Amine dehydrogenase, gene, recombinant bacteria, crude amine dehydrogenase enzyme solution, dehydrogenase composition, crude dehydrogenase enzyme solution composition and method for preparing (R)-3-aminobutanol

Abstract

The present invention belongs to the field of enzyme catalysis technology, and is specifically related to a kind of amine dehydrogenase, gene, recombinant bacteria, amine dehydrogenase crude enzyme liquid, dehydrogenase composition, dehydrogenase crude enzyme liquid composition and preparation method of (R) 3-aminobutanol. The present invention constructs an amine dehydrogenase with high selectivity and high activity, and the amine dehydrogenase is combined with formate dehydrogenase as a catalyst to provide a method for preparing chiral (R) 3-aminobutanol. The inventive method does not require a large amount of cofactors, improves the conversion rate and product yield of raw materials, and the chiral product prepared at the same time has high selectivity and ee value greater than 99.9%. In addition, the inventive method simplifies downstream separation and refining process, is a new process of simple process and green environmental protection, and has the potential for industrialization and good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme catalysis, and specifically relates to an amine dehydrogenase, a gene, a recombinant bacterium, a crude amine dehydrogenase enzyme solution, a dehydrogenase composition, a crude dehydrogenase enzyme solution composition and a method for preparing (R)-3-aminobutanol. Background Art

[0002] Dolutegravir is a human immunodeficiency virus type 1 (HIV-1) integrase strand transfer inhibitor (INSTI) used in combination with other antiretroviral drugs to treat HIV-1 infection. Preclinical study results showed that it has low toxicity, no genotoxicity and carcinogenicity, and no obvious reproductive toxicity and teratogenicity at 27 times the clinical dose. The drug was approved by the U.S. Food and Drug Administration in 2013 and approved by the European Commission for use throughout the European Union in 2014.

[0003] (R)-3-aminobutanol is the core intermediate for the preparation of dolutegravir, with a market demand of more than 500 tons. In recent years, many strategies for synthesizing high-purity (R)-3-aminobutanol have been developed through chemical or biocatalytic methods. Chemical methods, such as kinetic resolution and reduction of chiral sources, require expensive transition metal catalysts, complex protection and deprotection conditions, and high-pressure equipment. Compared with traditional chemical methods, biocatalysis has mild reaction conditions, reduces environmental impact, and is a more economical and environmentally friendly synthesis technology. In addition, it has strong enantioselectivity. Transaminase catalyzes the asymmetric reductive amination of 4-hydroxy-2-butanone, which is considered to be an effective method due to its high stereoselectivity and high atom efficiency. However, transaminase has some disadvantages, such as transaminase requires the coenzyme pyridoxal phosphate to participate in the transamination reaction, and this coenzyme is relatively expensive; the substrate binding region of transaminase has a special spatial structure, and there are many deficiencies in substrate specificity, stability, catalytic efficiency, etc. At present, transaminase that meets the needs of industrial applications is still relatively limited. To date, only a few studies have reported (R)-selective aminotransferases that can work with 4-hydroxy-2-butanone. This is mainly due to the resource limitations of (R)-selective aminotransferases and the fact that the substrate is not easily recognized by the enzyme. Therefore, discovering new biocatalysts that can catalyze this reaction and improving the enzyme activity through enzyme engineering remains a daunting challenge.

[0004] Recently, amine dehydrogenases (AmDHs) have been used as a promising class of biocatalysts for the synthesis of high optical purity chiral amines. AmDHs catalyze asymmetric reductive amination reactions, using cheap ammonia as an amino donor and NAD(P)H as a reducing agent to aminate prochiral ketones. Compared with other biocatalytic methods, the method of synthesizing chiral amines by asymmetric reductive amination of ketones with ammonia catalyzed by amine dehydrogenases not only uses cheap inorganic ammonia as an amino donor, but also produces water as a byproduct. Therefore, it has the advantages of high atom economy, high product optical purity, mild reaction conditions, environmental friendliness, and simple product post-processing, and shows good application prospects in the synthesis of various chiral amines. However, amine dehydrogenases, as a redox enzyme, mostly rely on the coenzyme NADPH or NADH for catalytic reactions, but the coenzyme will be consumed as the product is generated, and the high cost of NAD(P)H hinders its large-scale production.

[0005] The Chinese patent application with patent publication number CN117844772A discloses an amine dehydrogenase mutant, an engineered bacterium and the use thereof in the synthesis of (R)-3-aminobutanol. However, when the amine dehydrogenase mutant and the engineered bacterium are used in the reaction of synthesizing (R)-3-aminobutanol, the following problems exist: (1) the reaction belongs to a single enzyme reaction system and consumes a large amount of high-cost cofactor NAD(P)H, which is not conducive to large-scale production; (2) the yield of the product under the optimal system of the method is only 75.4%, which needs to be further improved.

[0006] In a word, the chemical method for synthesizing (R)-3-aminobutanol at present has the disadvantages of long steps, high cost, low yield and difficult operation of reaction conditions. And the method for synthesizing (R)-3-aminobutanol by enzyme catalysis at present needs to use high-cost catalyst enzyme, coenzyme, cosolvent buffer etc., and subsequent treatment is complicated, such as needing to process under low temperature conditions and use more reagents, which is not suitable for large-scale production. Therefore, for (R)-3-aminobutanol synthetic method, how to design new synthetic route, and select appropriate enzyme catalyst and synthesis conditions, this is a problem that needs to be solved in this area. Summary of the invention

[0007] In view of the problems of the prior art, the object of the present invention is to provide an amine dehydrogenase and an efficient preparation method of (R)-3-aminobutanol.

[0008] The present invention provides an amine dehydrogenase, whose amino acid sequence is shown in SEQ ID NO. 2.

[0009] The present invention also provides a gene, whose nucleotide sequence is shown in SEQ ID NO. 1.

[0010] The present invention also provides a recombinant bacterium, which comprises a gene with a nucleotide sequence as shown in SEQ ID NO. 1.

[0011] Furthermore, the recombinant bacteria is recombinant Escherichia coli.

[0012] The present invention also provides an amine dehydrogenase, whose amino acid sequence is shown in SEQ ID NO. 4.

[0013] The present invention also provides a gene, whose nucleotide sequence is shown in SEQ ID NO. 3.

[0014] The present invention also provides a recombinant bacterium, which comprises a gene with a nucleotide sequence as shown in SEQ ID NO. 3.

[0015] The present invention also provides a dehydrogenase composition, which is composed of the above-mentioned amine dehydrogenase and formate dehydrogenase. The amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO. 4.

[0016] Furthermore, the mass ratio of the amine dehydrogenase to the formate dehydrogenase is 1:(1-2).

[0017] Furthermore, the preparation method of the amine dehydrogenase or formate dehydrogenase comprises the following steps:

[0018] Step (1), culturing the recombinant bacteria containing the gene shown in SEQ ID NO. 1 or SEQ ID NO. 3 to a cell density OD 600 The value reaches 0.7-1.0;

[0019] Step (2), add isopropyl-β-D-thiogalactoside to a concentration of 0.8~1 mM and continue culturing until OD 600 The value reaches 7-9;

[0020] Step (3), collecting the bacterial cells by centrifugation, adding a buffer to resuspend the bacterial cells, ultrasonically disrupting the bacterial suspension, and centrifuging. The supernatant is a crude enzyme solution containing amine dehydrogenase or formate dehydrogenase.

[0021] The present invention also provides a crude amine dehydrogenase enzyme solution, and its preparation method comprises the following steps:

[0022] Step (1), culturing the recombinant bacteria containing the gene shown in SEQ ID NO. 1 to a cell density OD 600 The value reaches 0.7-1.0;

[0023] Step (2), add isopropyl-β-D-thiogalactoside to a concentration of 0.8~1 mM and continue culturing until OD 600 The value reaches 7-9;

[0024] Step (3), collecting the bacterial cells by centrifugation, adding buffer to resuspend, ultrasonically disrupting, centrifuging, and the supernatant is the crude amine dehydrogenase enzyme solution.

[0025] The present invention also provides a crude formate dehydrogenase enzyme solution, and its preparation method comprises the following steps:

[0026] Step (1), culturing the recombinant bacteria containing the gene shown in SEQ ID NO. 3 to a cell density OD 600 The value reaches 0.7-1.0;

[0027] Step (2), add isopropyl-β-D-thiogalactoside to a concentration of 0.8~1 mM and continue culturing until OD 600 The value reaches 7-9;

[0028] Step (3), collecting the bacterial cells by centrifugation, adding buffer to resuspend, ultrasonically disrupting, centrifuging, and the supernatant is the crude formate dehydrogenase enzyme solution.

[0029] Furthermore, in step (1), the culture medium is LB liquid culture medium, and the culture temperature is 20-37°C; in step (2), isopropyl-β-D-thiogalactoside is added to a concentration of 0.1-1 mM; in step (2), the culture temperature is 20-28°C; in step (3), the buffer is phosphate buffer or Tris-HCl buffer, and the pH value of the buffer is 7.5.

[0030] The present invention also provides a dehydrogenase crude enzyme liquid composition, characterized in that it is composed of the above-mentioned amine dehydrogenase crude enzyme liquid and the above-mentioned formate dehydrogenase crude enzyme liquid.

[0031] Furthermore, in the crude dehydrogenase enzyme liquid composition, the mass ratio of amine dehydrogenase to formate dehydrogenase is 1:(1-2).

[0032] The present invention also provides a gene composition, which consists of a gene with a nucleotide sequence as shown in SEQ ID NO. 1 and a gene with a nucleotide sequence as shown in SEQ ID NO. 3.

[0033] The present invention also provides a recombinant bacterial composition, which consists of recombinant bacteria 1 and recombinant bacteria 2, wherein recombinant bacteria 1 comprises a gene whose nucleotide sequence is shown in SEQ ID NO. 1, and recombinant bacteria 2 comprises a gene whose nucleotide sequence is shown in SEQ ID NO. 3.

[0034] The present invention also provides a method for preparing (R)-3-aminobutanol, comprising the following steps: adding 4-hydroxy-2-butanone and the above-mentioned dehydrogenase composition or the above-mentioned dehydrogenase composition crude enzyme solution composition into a buffer solution containing a cofactor for reaction to obtain (R)-3-aminobutanol.

[0035] Furthermore, the buffer solution is an ammonium formate buffer solution containing a cofactor, and the cofactor is NAD + , the concentration of the cofactor in the buffer solution is 0.5-2 mM; the pH value of the reaction is 8-9, the reaction temperature is 30-50°C, and the reaction time is 15-30 h; the mass ratio of the 4-hydroxy-2-butanone to the amine dehydrogenase is (0.5-2.0):1, and the feed mass volume ratio of the 4-hydroxy-2-butanone to the buffer solution is 1 g:20-100 mL.

[0036] Furthermore, the concentration of the cofactor in the buffer solution is 1 mM; the pH value of the reaction is 8.5, the reaction temperature is 40°C, and the reaction time is 24 h; the mass ratio of the 4-hydroxy-2-butanone to the amine dehydrogenase is 1.2:1, and the feed mass volume ratio of the 4-hydroxy-2-butanone to the buffer solution is 1 g:75 mL.

[0037] Furthermore, after the reaction is completed, the following purification step is further included: adding acid to adjust the pH to 2.5-3.5, extracting with dichloromethane, collecting the aqueous phase, adding alkali to adjust the pH to 7.5-8.5, extracting with dichloromethane, and collecting the organic phase to obtain (R)-3-aminobutanol.

[0038] The present invention constructs an amine dehydrogenase with high selectivity and high activity, and the amine dehydrogenase is combined with formate dehydrogenase as a catalyst to construct an efficient and regenerative circulation system, providing a preparation method of an important pharmaceutical intermediate chiral (R)-3-aminobutanol. The present invention adds formate dehydrogenase to the reaction system to recover the cofactor at the expense of a cheap substrate, thereby constructing an efficient and low-cost cofactor regeneration system.

[0039] The dual enzyme system of the present invention can catalyze the preparation of (R)-3-aminobutanol with high selectivity, high stability and high conversion rate. The molar yield of the product obtained by the preparation method of the present invention is as high as 85%, and the ee value is as high as 99.9% or more, which is significantly higher than the yield of the product obtained under the optimal system of the prior art CN117844772A method (75.4%).

[0040] The cofactor NAD used in the preparation method of the present invention + The cost is significantly lower than the coenzyme NADPH or NADH used in the prior art, thus reducing the cost.

[0041] In addition, the present invention uses 4-hydroxy-2-butanone as a raw material, and obtains (R)-3-aminobutanol through one-step enzyme catalysis. Only a buffer solution is needed in the process, and no cosolvent and other reagents are needed, which greatly saves costs. The preparation method of the present invention simplifies the downstream separation and refining process, and is a new process with simple process, green environmental protection and reduced cost.

[0042] In summary, the present invention has industrialization potential and good application prospects.

[0043] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0044] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Picture 1 .Synthetic route for the enzyme-catalyzed preparation of (R)-3-aminobutanol.

[0046] Picture 2 .NMR spectrum of (R)-3-aminobutanol. DETAILED DESCRIPTION

[0047] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0048] Example 1 Preparation of amine dehydrogenase and formate dehydrogenase

[0049] This embodiment provides a novel amine dehydrogenase and a novel formate dehydrogenase, wherein:

[0050] Amine dehydrogenase nucleotide sequence (SEQ ID NO. 1):

[0051]

[0052] Amino acid sequence of amine dehydrogenase (SEQ ID NO. 2):

[0053] MKVLVLGAGLMGKEAARDLVQCQDVEAVTLADVDLAKAEQTVRQLHSKKLAAVRVDCGDPQQLAAAMKGHDVVVNALFYQFNETVAKTAIETGVHSVDLGGHIGHITDRVLELHERAQAAGVTIIPDLGVAPGMINILSGYGASQLDEVESILLYVGGIPVRPEHPLEYNHVASLEGLLDHYTDPALIIRNGQKQEVPSLSEVEPIYFDRFGPLEAFHTSGETSTLSRSFPNLKRLEYKTIRYRGHAEKCKLLVDLTLTRHDVEVEINGCRVKPRDVLLSVLKPLLDLKGKDDVVLLRVIVGGNKDGKETVLEYETVTFNDRDNKVTAMARTTAYTISAVAQLIGRGVATKRGVYPPEQIVPGDVYMDEMKKRGVLISEKRTVHS;

[0054] Nucleotide sequence of formate dehydrogenase (SEQ ID NO. 3):

[0055]

[0056] Formate dehydrogenase amino acid sequence (SEQ ID NO. 4):

[0057] MASRGSHHHHHGAMKIVLVLYDAGKFAADEEKLYGATENKLGIANWLKDQGHELITTSDKEGETSELDKHIPDADIIITTPFHPAYITKERLDKAKNLKLVVVAGVGSDHIDLDYINQTGKKISVLEVTGLNVVSVAEHVVMTMLVLVRNFVPAHEQIINHDWEVAAIAKDAYDIEGKTIATIGAGRI GYRVLERLLPFNPKELLYYSFRALPKEAEEKVGCRRVENIEELVAQADIVTVNAPLHAGTKGLINKELLSKFKKGAWLVNTARGAICVAEDVAAALESGQLRGYGGDVWSPQPAPKDHPWRDMRNKYTAGNAMTPHYSGTTLDAQTRYAEGTKNILESFFTGSFDYRPQDIILLNGEYVTKAYGKHDKK.

[0058] The crude enzyme solution containing the above-mentioned amine dehydrogenase and formate dehydrogenase is prepared according to the following method:

[0059] (1) Construction of strains expressing amine dehydrogenase and formate dehydrogenase:

[0060] The nucleotide sequence shown in SEQ ID NO. 1 was synthesized according to the existing method, and constructed on the plasmid vector pET-28a (+) by sticky end ligation using T4 DNA ligase, with restriction sites EcoRI / BamHI and HindIII; then the constructed plasmid was introduced into the expression host E. coli BL21(DE3) strain, thus obtaining the amine dehydrogenase gene engineering bacteria E. coli BL21(DE3) / pET-28a(+)-AmDH.

[0061] The nucleotide sequence shown in SEQ ID NO. 3 was synthesized according to the existing method, and constructed on the plasmid vector pET-28a (+) by sticky end ligation using T4 DNA ligase, with restriction sites EcoRI / BamHI and HindIII; then the constructed plasmid was introduced into the expression host E. coli BL21(DE3) strain, and the formate dehydrogenase gene engineering bacteria were obtained. E. coli BL21(DE3) / pET-28a(+)-FDH.

[0062] (2) Production of amine dehydrogenase and formate dehydrogenase - shake flask procedure:

[0063] The above successfully constructed amine dehydrogenase genetic engineering bacteria E. coli BL21(DE3) / pET-28a(+)-AmDH was inoculated into LB liquid medium and cultured in a shaker at 37°C and 200 rpm for 4-5 h. 600 When the value reached 0.8, the temperature was lowered to 25 °C, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM. The flask was then transferred to a 28 °C shaker at 200 rpm for 16 h until the OD reached 0.8. 600 Reach 8. After the cultivation is completed, the obtained culture solution is centrifuged at 8000 rpm for 5 min, the supernatant is discarded, the bacterial cells are collected, and then resuspended with 100 mM, pH = 7.5 phosphate buffer, the resuspension ratio is 1 g:5 mL according to the mass volume ratio of bacteria and phosphate buffer, the bacterial suspension is ultrasonically disrupted, and the supernatant obtained after centrifugation is the crude enzyme solution of amine dehydrogenase.

[0064] According to the above method, the only difference is that the amine dehydrogenase gene engineering bacteria E. coli BL21(DE3) / pET-28a(+)-AmDH was replaced with formate dehydrogenase genetic engineering bacteria BL21(DE3) / pET-28a(+)-FDH to obtain a crude formate dehydrogenase enzyme solution.

[0065] (3) Production of amine dehydrogenase and formate dehydrogenase - expanded fermentation process:

[0066] The above successfully constructed amine dehydrogenase genetic engineering bacteria E. coli BL21(DE3) / pET-28a(+)-AmDH was inoculated into LB liquid medium and cultured in a shaker at 28°C and 200 rpm for 12-16 h. 600The value reaches 4-8 in preparation for tank fermentation. Use 6.0 L of growth medium in an aerated, stirred 15 L fermentor (the growth medium consists of 0.88 g / L ammonium sulfate, 0.98 g / L sodium citrate; 12.5 g / L potassium phosphate dibasic trihydrate, 6.25 g / L potassium phosphate monobasic, 3.3 g / L Tastone-154 yeast extract, 0.083 g / L ammonium ferric citrate, and 8.3 mL / L of trace element solution; the trace element solution contains 2 g / L calcium chloride dihydrate, 2.2 g / L zinc sulfate heptahydrate, 0.5 g / L manganese sulfate monohydrate, 1 g / L cuprous sulfate heptahydrate, 0.1 g / L ammonium molybdate tetrahydrate, and 0.02 g / L sodium tetraborate). Sterilize the container at 121 °C and 15 PSI for 30 minutes, and add 100 μM pyridoxine after sterilization. The fermentor is inoculated with OD 600 Values ​​of 4-8 E. coli Late seed culture, this culture contains a plasmid encoding the desired amine dehydrogenase gene. The fermenter is stirred at 250-1250 rpm and air is supplied to the fermentation vessel at a rate of 0.6-25 L / min to maintain a dissolved oxygen level of 50% saturation or higher. The pH of the culture is maintained at 0.2 by adding 7% v / v ammonium hydroxide. The growth of the culture is maintained by adding a feed solution containing 500 g / L glucose, 12 g / L ammonium chloride, and 5.1 g / L magnesium sulfate heptahydrate. The culture is grown until the bacterial concentration reaches OD 600 After 20, IPTG was added to a final concentration of 1 mM to induce the expression of amine dehydrogenase, and the fermentation was continued for 18 hours. The culture was then cooled to 4 °C and kept at this temperature until harvesting. The obtained culture solution was centrifuged at 6000 rpm for 5 min to collect the bacterial cells, and resuspended in 100 mM, pH = 7.5 phosphate buffer at a mass volume ratio of 1g:5 mL for the bacterial cells and phosphate buffer. The bacterial suspension was ultrasonically disrupted, and the supernatant obtained after centrifugation was the crude enzyme solution of amine dehydrogenase.

[0067] According to the above method, the only difference is that the amine dehydrogenase gene engineering bacteria E. coli BL21(DE3) / pET-28a(+)-AmDH replaced with formate dehydrogenase gene engineering bacteria E. coli BL21(DE3) / pET-28a(+)-FDH to obtain crude formate dehydrogenase enzyme solution.

[0068] The amino acid sequence of the recombinant protein in the crude amine dehydrogenase solution is shown in SEQ ID NO. 2, and the amino acid sequence of the recombinant protein in the crude formate dehydrogenase solution is shown in SEQ ID NO. 4.

[0069] Example 2 Preparation of amine dehydrogenase and formate dehydrogenase

[0070] The method of Example 1 was followed, except that the 100 mM, pH = 7.5 phosphate buffer used for resuspension in step (2) and step (3) was replaced with 100 mM, pH = 7.5 Tris-HCl buffer, to prepare crude amine dehydrogenase enzyme solutions and crude formate dehydrogenase enzyme solutions, respectively.

[0071] The concentration of the crude amine dehydrogenase solution obtained in Example 1 and Example 2 was adjusted to 0.2 g / mL, and the concentration of the crude formate dehydrogenase solution obtained in Example 1 and Example 2 was adjusted to 0.2 g / mL, which were used for the enzymatic preparation of (R)-3-aminobutanol in the following text.

[0072] Example 3: Enzymatic Preparation of (R)-3-aminobutanol

[0073] The synthetic route of the enzyme-catalyzed preparation of (R)-3-aminobutanol in this example is as follows Picture 1 shown.

[0074] (R)-3-aminobutanol was prepared using 4-hydroxy-2-butanone as a substrate. The preparation method was as follows: 1.2 g 4-hydroxy-2-butanone, 5 mL crude amine dehydrogenase solution, and 5 mL crude formate dehydrogenase solution (the crude enzyme solution was prepared according to step 2 of Example 1) were added to a mixture containing NAD + (1 mM) 2 mol / L ammonium formate buffer solution (total reaction system is 100 mL, pH of reaction system is adjusted to 8.5) at 40°C with open oscillation for 24 h. Then, concentrated hydrochloric acid (6 mL) was added to acidify the system to pH 3, unreacted starting material was extracted with dichloromethane (3×50 mL), phases were separated, the aqueous phase was collected, Na2CO3 aqueous solution (6 M, 12 mL) was added to the aqueous phase pH 8, amine product was extracted with dichloromethane (3×100 mL), and phases were separated. The organic phase was collected, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product (R)-3-aminobutanol. Liquid chromatography showed that the substrate conversion rate was 89%, the molar yield of the product (R)-3-aminobutanol was 85%, and the ee value was greater than 99.9%.

[0075] Example 4: Enzymatic Preparation of (R)-3-aminobutanol

[0076] (R)-3-aminobutanol was prepared using 4-hydroxy-2-butanone as a substrate. The preparation method was as follows: 1.2 g 4-hydroxy-2-butanone, 5 mL crude amine dehydrogenase enzyme solution, and 5 mL crude formate dehydrogenase enzyme solution (the crude enzyme solution was prepared according to step 2 of Example 1) were added to a 2 mol / L ammonium formate buffer solution containing NAD+ (1mM) (the total reaction system was 100 mL, and the reaction system pH was adjusted to 7.0) and the reaction was performed under open oscillation at 40°C for 24 h. Then, concentrated hydrochloric acid (6 mL) was added to acidify the system to pH 3, and unreacted starting materials were extracted with dichloromethane (3×50 mL). The phases were separated, the aqueous phase was collected, and Na2CO3 aqueous solution (10 M, 12 mL) was added to the aqueous phase to pH 8, and the amine product was extracted with dichloromethane (3×100 mL), and the phases were separated. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product (R)-3-aminobutanol. Detection by liquid chromatography showed that the substrate conversion rate was 48%, the molar yield of the product (R)-3-aminobutanol was 53%, and the ee value was greater than 96.5%.

[0077] Example 5: Enzymatic Preparation of (R)-3-aminobutanol

[0078] (R)-3-aminobutanol was prepared using 4-hydroxy-2-butanone as a substrate. The preparation method was as follows: 1.2 g 4-hydroxy-2-butanone, 5 mL crude amine dehydrogenase enzyme solution, and 5 mL crude formate dehydrogenase enzyme solution (the crude enzyme solution was prepared according to step 2 of Example 1) were added to a 2 mol / L ammonium formate buffer solution containing NAD+ (1 mM) (the total reaction system was 100 mL, and the reaction system pH was adjusted to 7.5) and the reaction was carried out at 40°C with open oscillation for 24 h. Then, concentrated hydrochloric acid (6 mL) was added to acidify the system to pH 3, and unreacted starting materials were extracted with dichloromethane (3×50 mL). The phases were separated, the aqueous phase was collected, Na2CO3 (10 M, 12 mL) was added to the aqueous phase to pH 8, and the amine product was extracted with dichloromethane (3×100 mL), and the phases were separated. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product (R)-3-aminobutanol. Detection by liquid chromatography showed that the substrate conversion rate was 56%, the molar yield of the product (R)-3-aminobutanol was 63%, and the ee value was greater than 98.0%.

[0079] Example 6: Enzymatic Preparation of (R)-3-aminobutanol

[0080] (R)-3-aminobutanol was prepared using 4-hydroxy-2-butanone as a substrate. The preparation method was as follows: 1.2 g 4-hydroxy-2-butanone, 5 mL crude amine dehydrogenase solution, and 10 mL crude formate dehydrogenase solution (the crude enzyme solution was prepared according to step 2 of Example 2) were added to a mixture containing NAD +The reaction mixture was stirred in an open air oscillation reaction at 40 °C for 24 h in a 2 mol / L ammonium formate buffer solution (1 mM) (total reaction system was 100 mL, and the reaction system pH was adjusted to 8.0), and then concentrated HCl (6 mL) was added to acidify the system pH to 3, and unreacted starting materials were extracted with dichloromethane (3×50 mL). The phases were separated, the aqueous phase was collected, Na2CO3 (10 M, 12 mL) was added to the aqueous phase pH to 8, and the amine product was extracted with dichloromethane (3×100 mL), and the phases were separated. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product (R)-3-aminobutanol. Liquid chromatography showed that the substrate conversion rate was 87%, the molar yield of the product (R)-3-aminobutanol was 83%, and the ee value was greater than 99.7%.

[0081] Example 7: Enzymatic Preparation of (R)-3-aminobutanol

[0082] (R)-3-aminobutanol was prepared using 4-hydroxy-2-butanone as a substrate. The preparation method was as follows: 1.2 g 4-hydroxy-2-butanone, 5 mL crude amine dehydrogenase solution, and 10 mL crude formate dehydrogenase solution (the crude enzyme solution was prepared according to step 2 of Example 2) were added to a mixture containing NAD + (1 mM) 2 mol / L ammonium formate buffer solution (total reaction system is 100 mL, the reaction system pH is adjusted to 9.0) at 40 ° C for 24 h of open shaking reaction, then add concentrated HCl (6 mL) to acidify the system pH to 3, and extract the unreacted starting material with dichloromethane (3×50 mL). Phase separation, collect the aqueous phase, add Na2CO3 (10 M, 12 mL) to the aqueous phase pH 8, extract the amine product with dichloromethane (3×100 mL), and phase separation. Collect the organic phase, add anhydrous sodium sulfate to dry and concentrate under reduced pressure to obtain the product (R)-3-aminobutanol. Liquid chromatography detection shows: substrate conversion rate 74%, product (R)-3-aminobutanol molar yield 82%, ee value greater than 99.5%.

[0083] Example 8: Enzymatic Preparation of (R)-3-aminobutanol

[0084] (R)-3-aminobutanol was prepared using 4-hydroxy-2-butanone as a substrate. The preparation method was as follows: 1.2 g 4-hydroxy-2-butanone, 5 mL crude amine dehydrogenase solution, and 10 mL crude formate dehydrogenase solution (the crude enzyme solution was prepared according to step 2 of Example 2) were added to a mixture containing NAD +The reaction was carried out in an open oscillation reaction at 40 °C in a 2 mol / L ammonium formate buffer solution (1 mM) (total reaction system was 100 mL, and the reaction system pH was adjusted to 9.5) for 24 h, and then concentrated HCl (6 mL) was added to acidify the system pH to 3, and unreacted starting materials were extracted with dichloromethane (3×50 mL). The phases were separated, the aqueous phase was collected, Na2CO3 (10 M, 12 mL) was added to the aqueous phase pH to 8, and the amine product was extracted with dichloromethane (3×100 mL), and the phases were separated. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the product (R)-3-aminobutanol. Liquid chromatography showed that the substrate conversion rate was 45%, the molar yield of the product (R)-3-aminobutanol was 40%, and the ee value was greater than 93%.

[0085] The structure of the product (R)-3-aminobutanol obtained in Example 3-8 was verified by NMR characterization. Picture 2 The representative characterization data are shown as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 3.77 – 3.61(m, 2H), 3.11 – 2.97 (m, 1H), 1.61 – 1.34 (m, 2H), 1.06 (dd, J = 6.4, 1.3 Hz, 3H).

[0086] In summary, the present invention constructs an amine dehydrogenase with high selectivity and high activity, and the amine dehydrogenase is combined with formate dehydrogenase as a catalyst to construct an efficient and renewable circulation system, and provides an important preparation method of chiral (R)-3-aminobutanol of pharmaceutical intermediate. The present invention uses 4-hydroxy-2-butanone as a raw material, and obtains (R)-3-aminobutanol through a one-step enzyme catalysis method of amine dehydrogenase and formate dehydrogenase. Only substrates, enzymes and buffers containing cofactors are required in the preparation process, and no other reagents are required. The operation process is simple and easy, and the reaction conditions are mild. This method not only improves the conversion rate and product yield of raw materials, but also has high optical selectivity of the chiral product prepared at the same time, and the ee value is greater than 99.9%. In addition, the green and clean production process achieved by industrial biotechnology is the "green power" for the transformation and upgrading of the chiral compound manufacturing industry. The preparation method of the present invention simplifies the downstream separation and refining process, and is a new process with simple process, low cost and green environmental protection, with the potential for industrialization and good application prospects.

Claims

1. An amine dehydrogenase, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

2. A gene, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

3. A recombinant bacterium comprising a gene with a nucleotide sequence as shown in SEQ ID NO.

1.

4. A crude amine dehydrogenase enzyme solution, characterized in that: Its preparation method comprises the following steps: Step (1), culturing the recombinant bacteria containing the gene shown in SEQ ID NO. 1 to a cell density OD 600 The value reaches 0.7-1.0; Step (2), add isopropyl-β-D-thiogalactoside to a concentration of 0.8~1 mM and continue culturing until OD 600 The value reaches 7-9; Step (3), collect the bacterial cells by centrifugation, add buffer to resuspend, ultrasonically disrupt, and centrifuge. The supernatant is the crude enzyme solution of amine dehydrogenase.

5. A dehydrogenase composition, characterized in that It consists of the amine dehydrogenase according to claim 1 and formate dehydrogenase, and the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.

4.

6. The dehydrogenase composition according to claim 5, characterized in that The mass ratio of the amine dehydrogenase to the formate dehydrogenase is 1:(1-2).

7. A crude dehydrogenase enzyme liquid composition, characterized in that: It consists of the crude amine dehydrogenase solution and the crude formate dehydrogenase solution as claimed in claim 4, wherein the amino acid sequence of the formate dehydrogenase in the crude formate dehydrogenase solution is shown as SEQ ID NO.

4.

8. A method for preparing (R)-3-aminobutanol, characterized in that: The method comprises the following steps: adding 4-hydroxy-2-butanone and the dehydrogenase composition according to any one of claims 5 to 6 or the crude enzyme solution of the dehydrogenase composition according to claim 7 to a buffer solution containing a cofactor for reaction to obtain (R)-3-aminobutanol.

9. The method according to claim 8, characterized in that The buffer solution is an ammonium formate buffer solution containing a cofactor, and the cofactor is NAD + , the concentration of the cofactor in the buffer solution is 0.5-2 mM; the pH value of the reaction is 8-9, the reaction temperature is 30-50°C, and the reaction time is 15-30 h; the mass ratio of the 4-hydroxy-2-butanone to the amine dehydrogenase is (0.5-2.0):1, and the feed mass volume ratio of the 4-hydroxy-2-butanone to the buffer solution is 1 g:20-100 mL.

10. The method according to claim 9, characterized in that The concentration of the cofactor in the buffer solution is 1 mM; the pH value of the reaction is 8.5, the reaction temperature is 40° C., and the reaction time is 24 h; the mass ratio of the 4-hydroxy-2-butanone to the amine dehydrogenase is 1.2:1, and the feed mass volume ratio of the 4-hydroxy-2-butanone to the buffer solution is 1 g:75 mL.

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