T 、GKGBAmDH F100A / N206Q 、GKGBAmDH F100A / N206G , the results are shown in Table 3.

[0061] Table 3 Enzyme activity of double mutants

[0062]

[0063]

[0064] 6. Determination of thermal stability of mutants

[0065] The dominant mutant GKGBAmDH obtained above F100A 、GKGBAmDH N206A GKGBAmDH F100A / N206AWet cells were prepared using the method in step 3 for the original amine dehydrogenase GKGBAmDH and were tested for thermal stability. Specifically, 1 g of wet cells was added to 10 mL of 1 M ammonium chloride buffer (pH 10.0) and incubated at 40°C for 80 hours. The initial enzyme activity was 100%. Samples were then taken every 2 hours and tested using the method in step 4.

[0066] The curve of enzyme activity changing with time is shown in Figure 2 The results showed that the enzyme activity gradually decreased with time, among which GKGBAmDH F100A / N206A The slowest decrease rate, followed by GKGBAmDH N206A 、GKGBAmDH F100A , GKGBAmDH, indicating that the thermal stability of the mutants has been generally improved. F100A 、GKGBAmDH N206A GKGBAmDH F100A / N206A The half-lives are 9.0h, 14.59h, 20.79h and 33.6h respectively.

[0067] Example 2: Preparation of Amine Dehydrogenase Recombinant Genetically Engineered Bacteria Catalyst

[0068] The original strain E. coli BL21 (DE3)-pET28a-GKGBAmDH constructed in Example 1 and the recombinant genetic engineering strain E. coli BL21 (DE3)-pET28a-GKGBAmDH containing the amine dehydrogenase mutant gene were F100A , E.coli BL21(DE3)-pET28a-GKGBAmDH N206A , E.coliBL21(DE3)-pET28a-GKGBAmDH F100A / N206A

[0069] Fermentation was carried out as follows:

[0070] (1) Plate culture: The recombinant genetically engineered bacteria were inoculated into LB solid medium containing 50 μg / mL kanamycin resistance and cultured at 37°C for 12-14 h to obtain slant bacteria on the plate.

[0071] (2) Seed culture: Inoculate the slant bacteria into LB liquid culture medium containing 50 μg / mL kanamycin resistance, and culture at 37°C and 180 rpm for 8-10 h to obtain seed solution.

[0072] (3) Fermentation culture: The seed liquid was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance at an inoculum concentration of 1% by volume, and cultured at 37°C and 180 rpm until the OD 600The value was 0.4, and 0.1 mM isopropylthiogalactoside (IPTG) was added, and the cells were cultured at 28 °C for 12 h. The wet cells were collected by centrifugation to obtain GKGBAmDH and GKGBAmDH. F100A 、GKGBAmDH N206A 、GKGBAmDH F100A / N206A The wet bacteria were stored in a -20℃ refrigerator for later use.

[0073] Example 3: Amine dehydrogenase mutant catalyzes the synthesis of (R)-3-aminobutanol from 4-hydroxy-2-butanone

[0074] The wet bacteria prepared by the method of Example 2 were used as catalyst, 4-hydroxy-2-butanone was used as substrate, glucose was used as cosubstrate, GDH was used as coenzyme, and NAD was used as catalyst. + As a cofactor, the following reaction was carried out using pH 10.0, 1M NH4Cl / NH3·H2O buffer as the reaction medium:

[0075] (1) Reaction system 1: 100 g / L wet bacteria, 50 mM substrate concentration, 200 mM glucose, 2 mg / mL GDH lyophilized powder, 3.0 mM NAD+, 1.0 mL reaction system was prepared with NH4Cl / NH3·H2O buffer (1 M, pH 10.0). The reaction was carried out at 40°C and 1100 rpm for 24 h. The reaction solution was boiled for 15 min and centrifuged. The yield of the product (R)-3-aminobutanol was determined by the method of Example 1. The analysis showed that the yield of wet bacteria GKGBAmDH was 79.3%; the yield of GKGBAmDH was 79.3%. F100A As catalyst, the yield was 83.26% and the ee value was 99.9%. N206A As the catalyst, the yield was 86.55% and the ee value was 99.9%. F100A / N206A As a catalyst, the yield is 96.08% and the ee value is 99.9%.

[0076] (2) Reaction system 2: 100 g / L wet bacteria, 100 mM substrate concentration, 200 mM glucose, 2 mg / mL GDH lyophilized powder, 3.0 mM NAD+, 1.0 mL reaction system was prepared with NH4Cl / NH3·H2O buffer (1 M, pH 10.0). The reaction was carried out at 40°C and 1100 rpm for 24 h. The reaction solution was boiled for 15 min and centrifuged. The yield of the product (R)-3-aminobutanol was determined by the method of Example 1. The results showed that the yield of wet bacteria GKGBAmDH was 75.67% and the ee value was 99.9% with GKGBAmDH as the catalyst. F100AAs catalyst, the yield was 77.42% and the ee value was 99.9%. N206A As catalyst, the yield was 75.67% and the ee value was 99.9%. F100A / N206A As a catalyst, the yield is 93.22% and the ee value is 99.9%.

[0077] (3) Reaction system 3: 100 g / L wet cells, 200 mM substrate concentration, 200 mM glucose, 2 mg / mL GDH lyophilized powder, 3.0 mM NAD+, 1.0 mL reaction system was prepared with NH4Cl / NH3·H2O buffer (0.1 M, pH 10.0). The reaction was carried out at 40°C and 1100 rpm for 24 h. The reaction solution was boiled for 15 min and centrifuged. The yield of the product (R)-3-aminobutanol was determined by the method of Example 1. HPLC analysis showed: Figure 3 As shown in a, the analysis showed that the yield was 69.67% and the ee value was 99.9% when wet bacteria GKGBAmDH was used as catalyst; F100A As catalyst, the yield was 77.78% and the ee value was 99.9%. N206A As catalyst, the yield was 71.73% and the ee value was 99.9%. F100A / N206A As a catalyst, the yield is 89.04% and the ee value is 99.9%.

[0078] The present invention is not limited to the above description. The present invention can be modified in various ways within the scope of the claims, and these modifications are all within the scope of the present invention.

Claims

1. An amine dehydrogenase mutant with improved enzyme activity and thermal stability, characterized in that: The amine dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID No. 1 is mutated into one of the following: (1) the phenylalanine at position 100 is mutated into alanine; (2) the asparagine at position 206 is mutated into alanine; (3) the phenylalanine at position 100 is mutated into alanine, and the asparagine at position 206 is mutated into alanine.

2. A recombinant genetically engineered bacterium containing a gene encoding the amine dehydrogenase mutant according to claim 1.

3. Use of the amine dehydrogenase mutant according to claim 1 in catalyzing the synthesis of (R)-3-aminobutanol from 4-hydroxy-2-butanone.

4. The use according to claim 3, characterized in that The application method is: using the wet bacteria obtained by fermentation culture of the recombinant genetic engineering bacteria containing the amine dehydrogenase mutant encoding gene of claim 1 as a catalyst, glucose dehydrogenase as a coenzyme, 4-hydroxy-2-butanone as a substrate, glucose as a cosubstrate, and NAD + The invention relates to a method for preparing (R)-3-aminobutanol by using a cofactor and a buffer solution with a pH of 8.5 to 11.0 as a reaction medium to form a reaction system. The reaction is carried out at a temperature of 20 to 45° C. and a rotation speed of 1000 to 1500 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain (R)-3-aminobutanol.

5. The use according to claim 4, characterized in that In the reaction system, the catalyst dosage is 10-100 g / L based on the weight of wet cells, the final substrate concentration is 50-300 mM, the final cofactor concentration is 1-3 mM, the final glucose concentration is 100-300 mM, and the final glucose dehydrogenase concentration is 1-5 g / L.

6. The use according to claim 4, characterized in that The glucose dehydrogenase was added in the form of freeze-dried enzyme powder, and the specific enzyme activity was 56.12 U / mg.

7. The use according to claim 4, characterized in that The reaction medium is a 0.1M NH4Cl / NH3·H2O buffer solution at pH 10.

0.

8. The use according to claim 4, characterized in that The catalyst is prepared according to the following method: a recombinant genetically engineered bacterium containing the gene encoding the amine dehydrogenase mutant according to claim 1 is inoculated into an LB solid culture medium containing 50 μg / mL kanamycin, and cultured on a shaker at 37° C. and 180 rpm. When the OD600 of the culture solution reaches 0.6 to 0.8, isopropyl-β-D-thiogalactoside with a final concentration of 0.1 to 0.5 mmol / L is added as an inducer. After induction at 28° C. for 12 hours, the culture solution is centrifuged and the precipitate is collected to obtain the catalyst.

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