Amine dehydrogenase mutant, engineering bacterium and application in synthesis of (R)-3-aminobutanol

Abstract

The invention discloses an amine dehydrogenase mutant, an engineering bacterium and application of the amine dehydrogenase mutant in synthesis of (R)-3-aminobutanol. The mutant is obtained by performing single mutation or multiple mutation on 100th and 206th amino acids of an amino acid sequence as shown in SEQ ID No.1. The invention further discloses an application of the amine dehydrogenase mutant in synthesis of (R)-3-aminobutanol. The enzyme activity of the amine dehydrogenase mutant provided by the invention is remarkably improved from 55.3 U / g before mutation to 93.9 U / g, the thermal stability is also remarkably improved, the half-life period is prolonged from 9.0 h before mutation to 33.6 h, and the defects that existing amine dehydrogenase is relatively low in activity and low in thermal stability are effectively overcome.

Description

(I) Technical field

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

[0002] (R)-3-Aminobutanol is an important pharmaceutical intermediate with important 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 synthesized into penicillin antibiotics. It has wide application value in the pharmaceutical field.

[0003] At present, the synthesis of (R)-3-aminobutanol is mainly achieved by chemical methods. Chemical synthesis methods mainly include chiral raw material synthesis method, chemical induction method, chiral separation method, preparative chromatography, etc. These chemical synthesis methods have the disadvantages of complex procedures, serious pollution, harsh reaction conditions, and low regional / enantioselectivity.

[0004] Compared with chemical methods, bioenzymatic catalytic synthesis has the advantages of stereoselectivity, high catalytic efficiency, mild catalytic conditions and high atomic 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 proess reswarch & development. 2019; 23: 1262-1268), 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, and the limitations of enzyme affinity, coenzyme and the generation of reaction by-products have severely restricted the application of transaminases. Lyases are also widely used in the preparation of chiral amines, such as the synthesis of L-aspartic acid and alanine. However, lyases cannot mediate the formation of CN bonds in certain reactions, such as the synthesis of L-phenylalanine (Angewandte Chemie (Internationled.). 2004; 43: 788-824). In addition, the optimal pH of lyases is too different from that of cascade enzymes, which seriously limits their wide application. Imine reductases have low activity, unclear catalytic mechanisms, cannot be effectively semi-rationally designed, and have a small substrate range, making them difficult to apply to industrial production (Angewandte Chemie (Internationaled.). 2012; 51: 2969-3972).

[0005] Compared with other biocatalytic methods, (R)-selective amine dehydrogenase can use 4-hydroxy-2-butanone as substrate, inorganic ammonia (such as ammonium chloride) as amino donor, coupled with glucose dehydrogenase (GDH) cofactor regeneration system for hydrogen supply, and synthesize (R)-3-aminobutanol in one pot reaction, only producing H2O and gluconic acid as by-products. The products are easy to separate, meeting the requirements of green and sustainable production. It shows good application prospects in the synthesis of various chiral amines and is expected to be applied in industrial production. However, the enzyme activity of current amine dehydrogenases is generally low, and the thermal stability is poor, which cannot meet the actual application needs.

[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. (III) 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 activity and thermal stability of amine dehydrogenase.

[0008] The technical solution adopted by 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 100th and 206th amino acids 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 mutation or multiple mutation.

[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 to one of the following: (1) the phenylalanine at position 100 is mutated to alanine, 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 are not involved 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 connecting the nucleic acid encoding the amine dehydrogenase mutant gene of the present invention to various suitable vectors by conventional methods in the art. The vector can be various conventional vectors in the art, such as commercially available plasmids, cosmids, phages or viral vectors, etc., as long as the recombinant expression vector can be normally replicated in the corresponding expression host and express the amine dehydrogenase mutant. The amine dehydrogenase mutant gene can be operably connected to the downstream of a suitable regulatory sequence in the vector to achieve constitutive or inducible expression of the amine dehydrogenase mutant. The vector is preferably a plasmid, more preferably plasmid pET-28a. The host cell is various conventional host cells in the art, as long as the recombinant expression vector can stably replicate itself and can effectively express the target protein after being induced by an inducer. In the present invention, Escherichia coli is preferably used as the host cell, and Escherichia coli BL21 (DE3) is more preferably used for efficiently expressing the amine dehydrogenase mutant of the present invention. The culture of the recombinant expression transformant is a conventional method and culture condition 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 genetic engineering 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 cofactor, a reaction system is formed with a buffer solution of pH 8.5 to 11.0 (preferably pH 10.0, 0.1M NH4Cl / NH3·H2O buffer solution) 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 amount of catalyst is 10-100 g / L (preferably 50 g-100 g / L, most preferably 50 g / L) based on the weight of wet cells, the final concentration of substrate is 50-300 mM (preferably 50-200 mM, more preferably 50 mM), the final concentration of cofactor is 1-3 mM (preferably 3 mM), the final concentration of glucose is 100-300 mM (preferably 200 mM), and the final concentration of glucose dehydrogenase is 1-5 g / L (preferably 2 g / L). The glucose dehydrogenase is added in the form of freeze-dried enzyme powder, and the specific enzyme activity is 56.12 U / mg.

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

[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 kanamycin, and cultured at 37° C. and 180 rpm on a shaker; when the OD600 of the culture solution reaches 0.6 to 0.8, isopropyl-β-D-thiogalactoside (IPTG) 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.

[0018] LB medium composition: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, solvent is water, pH is natural. LB solid medium is to add 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, which effectively solves 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. After the substrate and enzyme are added, the reaction begins, and the final product (R)-3-aminobutanol is directly obtained, which has low industrial cost. At the same time, the initial raw material is 4-hydroxy-2-butanone, and the amino donor is inorganic ammonia, which is cheap; the catalyst can be prepared in large quantities by constructing genetically engineered Escherichia coli bacteria and then fermenting, which is relatively easy to obtain and cheap.

[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 after 24 h of reaction is 89.04%, and the ee value is 99.9%, which provides a basis for the efficient preparation of (R)-3-aminobutanol. (IV) Description of the 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 in conjunction with 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 powder 5g / L, NaCl 10g / L, solvent is water, pH is natural. LB solid medium is to add 20g / L agar powder to the above LB liquid medium.

[0029] The specific enzyme activity of glucose dehydrogenase (GDH) lyophilized powder in the embodiment is 56.12 U / mg, wherein the enzyme activity is defined as: the amount of lyophilized enzyme powder required to convert 1 mmol of NADH per unit time, i.e., within 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 engineering 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, and then transferred 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 about 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 as shown in SEQ ID No. 1) was modeled using the AlphaFold2 (AlphaFold2 TIB Server (biodesign.ac.cn)) website, and predictions were made using 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 mutation 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 is 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, 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 repeated for 30 cycles, (5) extension at 72°C for 10 min, and (6) insulation at 16°C.

[0044] The PCR product was stored at 4°C. After the PCR product was verified by agarose gel electrophoresis analysis, the restriction endonuclease DpnI was added and digested at 37°C for 2-3 hours. The digestion product was transferred into E. coli BL21 (DE3) competent cells and spread on LB plates containing 50 μg / mL kanamycin resistance, and placed in a 37°C incubator for about 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, inoculated into LB liquid culture medium, cultured in an incubator at 37°C and 180 rpm for 12 h, transferred to a new LB liquid culture medium with a volume concentration of 1%, cultured at 37°C and 180 rpm for another 2 h, added with 0.1 mM isopropylthiogalactoside (IPTG), cultured at 28°C and 180 rpm for another 12 h, centrifuged, and the wet cells were collected, namely, amine dehydrogenase wet cells, 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 +, 2mg / mL GDH lyophilized powder and 100g / L amine dehydrogenase wet bacteria, 1M ammonium chloride buffer (pH10.0). After reacting at 40℃, 1100rpm for 24h, the reaction solution was boiled for 5-10min and centrifuged. After the supernatant was derivatized with FDAA, the peak area of ​​the product (R)-3-aminobutanol was determined by high performance liquid chromatography. The product concentration was calculated based on the standard curve prepared under the same conditions by the standard product, and then the specific enzyme activity was calculated. The results are shown in Table 2, and the recombinant bacteria with improved activity were screened.

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

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

[0052] The definition of enzyme activity is: 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 contained in a unit weight of protein, generally expressed as U / g protein.

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

[0055] Table 2 Enzyme activity of single mutants

[0056]

[0057] After sequencing analysis, mutants E23D, F100A, R102Q, N206A, P224T and D223R with active units were screened and obtained, showing enzyme activities of 62.3, 79.0, 67.9, 63.4, 69.1 and 66.1 U / g for 4-hydroxy-2-butanone. Single mutations at sites 92, 93, 277 and 270 did not produce mutants with higher activity than the parent. The single mutant F100A had the highest activity and was recorded 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. F100ARecombinant genetic engineering bacteria E. coli BL21 (DE3)-pET28a-GKGBAmDH F100A Perform the following reactions 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, and then the DNA template was removed by DpnI enzyme degradation, and the mutant plasmid was transformed into E. coli BL21 (DE3) to obtain the recombinant genetic engineering 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, 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.) The single mutant enzyme activity detection method and conditions were used for detection. The specific enzyme activity of the mutant wet bacteria in catalyzing the synthesis of (R)-3-aminobutanol from 4-hydroxy-2-butanone was 93.9 U / g.

[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 superior mutant GKGBAmDH F100A 、GKGBAmDH N206A GKGBAm F100A / N206AThe wet cells of the original amine dehydrogenase GKGBAmDH were prepared by the method in step 3 to determine their thermal stability. The specific method is: 1g of wet cells was added to 10mL of 1M ammonium chloride aqueous buffer (pH10.0), and kept at 40℃ for 80h. The initial enzyme activity was 100%, and then samples were taken every 2h, and the enzyme activity was tested by 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 GKGBAm F100A / N206A The half-lives are 9.0h, 14.59h, 20.79h and 33.6h respectively.

[0067] Example 2: Preparation of recombinant genetically engineered amine dehydrogenase 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 above-mentioned 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: The slant bacteria were inoculated into LB liquid culture medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C and 180 rpm for 8-10 h to obtain seed solution.

[0072] (3) Fermentation culture: The seed solution was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance at a volume concentration of 1%, and cultured at 37°C and 180 rpm until the OD 600The value was 0.4, and a final concentration of 0.1 mM isopropylthiogalactoside (IPTG) was added, and the cells were cultured at 28 °C for 12 h. After centrifugation, the wet cells were collected and GKGBAmDH and GKGBAmDH were obtained. 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 + As a cofactor, the following reaction was carried out with 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), and 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 detected by the method of Example 1. The analysis showed that the yield of wet bacteria GKGBAmDH was 79.3%; the yield of GKGBAmDH was 1.0%; the yield of (R)-3-aminobutanol ... F100A As catalyst, the yield reached 83.26% and the ee value reached 99.9%. N206A As catalyst, the yield reached 86.55% and the ee value reached 99.9%. F100A / N206A As a catalyst, the yield is 96.08% and the ee value is 99.9%.

[0076] (2) Reaction system 2: The amount of wet bacteria was 100 g / L, the substrate concentration was 100 mM, 200 mM glucose, 2 mg / mL GDH lyophilized powder, 3.0 mM NAD+, and 1.0 mL of the reaction system was prepared with NH4Cl / NH3·H2O buffer (1 M, pH 10.0). After reacting 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 detected by the method of Example 1. The analysis showed that the yield was 75.67% and the ee value was 99.9% when the wet bacteria GKGBAmDH was used 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 bacteria, 200 mM substrate concentration, 200 mM glucose, 2 mg / mL GDH lyophilized powder, 3.0 mM NAD+, 1.0 mL reaction system with NH4Cl / NH3·H2O buffer (0.1 M, pH 10.0), 40°C, 1100 rpm for 24 h, the reaction solution was boiled for 15 min, centrifuged, and the yield of the product (R)-3-aminobutanol was detected by the method of Example 1. HPLC analysis showed: Figure 3 As shown in a, the analysis shows that the yield is 69.67% and the ee value is 99.9% when the wet bacteria GKGBAmDH is used as the catalyst; F100A As catalyst, the yield reached 77.78% and the ee value reached 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 by the above textual description. The present invention can make various changes within the scope of the claims, and these changes 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 mutant is obtained by single mutation or multiple mutation of the 100th and 206th amino acids of the amino acid sequence shown in SEQ ID No.

1.

2. The amine dehydrogenase mutant according to claim 1, characterized in that The amine dehydrogenase mutant is obtained by mutating the amino acid sequence shown in SEQ ID No. 1 to one of the following: (1) the phenylalanine at position 100 is mutated to alanine; (2) the asparagine at position 206 is mutated to alanine; (3) the phenylalanine at position 100 is mutated to alanine, and the asparagine at position 206 is mutated to alanine.

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

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

5. The use according to claim 4, characterized in that The application method comprises: using wet bacteria obtained by fermentation culture of recombinant genetic engineering bacteria containing an amine dehydrogenase mutant encoding gene as a catalyst, glucose dehydrogenase as a coenzyme, 4-hydroxy-2-butanone as a substrate, glucose as a cosubstrate, and NAD + The reaction system is formed by using a buffer solution with a pH of 8.5 to 11.0 as a cofactor and a reaction medium 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.

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

7. The use according to claim 5, 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.

8. The use according to claim 5, characterized in that The reaction medium is a NH4Cl / NH3·H2O buffer solution with a pH of 10.0 and a 0.1M concentration.

9. The use according to claim 5, characterized in that The catalyst is prepared according to the following method: a recombinant genetic engineering bacterium containing a gene encoding a mutant of amine dehydrogenase is inoculated into an LB solid culture medium containing 50 μg / mL kanamycin, and cultured at 37° C. and 180 rpm on a shaker; 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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