Process for preparation of nitroreduction products

Abstract

The invention provides a method for preparing a nitroreduction product. The method comprises the following steps: carrying out reduction reaction on a nitro reduction substrate in the presence of nitroreductase so as to obtain a nitro reduction product; the nitroreductase has an amino acid sequence as shown in SEQ ID NO: 1. The method for preparing the nitro-reduction product is higher in catalytic efficiency, smaller in environmental pollution and lower in cost.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to a method for preparing a nitro-reduction product. Background Art

[0002] Ensitrelvir (S-217622) is the first orally active, non-covalent, non-peptide SARS-CoV-2 3CL protease inhibitor developed by Shionogi of Japan.

[0003] 6-Chloro-2-methyl-2H-indazole-5-amine is a key intermediate in the synthesis of Ensitrelvir (S-217622). It is crucial to synthesize this intermediate with high yield and high selectivity.

[0004] Patent WO2019153080 discloses a method for preparing 6-chloro-2-methyl-2H-indazole-5-amine, and its specific synthetic route is as follows Figure 1 EST03 was used as a reaction substrate, 9.0 equivalents of iron powder and 9.0 equivalents of ammonium chloride were added to 43 volumes of 50% ethanol aqueous solution and reacted at 80°C for 6 hours. The use of iron powder to reduce the nitro group produced a large amount of iron sludge, which was difficult to recycle and produce a large amount of solid waste, causing environmental pollution.

[0005] Therefore, there is an urgent need in the art to develop a green and environmentally friendly method for preparing 6-chloro-2-methyl-2H-indazole-5-amine. Summary of the invention

[0006] This application is based on the inventor's discovery and understanding of the following facts and problems:

[0007] In the existing method for preparing 6-chloro-2-methyl-2H-indazole-5-amine, the amount of reagents used is large, which will increase the cost and generate waste. In addition, a large amount of iron mud and wastewater will be generated during the reaction process, which will cause great environmental pollution.

[0008] To this end, in a first aspect of the present application, a method for preparing a nitro-reduction product is provided. According to an embodiment of the present application, the method comprises: subjecting a nitro-reduction substrate to a reduction reaction in the presence of a nitroreductase to obtain a nitro-reduction product.

[0009] According to the embodiments of the present application, the inventor unexpectedly discovered that nitroreductase can be used to catalyze the conversion of nitroreduction substrates into nitroreduction products. Using nitroreductase to reduce nitroreduction substrates is more efficient, less polluting to the environment, and less costly.

[0010] According to an embodiment of the present application, the method for preparing a nitro-reduction product further includes at least one of the following technical features:

[0011] According to an embodiment of the present application, the nitroreductase has an amino acid sequence shown in SEQ ID NO: 1;

[0012] MTNDNFFNVLYERTSTRAFNPNKEISSTELHEILKAAAQAPSAWNLQHWKFLVFQGEDVQKRLHPIAYNQQQILDASAVVAILGDLEAYKNVEPVYGPIVEQGF MKEEAKERLAKNIESAYAREQFPRDAAFSNASLAAMQLMLAAEATGWDTCAIGGFNPQALTDEFNVSSRYVPIMLITIGESTLKGHPAPRMSVEQVSEWAK(SEQ ID NO:1).

[0013] According to an embodiment of the present application, the nitroreductase has a nucleotide sequence shown in SEQ ID NO: 2;

[0014] (SEQ ID NO:2).

[0015] According to embodiments of the application, the nitro reduction substrate comprises 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomer, tautomer and salt. The application also provides an intermediate product synthesized by nitro reduction product: 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomer, tautomer and salt. These intermediate products can be used to prepare nitro reduction product equally.

[0016] According to an embodiment of the present application, the nitro reduction product is 6-chloro-2-methyl-2H-indazol-5-amine.

[0017] According to a specific embodiment of the present application, under normal temperature conditions, 6-chloro-2-methyl-5-nitro-2H-indazole can be synthesized by reducing 6-chloro-2-methyl-2H-indazole with the help of nitroreductase (NTR) under the action of a coenzyme. Figure 2 ).

[0018] According to an embodiment of the present application, the reduction reaction is carried out in a reduction reaction system.

[0019] According to an embodiment of the present application, the concentration range of the nitro reduction substrate in the reduction reaction system is 2 to 50 g / L. According to a specific embodiment of the present application, the nitro reduction substrate can react to generate a nitro reduction product when the concentration range of the nitro reduction substrate in the reduction reaction system is 2 to 50 g / L.

[0020] According to an embodiment of the present application, the concentration of the nitro-reduced substrate in the reduction reaction system is 10 g / L. According to a specific embodiment of the present application, when the concentration of the nitro-reduced substrate in the reduction reaction system is 10 g / L, the efficiency of nitroreductase in catalyzing the nitro-reduced substrate is the highest.

[0021] According to an embodiment of the present application, the reduction reaction system includes an additional enzyme.

[0022] According to an embodiment of the present application, the additional enzyme is selected from ketoreductase or glucose dehydrogenase. The inventors unexpectedly found that adding additional enzymes can enhance reaction efficiency and increase yield.

[0023] According to an embodiment of the present application, the additional enzyme is provided in the form of a bacterial cell expressing the additional enzyme. According to some specific embodiments of the present application, the reaction efficiency can also be enhanced by directly adding a pure additional enzyme.

[0024] According to an embodiment of the present application, the reduction reaction system further includes a coenzyme cycle reaction substrate. According to a specific embodiment of the present application, adding the coenzyme cycle reaction substrate can promote the catalytic efficiency and substrate conversion rate of the nitroreductase.

[0025] According to an embodiment of the present application, the coenzyme cycle reaction substrate is selected from isopropanol or glucose.

[0026] According to an embodiment of the present application, when the additional enzyme is ketoreductase, the coenzyme cycle reaction substrate is isopropanol. According to an embodiment of the present application, isopropanol binds to the active site of nitroreductase to form a complex, which can reduce the activation energy of the catalytic reaction of nitroreductase, thereby increasing the reaction rate.

[0027] According to an embodiment of the present application, when the additional enzyme is glucose dehydrogenase, the coenzyme cycle reaction substrate is glucose. According to a specific embodiment of the present application, the coenzyme cycle reaction substrate is selected as glucose to provide a reduction equivalent for the nitroreduction reaction to promote the catalytic activity and catalytic efficiency of the nitroreductase.

[0028] According to an embodiment of the present application, the reduction reaction system further includes a coenzyme. According to a specific embodiment of the present application, the addition of a coenzyme can further enhance the efficiency of the catalytic reaction.

[0029] According to an embodiment of the present application, the coenzyme is selected from nicotinamide adenine dinucleotide NAD+ or nicotinamide adenine dinucleotide phosphate NADP+.

[0030] According to an embodiment of the present application, the reduction reaction system further comprises a buffer. According to a specific embodiment of the present application, ensuring that the reduction reaction system is carried out in a buffer helps maintain the stability of the nitroreductase and the nitroreduction product.

[0031] According to an embodiment of the present application, the buffer is selected from at least one of a citric acid buffer, a Tris-HCl buffer solution, a phosphate buffer and a TEOA buffer. According to some specific embodiments of the present application, the buffer includes but is not limited to the aforementioned buffer, such as a disodium hydrogen phosphate / sodium dihydrogen phosphate buffer system.

[0032] According to the embodiments of the present application, the pH value of the buffer solution is in the range of 5 to 9. According to the specific embodiments of the present application, selecting a suitable buffer solution pH can improve the activity and catalytic efficiency of nitroreductase.

[0033] According to an embodiment of the present application, the pH value of the buffer solution is 8.5. According to a specific embodiment of the present application, the pH value of the buffer solution is selected to be 8.5, and the catalytic activity of the nitroreductase is higher.

[0034] According to an embodiment of the present application, the nitroreductase is provided in the form of a recombinant Escherichia coli cell expressing the nitroreductase. According to some specific embodiments of the present application, the nitroreductase can be added in the form of Escherichia coli cells or directly added as a pure nitroreductase.

[0035] According to an embodiment of the present application, the concentration range of the coenzyme in the reduction reaction system is 0.2 to 1 g / L.

[0036] According to an embodiment of the present application, the concentration of the coenzyme in the reduction reaction system is 1 g / L. According to a specific embodiment of the present application, when the concentration of the coenzyme in the reduction reaction system is 1 g / L, the reaction efficiency is higher.

[0037] According to an embodiment of the present application, the concentration range of the recombinant Escherichia coli in the reduction reaction system is 1 to 50 g / L, preferably 5 g / L. According to a specific embodiment of the present application, the recombinant Escherichia coli can achieve the conversion of nitro-reduced substrate to nitro-reduced product in a concentration range of 1 to 50 g / L.

[0038] According to an embodiment of the present application, the concentration of the recombinant Escherichia coli cells in the reduction reaction system is 20 g / L. According to a specific embodiment of the present application, when the concentration of the recombinant Escherichia coli cells in the reduction reaction system is 20 g / L, the catalytic efficiency is higher. Generally speaking, each milligram of recombinant Escherichia coli cells contains 0.1 mg of nitroreductase.

[0039] According to an embodiment of the present application, the concentration range of the nitroreductase in the reduction reaction system is 0.5 to 2 g / L.

[0040] According to an embodiment of the present application, the concentration range of the nitroreductase in the reduction reaction system is 0.3 to 1 g / L, preferably 0.5 g / L.

[0041] According to an embodiment of the present application, the concentration of the nitroreductase in the reduction reaction system is 2 g / L.

[0042] According to the embodiment of the present application, the concentration of ketoreductase bacteria in the reduction reaction system ranges from 0.2 to 10 g / L. According to the specific embodiment of the present application, the concentration of ketoreductase bacteria in the reduction reaction system ranges from 0.2 to 10 g / L, which can increase the nitro reduction reaction rate.

[0043] According to an embodiment of the present application, the concentration of ketoreductase bacteria in the reduction reaction system is 1 g / L. According to a specific embodiment of the present application, when the concentration of ketoreductase bacteria in the reduction reaction system is 1 g / L, the nitro reduction reaction rate is higher.

[0044] According to an embodiment of the present application, the volume fraction of the isopropanol in the reduction reaction system is in the range of 5 to 20%. According to a specific embodiment of the present application, selecting isopropanol in the range of 5 to 20% can increase the rate of the reduction reaction.

[0045] According to an embodiment of the present application, the volume fraction of the isopropanol in the reduction reaction system is 5%. According to a specific embodiment of the present application, when the volume fraction of isopropanol is 5%, the reduction reaction rate is higher.

[0046] According to the embodiment of the present application, the concentration of glucose dehydrogenase in the reduction reaction system is in the range of 0.5 to 25 g / L. According to the specific embodiment of the present application, the concentration of glucose dehydrogenase in the reduction reaction system is in the range of 0.5 to 25 g / L, which can increase the nitro reduction reaction rate.

[0047] According to an embodiment of the present application, the concentration of glucose dehydrogenase bacteria in the reduction reaction system is 2.5 g / L. According to a specific embodiment of the present application, when the concentration of glucose dehydrogenase bacteria in the reduction reaction system is 2.5 g / L, the nitro reduction reaction rate is higher.

[0048] According to an embodiment of the present application, the concentration of glucose in the reduction reaction system ranges from 7.5 to 375 g / L. According to a specific embodiment of the present application, selecting glucose with a concentration in the range of 7.5 to 375 g / L can increase the rate of the reduction reaction.

[0049] According to an embodiment of the present application, the concentration of glucose in the reduction reaction system is 300 g / L. According to a specific embodiment of the present application, when the concentration of glucose is 300 g / L, the reduction reaction rate is higher.

[0050] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0052] Figure 1 It is a schematic diagram of the synthesis of 6-chloro-2-methyl-2H-indazole-5-amine in the prior art of this application;

[0053] Figure 2 Schematic diagram of the synthesis of 6-chloro-2-methyl-2H-indazol-5-amine (ESTD04) using 6-chloro-2-methyl-5-nitro-2H-indazole (ESTD03) according to the examples of the present application. DETAILED DESCRIPTION

[0054] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0055] definition

[0056] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0057] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0058] In this application, unless otherwise specified, the term "nitroreductase" refers to an enzyme that can catalyze the reduction reaction of nitroaromatic compounds to aromatic amines. In this application, "homology" has the conventional meaning in the art and refers to the "identity" between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared after the best alignment, and the differences between the two sequences are randomly distributed over their entire length.

[0059] In this application, unless otherwise specified, the term "stereoisomer" refers to compounds with the same chemical constitution but different arrangements of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, etc.

[0060] In this application, unless otherwise indicated, the term "tautomer" refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0061] In the present application, unless otherwise specified, the term "salt" refers to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as described in the literature: SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.

[0062] In this application, unless otherwise specified, "recombinant E. coli wet cells" or "recombinant E. coli cells" have the same meaning. The preparation method may be slightly different in different experimental platforms. The following exemplary shows the specific preparation steps of recombinant E. coli wet cells: the expression vector pET-26b that has been digested with two endonucleases and the cloned nitroreductase nucleotide sequence are connected with T4 ligase overnight, and the connection product is added to the electroporation cup containing E. coli BL21 (DE3) electrocompetent cells, and immediately electroporated on the electroporator, and then immediately transferred to ice, and LB agar medium preheated to 37°C is added to mix; the mixed culture medium is transferred to a culture tube and cultured at 37°C for 1 hour at 200 rpm; the incubated transformation liquid is spread on an LB agar plate containing 100 microliters per milliliter of kanamycin for streak culture, and cultured in a 37°C incubator overnight for 16 hours; the next day, the transformants are picked on the coated plate for PCR verification, and the positive strains are sent for sequencing verification. The recombinant engineering strain with the correct sequencing result is picked on the inoculation plate and activated on the plate. On the next day, a single colony and a single clone are picked and inoculated into a conical flask containing a culture medium containing kanamycin, and cultured in a 37°C incubator overnight; the overnight culture is transferred to a fresh culture medium containing the same resistance at a ratio of 12 to 100, cultured at 37°C, and shaken at 220 rpm until the absorbance OD600 value of the bacterial solution at a wavelength of 600 nanometers reaches 0.6 to 0.8, then isopropyl-β-D-thiogalactoside is added, and culture is continued at 25°C overnight; centrifugation is performed, the supernatant is discarded, and the recombinant nitroreductase wet bacteria are collected.

[0063] In addition, the recombinant glucose dehydrogenase engineering strain and the recombinant ketoreductase engineering strain constructed in the laboratory were also activated on the corresponding resistance plates. On the second day, single colonies were picked and transferred to the 15mL culture medium of the corresponding resistance, and cultured at 37°C for 16h. Then the seed liquid was transformed into fresh LB culture medium at a volume ratio of 2%, and cultured at 37°C and 220rpm. When the absorbance OD600 value of each bacterial solution at a wavelength of 600 nanometers reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2mM to induce protein expression, and cultured at 25°C and 220rpm for 18h. Centrifuge each (9000rpm, 10min), discard the supernatant, and collect the wet bacterial cells of the recombinant glucose dehydrogenase engineering strain and the recombinant ketoreductase engineering strain respectively.

[0064] The following is a detailed description of the embodiments. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0065] Example 1: Construction of recombinant nitroreductase genetically engineered bacteria

[0066] The nitroreductase gene (its amino acid sequence is shown in SEQ: 1, and its nucleotide sequence is shown in SEQ: 2) was amplified from the Bacillus genome preserved in the laboratory. By comparing with the NCBI database, it was found that the amino acid sequence of the nitroreductase had 100% similarity with the nitroreductase protein sequence WP_000180387.1 reported on NCBI. Restriction sites Nde I and Xho I were added to both ends of the gene, and a gene fragment with restriction sites was amplified. Subsequently, the gene fragment and the expression vector pet26b were double-digested with Nde I and Xho I, and the target gene fragment and the vector pet26b fragment were recovered by gel cutting. T4 DNA ligase was then used to connect the target gene fragment and the pet26b vector fragment overnight. 5 microliters of the ligation product was transferred to an electroporation cup containing 50 microliters of Escherichia coli electrocompetent cells BL21 (DE3). Perform electroporation on an electroporator, then immediately transfer to ice, add 1 mL of LB agar medium preheated to 37°C, and mix well; then transfer the mixed medium to a 2 mL culture tube and culture at 200 rpm at 37°C for one hour; take 150 microliters of the incubated transformation liquid and spread it on an LB agar plate containing 100 microliters per mL of kanamycin, and culture it in a 37°C incubator overnight for 16 hours. The next day, pick the transformants on the coated plate for PCR verification, and send the positive strains for sequencing verification. The strains whose sequencing results are consistent with the SEQ:2 sequence are selected as genetic engineering bacteria for recombinant nitroreductase and as subsequent research objects.

[0067] Example 2: Preparation of recombinant Escherichia coli wet cells

[0068] The recombinant nitroreductase engineering strain constructed in Example 1 was activated on a plate containing kanamycin resistance. On the second day, a single colony was picked and transferred to a conical flask containing 15mL culture medium containing kanamycin, and cultured at 37°C for 16h. Subsequently, the seed solution was transformed into fresh LB culture medium at a volume ratio of 2%, and cultured at 37°C and 220rpm. When the absorbance OD600 value of the bacterial solution at a wavelength of 600 nanometers reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2mM to induce protein expression, and cultured at 25°C and 220rpm for 18h. Centrifuge (9000rpm, 10min), discard the supernatant, collect the wet bacterial cells of the recombinant nitroreductase engineering strain, and store at -20°C.

[0069] The recombinant glucose dehydrogenase engineering strain and recombinant ketoreductase engineering strain constructed in the laboratory were also activated on the corresponding resistance plates. On the second day, single colonies were picked and transferred to the 15mL conical flasks of the corresponding resistance culture medium, and cultured at 37°C for 16h. Then the seed liquid was transformed into fresh LB culture medium at a volume ratio of 2%, and cultured at 37°C and 220rpm. When the absorbance OD600 value of each bacterial solution at a wavelength of 600 nanometers reached 0.6-0.8, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.2mM to induce protein expression, and cultured at 25°C and 220rpm for 18h. Centrifuge each (9000rpm, 10min), discard the supernatant, collect the wet bacterial cells of the recombinant glucose dehydrogenase engineering strain and the recombinant ketoreductase engineering strain, and store them separately at -20°C.

[0070] Example 3: Preparation of 6-chloro-2-methyl-2H-indazol-5-amine

[0071] In a 25mL reaction bottle, add 100mg 6-chloro-2-methyl-5-nitro-2H-indazole, 50mg wet cells of recombinant Escherichia coli expressing the nitroreductase (the concentration of the wet cells is 5g / L, and each mg of wet cells contains 0.1mg of enzyme), 10mg wet cells of ketoreductase (the concentration of the wet cells is 1g / L, and each mg of wet cells contains 0.2mg of enzyme), 0.5ml isopropanol, 10mg nicotinamide adenine dinucleotide NADP+; add sodium phosphate buffer solution (0.1M, pH8.5) to a volume of 10ml. After magnetic stirring at 35°C for 24h, use ethyl acetate to inactivate the reaction and extract, dry the organic phase with excess anhydrous sodium sulfate, filter to remove the sodium sulfate solid, and concentrate to obtain 6-chloro-2-methyl-2H-indazole-5-amine.

[0072] Example 4: Preparation of 6-chloro-2-methyl-2H-indazol-5-amine

[0073] In a 25mL reaction bottle, add 100mg 6-chloro-2-methyl-5-nitro-2H-indazole, 200mg wet cells of recombinant Escherichia coli expressing the nitroreductase (the concentration of the wet cells is 20g / L, and each mg of wet cells contains 0.1mg of nitroreductase), 25mg wet cells of glucose dehydrogenase (the concentration of the wet cells is 2.5g / L, and each mg of wet cells contains 0.2mg of enzyme), 300mg glucose, and 10mg nicotinamide adenine dinucleotide NADP+; add sodium phosphate buffer solution (0.1M, pH7.5) to a volume of 10mL. After magnetic stirring at 30°C for 24 hours, use ethyl acetate to inactivate the reaction and extract, dry the organic phase with excess anhydrous sodium sulfate, filter to remove the sodium sulfate solid, and concentrate to obtain 6-chloro-2-methyl-2H-indazole-5-amine.

[0074] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0075] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a nitro reduction product, characterized in that: include: The nitro-reduced substrate is subjected to a reduction reaction in the presence of a nitroreductase to obtain a nitro-reduced product.

2. The method according to claim 1, characterized in that The nitroreductase has an amino acid sequence shown in SEQ ID NO:

1.

3. The method according to claim 2, characterized in that The nitro-reduced substrate includes 6-chloro-2-methyl-5-nitro-2H-indazole or its stereoisomers, tautomers and salts; Optionally, the nitro reduction product is 6-chloro-2-methyl-2H-indazol-5-amine.

4. The method according to claim 2, characterized in that: The reduction reaction is carried out in a reduction reaction system; Optionally, the concentration of the nitro-reduced substrate in the reduction reaction system is in the range of 2 to 50 g / L, preferably 10 g / L; Optionally, the reduction reaction system includes an additional enzyme; Optionally, the additional enzyme is selected from ketoreductase or glucose dehydrogenase; Optionally, the additional enzyme is provided in the form of a bacterium expressing the additional enzyme; Optionally, the reduction reaction system further comprises a coenzyme cycle reaction substrate; Optionally, the coenzyme cycle reaction substrate is selected from isopropanol or glucose.

5. The method according to claim 4, characterized in that When the additional enzyme is ketoreductase, the substrate of the coenzyme cycle reaction is isopropanol.

6. The method according to claim 4, characterized in that When the additional enzyme is glucose dehydrogenase, the substrate of the coenzyme cycle reaction is glucose.

7. The method according to claim 4, characterized in that The reduction reaction system further includes a coenzyme; Optionally, the coenzyme is selected from nicotinamide adenine dinucleotide NAD+ or nicotinamide adenine dinucleotide phosphate NADP+.

8. The method according to claim 4, characterized in that The reduction reaction system further comprises a buffer; Optionally, the buffer is selected from at least one of a citrate buffer, a Tris-HCl buffer solution, a phosphate buffer and a TEOA buffer; Optionally, the pH value of the buffer solution ranges from 5 to 9, preferably has a pH value of 8.

5.

9. The method according to claim 2, characterized in that: The nitroreductase is provided in the form of a recombinant Escherichia coli cell expressing the nitroreductase.

10. The method according to claim 4, 7 or 9, characterized in that: The concentration of the coenzyme in the reduction reaction system is in the range of 0.2 to 1 g / L, preferably 1 g / L; Optionally, the concentration of the recombinant Escherichia coli in the reduction reaction system is in the range of 1 to 50 g / L, preferably 5 g / L, more preferably 20 g / L; Optionally, the concentration of the nitroreductase in the reduction reaction system is in the range of 0.3 to 1 g / L, preferably 0.5 g / L.

11. The method according to claim 5, characterized in that The concentration of ketoreductase bacteria in the reduction reaction system is in the range of 0.2 to 10 g / L, preferably 1 g / L; Optionally, the volume fraction of the isopropanol in the reduction reaction system is in the range of 5 to 20%, preferably 5%.

12. The method according to claim 6, characterized in that The concentration of glucose dehydrogenase bacteria in the reduction reaction system is in the range of 0.5 to 25 g / L, preferably 2.5 g / L; Optionally, the concentration of glucose in the reduction reaction system is in the range of 7.5 to 375 g / L, preferably 300 g / L.

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