Two-step enzyme-catalyzed reaction system and its application in synthesis of (s)-1-methoxy-2-propylamine

Abstract

The application belongs to the technical field of biological catalysis, and discloses a two-step enzyme-coupled catalytic reaction system and application thereof in synthesis of (S)-1-methoxy-2-propylamine. Through in-vitro recombinant expression of alcohol dehydrogenase and transaminase, a two-step enzyme-coupled catalytic reaction system containing the above-mentioned enzymes is constructed, and the two-step enzyme-coupled catalytic reaction system is used to catalyze 1-methoxy-2-propanol to synthesize (S)-1-methoxy-2-propylamine, and the ee value of the product obtained by the reaction reaches more than 99%. The raw material cost of the scheme is low and the utilization rate is high, and the by-product acetone generated simultaneously can realize the internal circulation of coenzyme NAD + / NADH, and the conversion rate of the target product (S)-1-methoxy-2-propylamine reaches 77%. The process is simple, the reaction condition is mild, the cost is low, the process is green and efficient, and the application lays a foundation for industrial synthesis of (S)-1-methoxy-2-propylamine.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis, specifically relating to a two-step enzyme-linked catalytic reaction system and its application in the synthesis of (S)-1-methoxy-2-propane. Background Technology

[0002] (S)-1-Methoxy-2-propylamine, with the molecular formula C4H 11 NO is an important chiral amine intermediate. The compound contains a chiral center in its molecular structure, and its optical purity directly affects the performance of downstream products. The most important application of (S)-1-methoxy-2-propane is as a key chiral intermediate in the chloroacetamide herbicides S-Metolachlor and Dimethenamid. Amide herbicides are a class of pre-emergence, selective herbicides commonly used for soil treatment. They penetrate the soil to inhibit important biological processes in roots or shoots, particularly disrupting protein synthesis or fatty acid biosynthesis, thereby interfering with cell membrane formation, leading to inhibited growth of weed shoots and coleoptiles, and ultimately plant death. The herbicide S-Metolachlor was discovered by Syngenta in 1970 and commercialized in 1975. S-Metolachlor is the active S-form isolated from S-Metolachlor, possessing higher activity, lower dosage, better crop safety, and superior environmental compatibility. This product was first launched in the United States in 1997 and has now become the largest-selling amide herbicide. S-methylphenidate is also an important cornfield herbicide, with global sales of $219 million in 2018, and its EU registration renewal has been extended to 2034. Therefore, developing an efficient, economical, and environmentally friendly synthetic method for (S)-1-methoxy-2-propylamine has significant industrial application value.

[0003] Currently, the synthesis methods of (S)-1-methoxy-2-propane are mainly divided into two categories: chemical methods and biocatalytic methods. In terms of chemical synthesis, the traditional route typically uses 1-methoxy-2-propanone as a raw material, employing a metal catalyst under high temperature and high pressure conditions for reductive amination. For example, the method disclosed in Chinese patent application CN110066223A requires a metal catalyst, has harsh reaction conditions, and results in low product yield and unclear optical purity data, making it difficult to meet the development trend of green manufacturing in pesticide chemicals. Another strategy uses natural chiral sources such as L-alanine as raw materials, constructing chiral centers through multiple steps such as esterification and reduction. However, this method is cumbersome, uses expensive and difficult-to-remove metal hydride reducing agents (such as lithium aluminum hydride), has poor atom economy, and is not conducive to large-scale industrial production.

[0004] Biocatalysis is an environmentally friendly alternative that has emerged in recent years. Among them, the asymmetric reductive amination reaction catalyzed by ω-transaminase has attracted much attention. This method uses 1-methoxy-2-propanone as a prochiral substrate, and under the condition that isopropylamine or isopropylamine salt is used as an amino donor, an amino group transfer reaction is catalyzed by ω-transaminase to directly generate chiral pure (S)-1-methoxy-2-propanamine. This route has outstanding advantages such as mild reaction conditions (room temperature and pressure), extremely high optical purity of the product (ee value can reach over 99%), and environmental friendliness. The method disclosed in patent CN114134126A uses 1-methoxy-2-propanone as a substrate, relies on wild-type ω-transaminase or its mutant from Bacillus, uses isopropylamine / isopropylamine salt as an amino donor, and completes a single transamination reaction under the action of the cofactor pyridoxal phosphate (PLP) to generate (S)-1-methoxy-2-propanamine. However, the high cost of the substrate 1-methoxy-2-propanone limits the further large-scale application of this method.

[0005] The method disclosed in patent CN121931193 mentions that 1-methoxy-2-propanol reacts in the presence of an amino donor (ammonium chloride, formic acid, or chlorine water, etc.) under the catalysis of alcohol dehydrogenase, amine dehydrogenase, and coenzyme. During the reaction, glucose dehydrogenase and glucose are added to continue the reaction, yielding (S)-1-methoxy-2-propanol. This method effectively reduces raw material costs and improves product conversion rate, but it requires three enzymes for catalytic reaction, making the system relatively complex.

[0006] In summary, existing methods for synthesizing (S)-1-methoxy-2-propane still face numerous technical challenges in terms of catalyst cost, mild reaction conditions, atom economy, optical purity control, and feasibility for large-scale production. Therefore, developing a method for synthesizing (S)-1-methoxy-2-propane that features low-cost catalysts, mild reaction conditions, high optical purity, and suitability for industrial production is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0007] In view of the shortcomings of existing methods for synthesizing (S)-1-methoxy-2-propanamine, this invention provides a two-step enzyme-linked catalytic reaction system and its application in the synthesis of (S)-1-methoxy-2-propanamine. By recombinantly expressing alcohol dehydrogenase and transaminase in vitro, a two-step enzyme-linked catalytic reaction system containing these enzymes was constructed, and the synthesis of (S)-1-methoxy-2-propanamine from 1-methoxy-2-propanol using this two-step enzyme-linked catalytic reaction system was achieved. Specifically, the alcohol dehydrogenase uses 1-methoxy-2-propanol as a substrate, and the cofactor NAD+... +The enzyme participates in catalysis, oxidizing to 1-methoxy-2-propanone and NADH; transaminase uses 1-methoxy-2-propanone and isopropylamine as substrates, and PLP as a cofactor, to convert to (S)-1-methoxy-2-propanone and acetone, with the product yielding an ee value exceeding 99%. This method features low raw material costs and high utilization rates, while the generated byproduct acetone can assist the coenzyme NAD. + The NADH-based internal circulation system achieved a 77% conversion rate of the target product (S)-1-methoxy-2-propane. The process is simple, with mild reaction conditions, low cost, and is green and efficient, laying the foundation for the industrial synthesis of (S)-1-methoxy-2-propane.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: First, the present invention provides a two-step enzyme-linked catalytic reaction system, wherein the two-step enzyme-linked catalytic system comprises an alcohol dehydrogenase and a transaminase, wherein the alcohol dehydrogenase comprises an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 97% or higher homology with the amino acid sequence shown in SEQ ID NO.1; the transaminase comprises an amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence having at least 97% or higher homology with the amino acid sequence shown in SEQ ID NO.3.

[0009] More specifically, the alcohol dehydrogenase (ADH) mentioned was selected from the National Center for Biotechnology Information database, searching for alcohol dehydrogenases from different sources, including but not limited to Aspergillus fumigatus, Brucella suis, Rhodococcus ruber, Rhodococcus sp., Desulfurobacterium thermolithotrophum, Rubinisphaera brasiliensis, Streptomyces laurentii, Thermodesulfobium narugense, Niastella koreensis, and Brevibacterium sp.

[0010] In some preferred embodiments, the alcohol dehydrogenase is derived from Rhodococcus ruber. Specifically, the alcohol dehydrogenase comprises the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 97% or higher homology with the amino acid sequence shown in SEQ ID NO.1; exemplary, the nucleotide sequence encoding ADH is shown in SEQ ID NO.2.

[0011] Furthermore, the transaminase (ω-TA) is selected from gene databases (National Center for Biotechnology Information) or protein databases (Protein Data Bank), and transaminases from different sources are searched, including but not limited to Sporosarcina globispora, Rhizobium sp., Ancylobacter lacus, Domibacillus robiginosus, Chromobacterium violaceum, Aspergillus terreus, Pseudomonas, and Vibrio fluvialis.

[0012] In some preferred embodiments, the ω-TA is derived from *Sporosarcina globispora*. Specifically, the transaminase comprises the amino acid sequence shown in SEQ ID NO. 3, or an amino acid sequence having at least 97% or higher homology with the amino acid sequence shown in SEQ ID NO. 3; exemplary, the nucleotide sequence encoding ω-TA is shown in SEQ ID NO. 4.

[0013] The alcohol dehydrogenase and transaminase mentioned above were obtained by expressing engineered bacteria obtained through existing genetic engineering.

[0014] The alcohol dehydrogenase and transaminase mentioned above can be used to construct a two-step enzyme-linked catalytic reaction system for the synthesis of (S)-1-methoxy-2-propylamine. The amount of enzymes added to the system is as follows: 10-30 U / ml for alcohol dehydrogenase and 5-15 U / ml for transaminase.

[0015] Further preferably, the amount of alcohol dehydrogenase added is 10-20 U / ml, and the amount of transaminase added is 5-10 U / ml.

[0016] In addition, the two-step enzyme-linked catalytic reaction system for synthesizing (S)-1-methoxy-2-propane also includes: using 1-methoxy-2-propanol and isopropylamine as substrates, and nicotinamide adenine dinucleotide and pyridoxal phosphate as cofactors.

[0017] Furthermore, it also includes using Tris-HCl buffer solution or phosphate buffer solution as buffer solution, wherein the pH of the buffer solution is 6.0-9.0; In some preferred embodiments, the phosphate buffer solution has a concentration of 5-300 mM and a pH of 6.0-8.0; the Tris-HCl buffer solution has a concentration of 10-200 mM and a pH of 8.0-9.0.

[0018] More preferably, the concentration of 1-methoxy-2-propanol in the reaction system is 200-600 mM; the concentration of the amino donor isopropylamine in the reaction system is 1-3 times the concentration of 1-methoxy-2-propanol; more preferably 1.5-2 times.

[0019] The cofactor nicotinamide adenine dinucleotide (NAD) in the reaction system + The concentration of the cofactor pyridoxal phosphate (PLP) is 0.5-2 mM.

[0020] Based on the above system, this invention further discloses the application of this two-step enzyme-linked catalytic reaction system in the synthesis of (S)-1-methoxy-2-propane. The system is reacted in a constant-temperature shaker at 30-40°C, with the rotation speed controlled at 100-300 rpm, and the reaction time controlled at 18-24 hours to obtain (S)-1-methoxy-2-propane. Preferably, heating is used after the reaction to terminate the reaction and precipitate the protein. Results show that the conversion rate of (S)-1-methoxy-2-propane using low-cost 1-methoxy-2-propanol as a substrate is 45-77%.

[0021] In the above synthesis process, alcohol dehydrogenase uses 1-methoxy-2-propanol as a substrate and NAD+ as a cofactor. + It participates in catalysis, oxidizing to 1-methoxy-2-propanone and NADH; transaminase uses 1-methoxy-2-propanone and isopropylamine as substrates, and PLP as a cofactor, to convert to (S)-1-methoxy-2-propanone and acetone; moreover, alcohol dehydrogenase can utilize NADH to catalyze the production of NAD from the byproduct acetone. + This provides more NAD coenzyme for the first step of the catalytic reaction. + To achieve NAD + / NADH system internal circulation.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. Low cost and high yield: In the reaction catalysis system of the present invention, (S)-1-methoxy-2-propanol is synthesized efficiently using low-cost 1-methoxy-2-propanol as a substrate, with a conversion rate of up to 77%, effectively solving the problem of high production cost.

[0023] 2. In addition, alcohol dehydrogenase in the reaction system is NAD+. + As a cofactor, it is oxidized to NADH. Acetone, a byproduct of the enzyme-linked immunosorbent assay (ELISA) system, can assist the coenzyme NAD. + / NADH self-equilibrium: In a reaction system containing only ADH, the addition of acetone significantly increased the molar conversion of the product 1-methoxy-2-propanone by 23.5%. ADH itself is reversible; the generated NADH and the byproduct acetone produced by transamine reaction can be used to reverse the reaction to produce the cofactor NAD required in the system. + To achieve NAD + The regeneration cycle of NADH also promotes the positive catalytic reaction of the entire enzyme-linked system. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the two-step enzyme-linked catalytic reaction system described in this invention; Figure 2 The image shows the SDS-PAGE detection results of the crude enzyme solution described in Example 1. Detailed Implementation

[0025] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0026] In the following embodiments, unless otherwise specified, all reagents, culture media, etc., used are commercially available, and all experimental methods are performed using conventional existing techniques in the field.

[0027] The detection method for (S)-1-methoxy-2-propylamine in the following examples is as follows: The optical purity of the product was determined by gas chromatography. The specific method was as follows: column model: DB-1701 30 m × 0.32 mm × 0.25 μm; column temperature: 40℃; injection port and detector: 300℃; time: 20 min; pressure: 40 kPa; split ratio: 20; injection volume: 0.5 μL; hydrogen flow rate: 30 mL / min; air flow rate: 300 mL / min; make-up purge: 30 mL / min.

[0028] Example 1: Construction and expression method of relevant recombinant engineered bacteria in a two-step enzyme-linked system (1) Construction of recombinant plasmids In this embodiment, the alcohol dehydrogenase ADH used is derived from Rhodococcus ruber, GenBank accession number WZU35438.1; the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; In this embodiment, the transaminase ω-TA was derived from Sporosarcina globispora, GenBank accession number WP_053437037.1; the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0029] The gene sequence was designed using SnapGene software without altering the amino acid sequence encoded by the original gene. The codons were optimized based on E. coli’s preferences, and the optimized gene sequence was then synthesized by a commercial company.

[0030] In this invention, the gene ADH (SEQ ID NO. 2) encoding alcohol dehydrogenase, derived from Rhodococcus ruber, was cloned into the BamHⅠ / XhoⅠ restriction site of the expression vector pET28a; The transaminase ω-TA (SEQ ID NO.4) derived from Sporosarcina globispora was cloned into the BamHⅠ / XhoⅠ restriction site of the expression vector pET28a.

[0031] Recombinant plasmids pET28a-ADH and pET28a-ωTA were obtained, and the construction of the above plasmids was carried out using general techniques well known in the art.

[0032] (2) Construction of recombinant expression strains: The constructed plasmids pET28a-ADH and pET28a-ωTA were transformed into Escherichia coli BL21(DE3) competent cells, respectively. The specific process is as follows: Remove E. coli BL21(DE3) competent cells (purchased from Beyotime) from a -80℃ freezer and thaw them slowly on ice. Immediately add 5 μL of the recombinant plasmid, mix gently, and incubate on ice for 30 min. Then, heat shock the cells in a 42℃ water bath for 60 s, followed by incubation on ice for 2 min. Under aseptic conditions, add 1 ml of LB liquid antibiotic-free medium and incubate at 37℃ and 1000 rpm for 60 min to allow the cells to recover. Centrifuge at 8000 rpm for 1 min, discard the supernatant, and resuspend the cells in 100 μL of liquid. Streak the recombinant cells onto LB solid medium containing kanamycin (working concentration 50 μg / ml) in a triadic pattern. Seal the plates with sealing film and incubate them overnight at 37℃. Recombinant bacteria ADH and ω-TA carrying the recombinant plasmid were obtained.

[0033] (3) Protein expression of recombinant bacteria From the LB agar plates of the recombinant strains ADH and ω-TA, single clones of transformants were picked and inoculated into 100 ml shake flasks containing 20 ml of LB liquid medium (containing a working concentration of 50 μg / ml kanamycin) and cultured overnight at 37°C and 220 rpm until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.2 mM, and the culture was continued at 16°C for 16-20 h. The bacterial cells were disrupted using an ultrasonic homogenizer, and after lysis, the supernatant (crude enzyme solution) was collected by centrifugation (12000 rpm, 10 min). The crude enzyme solution was analyzed by protein gel electrophoresis (SDS-PAGE). Figure 2 ).

[0034] Figure 2 In the middle lane, M represents the protein molecular weight standard; lane 1 contains the transaminase (ω-TA) of recombinant bacteria ω-TA, with a protein size of 53kb; lane 2 contains the alcohol dehydrogenase (ADH) of recombinant bacteria ADH, with a protein size of 45kb.

[0035] Example 2 Enzyme activity detection of relevant enzymes in a two-step enzyme-linked system The crude enzyme solution prepared in Example 1 was used to detect enzyme activity. The reaction system for detecting alcohol dehydrogenase (ADH) activity in recombinant bacterial ADH was 1 ml, and the composition of the reaction system was: 50 mM 1-methoxy-2-propanol, 1 mM NAD. +100 μl of crude ADH enzyme solution was reacted in a constant temperature metal bath at 35℃ and 700 rpm for 15 min. After the reaction, the reaction was terminated by heating at 60℃ for 10 min, and the protein was precipitated. The supernatant was collected after centrifugation at 12000 rpm for 2 min and used for chromatographic detection. The concentration of 1-methoxy-2-propanone was determined for enzyme activity calculation. Enzyme activity unit (U / ml) is defined as the amount of enzyme required to produce 1 μmol of 1-methoxy-2-propanone in 1 min under standard conditions. The activity of alcohol dehydrogenase (ADH) was determined to be 66 U / ml.

[0036] The reaction system for detecting the transaminase (ω-TA) activity of recombinant bacterial ω-TA was 1 ml, and the composition was: 100 mM 1-methoxy-2-propanone, 500 mM isopropylamine, 0.5 mM PLP, and 500 μl of crude ω-TA enzyme solution. The reaction was carried out at 35℃ and 700 rpm in a constant temperature metal bath for 15 min. After the reaction, the reaction was terminated by heating at 60℃ for 10 min, and the protein was precipitated. The supernatant was collected after centrifugation at 12000 rpm for 2 min and used for chromatographic detection. The concentration of (S)-1-methoxy-2-propanamine was determined for enzyme activity calculation. The enzyme activity unit (U / ml) is defined as the amount of enzyme required to generate 1 μmol of (S)-1-methoxy-2-propanamine in 1 mL of enzyme solution under standard conditions in 1 min. The transaminase (ω-TA) activity was detected and calculated to be 30 U / ml.

[0037] Example 3: Construction of a two-step enzyme-linked catalytic reaction system for the synthesis of (S)-1-methoxy-2-propane. The crude enzyme solution prepared in Example 1 was used to construct the two-step enzyme-linked catalytic reaction system. The composition of the two-step enzyme-linked catalytic reaction system is as follows: The reaction system contained 200 mM PB buffer (pH 8.0), 200 mM 1-methoxy-2-propanol, and 1 mM NAD. + The system contained 400 mM isopropylamine and 1 mM PLP. The enzyme activity of alcohol dehydrogenase (ADH) was 20 U / ml and the enzyme activity of transaminase (ω-TA) was 10 U / ml.

[0038] The reaction was carried out in a constant-temperature shaker at a reaction temperature of 37°C and a rotation speed of 200 rpm for approximately 24 hours. After the reaction was completed, the reaction was terminated by heating at 60°C for 10 minutes, and the protein was precipitated. The supernatant was collected after centrifugation at 12000 rpm for 2 minutes for chromatographic analysis. The reaction process of the above system is as follows. Figure 1As shown in the figure. The results show that the yield of (S)-1-methoxy-2-propylamine obtained in this example was 12.02 g / L, the molar concentration was 135 mM, the molar conversion was 67.5%, and the ee value was 99%.

[0039] Example 4: Construction of a two-step enzyme-linked catalytic reaction system for the synthesis of (S)-1-methoxy-2-propane. The crude enzyme solution prepared in Example 1 was used to construct the two-step enzyme-linked catalytic reaction system. The composition of the two-step enzyme-linked catalytic reaction system is as follows: The reaction system contained 200 mM PB buffer (pH 8.0), 400 mM 1-methoxy-2-propanol, and 1 mM NAD. + The system contained 600 mM isopropylamine and 1 mM PLP. The enzyme activity of alcohol dehydrogenase (ADH) was 20 U / ml and the enzyme activity of transaminase (ω-TA) was 10 U / ml.

[0040] The reaction was carried out in a constant-temperature shaker at a reaction temperature of 37°C and a rotation speed of 200 rpm for approximately 24 hours. After the reaction was completed, the reaction was terminated by heating at 60°C for 10 min, and the protein was precipitated. The supernatant was collected after centrifugation at 12000 rpm for 2 min for chromatographic analysis. The results showed that the yield of (S)-1-methoxy-2-propylamine obtained in this example was 27.4 g / L, the molar concentration was 308 mM, the molar conversion rate was 77%, and the ee value was 99%.

[0041] Example 5: Construction of a two-step enzyme-linked catalytic reaction system for the synthesis of (S)-1-methoxy-2-propane. The crude enzyme solution prepared in Example 1 was used to construct the two-step enzyme-linked catalytic reaction system. The composition of the two-step enzyme-linked catalytic reaction system is as follows: The reaction system contained 200 mM PB buffer (pH 8.0), 600 mM 1-methoxy-2-propanol, and 1 mM NAD. + The system contained 1000 mM isopropylamine and 1 mM PLP. The enzyme activity of alcohol dehydrogenase (ADH) was 20 U / ml and the enzyme activity of transaminase (ω-TA) was 10 U / ml.

[0042] The reaction was carried out in a constant-temperature shaker at a reaction temperature of 37°C and a rotation speed of 200 rpm for approximately 24 hours. After the reaction was completed, the reaction was terminated by heating at 60°C for 10 min, and the protein was precipitated. The supernatant was then collected after centrifugation at 12,000 rpm for 2 min for chromatographic analysis. The results showed that the yield of (S)-1-methoxy-2-propylamine obtained in this example was 29.4 g / L, the molar concentration was 331 mM, the molar conversion was 55%, and the ee value was 99%.

[0043] Example 6: Construction of a two-step enzyme-linked catalytic reaction system for the synthesis of (S)-1-methoxy-2-propane. The crude enzyme solution prepared in Example 1 was used to construct the two-step enzyme-linked catalytic reaction system. The composition of the two-step enzyme-linked catalytic reaction system is as follows: The reaction system contained 200 mM PB buffer (pH 8.0), 400 mM 1-methoxy-2-propanol, and 1 mM NAD. + The system contained 600 mM isopropylamine and 1 mM PLP. The enzyme activity of alcohol dehydrogenase (ADH) was 20 U / ml and the enzyme activity of transaminase (ω-TA) was 5 U / ml.

[0044] The reaction was carried out in a constant-temperature shaker at a reaction temperature of 37°C and a rotation speed of 200 rpm for approximately 24 hours. After the reaction was completed, the reaction was terminated by heating at 60°C for 10 min, and the protein was precipitated. The supernatant was then collected after centrifugation at 12,000 rpm for 2 min for chromatographic analysis. The results showed that the yield of (S)-1-methoxy-2-propylamine obtained in this example was 20.6 g / L, the molar concentration was 232 mM, the molar conversion rate was 58%, and the ee value was 99%.

[0045] Example 7: Construction of a two-step enzyme-linked catalytic reaction system for the synthesis of (S)-1-methoxy-2-propane. The crude enzyme solution prepared in Example 1 was used to construct the two-step enzyme-linked catalytic reaction system. The composition of the two-step enzyme-linked catalytic reaction system is as follows: The reaction system contained 200 mM PB buffer (pH 8.0), 400 mM 1-methoxy-2-propanol, and 1 mM NAD. + The system contained 600 mM isopropylamine and 1 mM PLP, with 10 U / ml of alcohol dehydrogenase ADH and 10 U / ml of transaminase ω-TA.

[0046] The reaction was carried out in a constant-temperature shaker at a reaction temperature of 37°C and a rotation speed of 200 rpm for approximately 24 hours. After the reaction was completed, the reaction was terminated by heating at 60°C for 10 minutes, and the protein was precipitated. The supernatant was then collected after centrifugation at 12000 rpm for 2 minutes for chromatographic analysis. The reaction process of the above system is as follows: Figure 1 As shown in the figure. The results show that the yield of (S)-1-methoxy-2-propane obtained in this example was 17.6 g / L, the molar concentration was 198 mM, the molar conversion was 49.5%, and the ee value was 99%.

[0047] Example 8 Construction of coenzyme NAD + / NADH Self-Balancing Verification System The crude alcohol dehydrogenase solution prepared in Example 1 was used to construct the coenzyme NAD. + The NADH self-equilibrium verification system has the following reaction system composition: The reaction system contained 200 mM PB buffer (pH 8.0), 400 mM 1-methoxy-2-propanol, and 1 mM NAD. + The enzyme activity of alcohol dehydrogenase (ADH) added to the system was 20 U / ml. The reaction was carried out in a constant-temperature shaker at 37℃ and 200 rpm for approximately 24 hours. After the reaction was completed, the reaction was terminated by heating at 60℃ for 10 min, and the protein was precipitated. The supernatant was collected after centrifugation at 12000 rpm for 2 min for chromatographic analysis. The results showed that the final yield of 1-methoxy-2-propanone in this system was 22.5 g / L, the molar concentration was 256 mM, and the molar conversion rate was 64%.

[0048] The reaction system was supplemented with acetone containing 200 mM PB buffer (pH 8.0), 400 mM 1-methoxy-2-propanol, and 1 mM NAD. + The system contained 200 mM acetone, with an added amount of alcohol dehydrogenase (ADH) of 20 U / ml. The reaction was carried out in a constant-temperature shaker at 37°C and 200 rpm for approximately 24 hours. After the reaction was complete, the reaction was terminated by heating at 60°C for 10 min, and the protein was precipitated. The supernatant was collected after centrifugation at 12000 rpm for 2 min for chromatographic analysis. The results showed that the final yield of 1-methoxy-2-propanone was 30.8 g / L, with a molar concentration of 351 mM and a molar conversion rate of 87.5%.

[0049] The comparison of the above reaction systems shows that the conversion rate of 1-methoxy-2-propanone is significantly improved after the addition of acetone. In the technical solution of this application, the transaminase in the enzyme-linked catalytic reaction system uses 1-methoxy-2-propanone and isopropylamine as substrates to convert them into (S)-1-methoxy-2-propanone and acetone, thus increasing the acetone content in the system. Acetone then undergoes a reverse reaction to produce the cofactor NAD required by the system. + Thus achieving NAD + The regeneration cycle of NADH also promotes the positive catalytic reaction of the entire enzyme-linked system.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent substitutions or modifications made by those skilled in the art within the spirit and principle of the present invention without any creative effort should be included within the protection scope of the present invention.

Claims

1. A two-step enzyme-linked catalytic reaction system, characterized in that, The two-step enzyme-linked catalytic reaction system contains alcohol dehydrogenase and transaminase. The alcohol dehydrogenase comprises an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 97% or higher homology with the amino acid sequence shown in SEQ ID NO.

1. The transaminase comprises an amino acid sequence as shown in SEQ ID NO.3, or an amino acid sequence having at least 97% or higher homology with the amino acid sequence shown in SEQ ID NO.

3.

2. The two-step enzyme-linked catalytic reaction system according to claim 1, characterized in that, The enzyme addition amounts in the system were as follows: alcohol dehydrogenase 10-30 U / ml and transaminase 5-15 U / ml.

3. The two-step enzyme-linked catalytic reaction system according to claim 1 or 2, characterized in that, The enzyme addition amounts in the system were as follows: alcohol dehydrogenase 10-20 U / ml and transaminase 5-10 U / ml.

4. The two-step enzyme-linked catalytic reaction system according to claim 1, characterized in that, The system also includes: 1-methoxy-2-propanol and isopropylamine as substrates, and nicotinamide adenine dinucleotide and pyridoxal phosphate as cofactors.

5. The two-step enzyme-linked catalytic reaction system according to claim 1 or 4, characterized in that, The system also includes a Tris-HCl buffer solution or a phosphate buffer solution with a pH of 6.0-9.

0.

6. The two-step enzyme-linked catalytic reaction system according to claim 4, characterized in that, The concentration of 1-methoxy-2-propanol in the reaction system is 200-600 mM; the concentration of the amino donor isopropylamine in the reaction system is 1-3 times the concentration of 1-methoxy-2-propanol.

7. The two-step enzyme-linked catalytic reaction system according to claim 6, characterized in that, The concentration of the amino donor isopropylamine in the reaction system is 1.5 to 2 times the concentration of 1-methoxy-2-propanol.

8. The two-step enzyme-linked catalytic reaction system according to claim 4, characterized in that, The concentration of the cofactor nicotinamide adenine dinucleotide in the reaction system was 0.5-2 mM; the concentration of the cofactor pyridoxal phosphate was 0.5-2 mM.

9. The two-step enzyme-linked catalytic reaction system according to claim 5, characterized in that, The phosphate buffer solution has a concentration of 5-300 mM and a pH of 6.0-8.0; the Tris-HCl buffer solution has a concentration of 10-200 mM and a pH of 8.0-9.

0.

10. The use of the two-step enzyme-linked catalytic reaction system according to any one of claims 1-9 in the synthesis of (S)-1-methoxy-2-propylamine, characterized in that, The specific process is as follows: The two-step enzyme-linked catalytic reaction system is carried out in a constant temperature shaker. The temperature of the reaction system is controlled at 30-40℃ and the reaction time is controlled at 18-24 h to obtain (S)-1-methoxy-2-propylamine.

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