A process for the preparation of 3-ketotetrahydrofuran

Abstract

The application discloses a preparation method of 3-ketotetrahydrofuran and belongs to the technical field of biological catalysis and pharmaceutical engineering. The application aims to provide an enzymatic oxidation reaction method for obtaining 3-ketotetrahydrofuran with high conversion rate and high yield. In the application, alcohol dehydrogenase mutants are used to catalyze the oxidation of chiral 3-hydroxytetrahydrofuran in a whole cell and a pure enzyme, so that the biosynthesis of 3-ketotetrahydrofuran is realized, the highest yield is 29.73%, and the selectivity is 95.3%. The method has the advantages of mild conditions, environmental friendliness and low cost, and has high application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biocatalysis and pharmaceutical engineering, and particularly relates to a preparation method of 3-ketotetrahydrofuran. BACKGROUND

[0002] 3-ketotetrahydrofuran is an important pharmaceutical intermediate, which is mainly involved in the synthesis of drugs for treating diseases such as cancer, rheumatoid arthritis and AIDS. At present, there are many reports on the chemical synthesis of 3-ketotetrahydrofuran, but there are problems such as the use of strong oxidants, safety hazards, and large amounts of waste. Most of the synthesis routes use 3-hydroxytetrahydrofuran as a raw material, and the product is obtained by direct oxidation with strong oxidants such as PCC and PDC. The reagents are toxic and seriously pollute the environment. Therefore, it is urgent to develop a green and mild reaction condition method for preparing 3-ketotetrahydrofuran.

[0003] Patent CN102321054A uses 1,4-butynediol as a raw material, and Ce(SO4)2 to catalyze the dehydration and hydration reaction to synthesize 3-ketotetrahydrofuran. The advantage of the synthesis route is that after the product is refined by distillation, the water layer containing H2SO4 and Ce(SO4)2 can be repeatedly used, which saves the cost. However, the production needs to be carried out in a reaction kettle, and cerium sulfate and sulfuric acid aqueous solution not only cause corrosion to the equipment, but also cause serious pollution to the environment.

[0004] Patent CN105051021A uses 1,2,4-butanetriol as a raw material, which is ring-closed by p-toluenesulfonic acid to obtain 3-hydroxytetrahydrofuran, and then the latter is oxidized by TEMPO / TCCA (trichloroisocyanuric acid) to obtain the target product 3-ketotetrahydrofuran. The advantage of the synthesis route is that the use of the oxidant is less polluting to the environment than reagents such as pyridine chlorochromate, and the disadvantage is that the strong oxidant required by the reaction is in a large amount, which has a safety hazard, and the yield of this step is only about 50%.

[0005] Literature (Journal of Organic Chemistry 54 (2002): 1249-1256) and patent WO2006067430 use 3-hydroxytetrahydrofuran as a raw material, which can be directly oxidized to 3-ketotetrahydrofuran by pyridine chlorochromate (PCC) or pyridine dichromate (PDC). The reaction yield is only 40%-70% (crude product yield), contains many impurities, and the metal chromium compounds seriously pollute the environment.

[0006] Patent CN113185481A catalyzes hydroxyacetic acid methyl ester and methyl acrylate to obtain 4-methyl ester-tetrahydrofuran-3-ketone, which can be decarboxylated under the catalysis of HCl to obtain 3-ketotetrahydrofuran. The advantage of the synthesis route is that the total yield is relatively high, about 81%, and the disadvantage is that the raw material methyl acrylate has strong toxicity, and the strong base sodium hydride is flammable, which has great safety hidden danger.

[0007] The literature (Advanced Synthesis & Catalysis 346 (2004): 268-274) uses N2O as a catalyst to oxidize the olefinic bond of 2,5-dihydrofuran to synthesize 3-ketotetrahydrofuran at 10 atm and 220℃. The conversion rate of the raw material is only 16% after 12 h of reaction, and the reaction process requires high temperature and continuous N2O pressure, which makes the synthesis route difficult to apply to actual production. SUMMARY

[0008] The purpose of the present application is to provide an enzymatic oxidation reaction method for obtaining 3-ketotetrahydrofuran with high conversion rate and high yield.

[0009] The present application provides an application of an alcohol dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO. 3 or a thermophilic anaerobic bacillus containing a gene sequence as shown in SEQ ID NO. 2 in catalyzing the synthesis of 3-ketotetrahydrofuran.

[0010] Further limited, the alcohol dehydrogenase mutant is obtained by mutating isoleucine at position 86, cysteine at position 295, and serine at position 39 in the amino acid sequence shown in SEQ ID NO. 1 to asparagine, asparagine, and threonine, respectively.

[0011] The present application provides a preparation method of 3-ketotetrahydrofuran, which uses (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran as a substrate and utilizes an alcohol dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO. 3 or a thermophilic anaerobic bacillus containing a gene sequence as shown in SEQ ID NO. 2 for catalysis to obtain 3-ketotetrahydrofuran.

[0012] Further limited, the concentration of (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran in the reaction system is 5-50 g / L.

[0013] Further limited, the conversion reaction is carried out in a phosphate buffer with pH 7.2-7.4 at 30℃ for 1-72 h.

[0014] Further limited, the reaction system contains coenzyme NADP+ with a concentration of 0.1-1 mM.

[0015] Further limitation, the reaction system contains a concentration of 10-100% of the co-substrate: any one of acetone, 2-pentanone, methyl isopropyl ketone, cyclopropyl methyl ketone, 2-hexanone, methyl isobutyl ketone, tetrahydrothiophene-3-ketone, 3-oxopyrrolidine-1-carboxylic acid tert-butyl ester, N-tert-butoxycarbonyl-3-piperidone and 1,3-dihydroxyacetone co-substrate.

[0016] Further limitation, the alcohol dehydrogenase mutant with amino acid sequence as shown in SEQ ID NO. 3 is added in an amount of 1.5-2.0 mg / mL; the whole cell of thermophilic anaerobic bacillus is used in an amount of 0.05-0.25 g / mL.

[0017] The present application provides a method for improving the yield, conversion rate and selectivity of 3-ketotetrahydrofuran, using (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran as the substrate, any one of acetone, 2-pentanone, methyl isopropyl ketone, cyclopropyl methyl ketone, 2-hexanone, methyl isobutyl ketone, tetrahydrothiophene-3-ketone, 3-oxopyrrolidine-1-carboxylic acid tert-butyl ester, N-tert-butoxycarbonyl-3-piperidone and 1,3-dihydroxyacetone co-substrate as the co-substrate, adding coenzyme NADP+, and using the alcohol dehydrogenase mutant with amino acid sequence as shown in SEQ ID NO. 3 to catalyze the improvement of the yield of 3-ketotetrahydrofuran.

[0018] The present application provides the application of the thermophilic anaerobic bacillus with the alcohol dehydrogenase mutant with amino acid sequence as shown in SEQ ID NO. 2 and the gene with nucleotide sequence as shown in SEQ ID NO. 3 in improving the yield, conversion rate and selectivity of 3-ketotetrahydrofuran.

[0019] Advantages: the biological preparation method of 3-ketotetrahydrofuran adopted by the present application has the following advantages compared with the chemical synthesis method: (1) the reversibility of the alcohol dehydrogenase (TbSADH) of the biological catalyst thermophilic anaerobic bacillus is utilized to realize the synthesis of high value-added 3-ketotetrahydrofuran from 3-hydroxytetrahydrofuran; (2) the biological catalyst has the advantages of easy availability, safety and green environmental protection in catalyzing the synthesis of 3-ketotetrahydrofuran from hydroxyl oxidation. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure is the result graph of the whole cell catalysis of 3-ketotetrahydrofuran under different substrate concentrations;

[0021] Figure 2 The figure is the result graph of the whole cell catalysis of 3-ketotetrahydrofuran under different substrate concentrations;

[0022] Figure 3Figure of 3-ketotetrahydrofuran yield and conversion rate catalyzed by whole cell at different time;

[0023] Figure 4 Figure of 3-ketotetrahydrofuran yield and conversion rate catalyzed by whole cell at different cell amount;

[0024] Figure 5 Figure of 3-ketotetrahydrofuran yield and conversion rate catalyzed by whole cell at different NADP + Figure of 3-ketotetrahydrofuran yield and conversion rate catalyzed by whole cell at different NADP

[0025] Figure 6 Figure of 3-ketotetrahydrofuran yield and conversion rate catalyzed by whole cell at different acetone concentration; A is the result of 6h reaction; B is the result of 48h reaction. DETAILED DESCRIPTION

[0026] The present application is further described in the following Examples. It should be understood that these Examples are included merely to further illustrate and explain the present application and should not be considered limiting of the scope of the application. Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the present application there is a full equivalency of means and materials as set forth above.

[0027] The materials, reagents, instruments and methods used in the following examples are all conventional in the art unless otherwise specified, and can be obtained or prepared by general methods by those skilled in the art.

[0028] Example 1: Preparation of whole cell catalyst

[0029] The single colony was picked and inoculated into 40 mL LB medium containing 80 mg / mL ampicillin sodium at 37℃, 200 rpm for 4 h. 2 mL of the culture was transferred into 200 mL fresh LB medium containing the same antibiotic for secondary culture (37℃, 200 rpm). When the OD600 of the culture reached about 0.6, 0.2 mM IPTG was added and the culture was incubated at 30℃, 200 rpm overnight. The fermentation broth was centrifuged (8000 rpm, 4℃, 10 min), and the supernatant was removed. The cell mass (containing the gene shown in SEQ ID NO. 3) was collected, washed with phosphate buffer (100 mM pH 7.2-7.4) and resuspended. The supernatant was discarded again and the washing was repeated. The cell mass was stored in a refrigerator at -20℃ for standby use.

[0030] Example 2: Whole cell catalyzed synthesis of 3-ketotetrahydrofuran at different substrate concentrations

[0031] In the reaction system, chiral R-3-hydroxytetrahydrofuran (concentration of 5-50 g / L), co-substrate acetone (concentration of 10%), whole cell catalyst (0.5 g), NADP+ (Concentration 0.1 Mm) and brought to a final volume of 5 mL with 0.1 M phosphate buffer. The reaction was carried out at 30 °C and 200 rpm for 6 h, centrifuged (8000 rpm, 10 min), and 100 μL of the supernatant was diluted with methanol. The reaction conversion and yield were determined by gas chromatography, and the molecular weight of the product 3-ketotetrahydrofuran was determined by gas chromatography-mass spectrometry.

[0032] Experimental results are as follows Figure 1 As shown, the conversion rate and yield generally decreased with increasing substrate concentration. Considering that the practical application effect is extremely limited when the substrate concentration is much less than 100 mM, a substrate concentration of 30 g / L (348.47 mM) was selected in subsequent experiments. The aim was to improve the whole-cell catalytic performance of the substrate by optimizing catalytic conditions such as reaction time and co-substrate concentration. The product mass spectrometry results are shown below. Figure 2 As shown, the molecular ion peak of the product 3-ketotetrahydrofuran is 86 (the calculated value of C4H6O2 is 86.09), which is consistent with the molecular ion peak of the standard 3-ketotetrahydrofuran, which is 86.

[0033] Example 3: Whole-cell catalytic synthesis of 3-ketotetrahydrofuran at different reaction times

[0034] R-3-hydroxytetrahydrofuran (30 g / L), acetone (10%), whole-cell catalyst (0.5 g), and NADP were added to the reaction system. + (Concentration 0.1 Mm) and brought to a final volume of 5 mL with 0.1 M phosphate buffer. React at 30 °C and 200 rpm for 1–48 h, centrifuge (8000 rpm, 10 min), take 100 μL of the supernatant, dilute with methanol, and determine the reaction conversion and yield using gas chromatography.

[0035] Experimental results are as follows Figure 3 As shown, the yield first increases and then slowly decreases with the extension of reaction time. When the reaction proceeds for 6 hours, the conversion rate is 15.39%, the yield is 9.2%, and the selectivity is 59.7%. Further extending the reaction time does not significantly increase the yield.

[0036] Example 4: Whole-cell catalytic synthesis of 3-ketotetrahydrofuran with different dosages

[0037] R-3-hydroxytetrahydrofuran (30 g / L), acetone (10%), whole-cell catalyst (0.25-1.25 g), and NADP were added to the reaction system. +(Concentration 0.1 Mm) and brought to a final volume of 5 mL with 0.1 M phosphate buffer. The reaction was carried out at 30 °C and 200 rpm for 6 h, centrifuged (8000 rpm, 10 min), and 100 μL of the supernatant was diluted with methanol and the reaction conversion and yield were determined by gas chromatography.

[0038] Experimental results are as follows Figure 4 As shown, excessive whole cells increase the difficulty of mass transfer, restricting the substrate from crossing the cell membrane and binding to the active pocket of TbSADH. Simultaneously, the generated product also has difficulty crossing the cell membrane in a timely manner, leading to a decrease in yield. Therefore, adding 0.5g of whole cells is more appropriate.

[0039] Example 5: Whole-cell catalytic synthesis of 3-ketotetrahydrofuran at different coenzyme concentrations

[0040] R-3-hydroxytetrahydrofuran (30 g / L), acetone (10%), whole-cell catalyst (0.5 g), and NADP were added to the reaction system. + (Concentration 0.1-1 Mm) and brought to a final volume of 5 mL with 0.1 M phosphate buffer. The reaction was carried out at 30 °C and 200 rpm for 6 h, centrifuged (8000 rpm, 10 min), and 100 μL of the supernatant was diluted with methanol and the reaction conversion and yield were determined by gas chromatography.

[0041] Experimental results are as follows Figure 5 As shown, the yield of 3-ketotetrahydrofuran first increased and then decreased with increasing coenzyme concentration, but the overall change was not significant. The yield increased by only 0.31% when the coenzyme concentration was 0.4 mM compared to 0.1 mM.

[0042] Example 6: Whole-cell catalytic synthesis of 3-ketotetrahydrofuran under different co-substrate conditions

[0043] R-3-hydroxytetrahydrofuran (concentration 30 g / L) was added to the reaction system, along with various co-substrates including but not limited to acetone, 2-pentanone, and methyl isopropyl ketone (concentration 10%), whole-cell catalyst (0.5 g), and NADP. + (Concentration 0.1 Mm) and brought to a final volume of 5 mL with 0.1 M phosphate buffer. The reaction was carried out at 30 °C and 200 rpm for 6 h, centrifuged (8000 rpm, 10 min), and 100 μL of the supernatant was diluted with methanol and the reaction conversion and yield were determined by gas chromatography.

[0044] The experimental results are shown in Table 1. Acetone was the most effective co-substrate for whole-cell catalysis, with a conversion rate of 20.8%, a yield of 8.9%, and a selectivity of 42.8%. Methyl isopropyl ketone was the second most effective co-substrate, with a conversion rate of 12.7%, a yield of 4.36%, and a selectivity of 21.0%. In contrast, N-tert-butoxycarbonyl-3-piperidinone had a yield of only 1.05%.

[0045] Table 1: Whole-cell catalysis results under different types of cosubstrate

[0046]

[0047] Yield formula: Actual product mass / Theoretical product mass;

[0048] Conversion rate formula: mass of remaining raw materials / mass of added raw materials;

[0049] The selective formula is: yield / conversion rate.

[0050] Example 7: Whole-cell catalytic synthesis of 3-ketotetrahydrofuran at different co-substrate concentrations

[0051] R-3-hydroxytetrahydrofuran (30 g / L), acetone (10-100%), whole-cell catalyst (0.5 g), and NADP were added to the reaction system. + (Concentration 0.1 Mm) and bring the volume to 5 mL with 0.1 M phosphate buffer. React at 30 °C and 200 rpm for 6 h or 48 h, centrifuge (8000 rpm, 10 min), take 100 μL of supernatant, dilute with methanol, and determine the reaction conversion and yield by gas chromatography.

[0052] Experimental results are as follows Figure 6 As shown, with increasing acetone concentration, both groups of reactions exhibited a trend of initial increase followed by decrease in conversion rate and yield. Specifically, when the reaction time was 6 hours and the acetone concentration was 20%, the conversion rate was 19.05%, and the yield reached its highest value of 11.10%. Extending the reaction time to 48 hours and using an acetone concentration of 60% significantly improved both conversion rate and yield, with the conversion rate reaching 39.15%, the yield reaching its highest value of 23.74%, and the selectivity at 60.6%.

[0053] Example 8: Preparation of pure enzyme catalyst

[0054] After the whole cell cultured in Example 1 was collected, it was resuspended in 50 mL of pre-cooled cell lysis solution, and the cells were broken using a high-pressure cell crusher. The broken bacterial solution was collected in a 50 mL centrifuge tube, centrifuged at 4°C and 13000 rpm for 1 h, and the supernatant was collected and filtered with a 0.22 μm microporous filter. After purification by nickel column affinity chromatography, the filtrate containing the target protein was concentrated and desalted to a final volume of 500 μL using an ultrafiltration tube (30 KDa cutoff), and finally the protein solution was aliquoted and stored at -80°C for standby.

[0055] I86N / C295N / S39T: the isoleucine at position 86, the cysteine at position 295, and the serine at position 39 in the amino acid sequence shown in SEQ ID NO. 1 were mutated to asparagine, asparagine, and threonine, respectively;

[0056] Amino acid sequence of alcohol dehydrogenase SEQ ID NO: 1:

[0057] MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFEGAIGERHNMILGHEAV

[0058] GEVVEVGSEVKDFKPGDRVVVPAITPDWRTSEVQRGYHQHSGGMLAGWKFSNVKDGVF

[0059] GEFFHVNDADMNLAHLPKEIPLEAAVMIPDMMTTGFHGAELADIELGATVAVLGIGPVGL

[0060] MAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTEGKGVDAAII

[0061] AGGNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMAHKTIKGGLCPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPVVILA;

[0062]

[0063] I86N / C295N / S39T amino acid sequence (SEQ ID NO. 3):

[0064] MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTTDIHTVFEGAIGERHNMILGHEAVGEVVEVGSEVKDFKPGDRVVVPANTPDWRTSEVQRGYHQHSGGMLAGWKFSNVKDGVFGEFFHVNDADMNLAHLPKEIPLEAAVMIPDMMTTGFHGAELADIELGATVAVLGIGPVGLMAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTEGKGVDAAIIAGGNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMAHKTIKGGLNPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPVVILA.

[0065] Example 9: pure enzyme catalyzed synthesis of 3-ketotetrahydrofuran

[0066] R-3-hydroxytetrahydrofuran (30 g / L), co-substrate acetone (10 or 60%), 43 μL of I86N / C295N / S39T pure enzyme (40 mg / mL), NADP + (1 mM) were added into the reaction system, and the volume was made to 1 mL with 0.1 M phosphate buffer. The reaction was carried out at 30°C, 200 rpm for 6 h or 48 h. 100 μL of supernatant was diluted with methanol, and the conversion rate and yield were determined by gas chromatography.

[0067] The experimental results are shown in Table 2. When the concentration of acetone was 60%, the conversion rate and yield were increased by about 19% compared with the concentration of 10%. When the concentration of acetone in the system was 60% and the reaction time was 48 h, the conversion rate and yield reached the highest values of 31.21% and 29.73%, respectively, and the selectivity was 95.3%.

[0068] Table 2: whole cell catalysis results under different types of co-substrates

[0069]

[0070] In summary, the present application provides a preparation method of 3-ketotetrahydrofuran, which uses alcohol dehydrogenase (TbSADH) to catalyze the oxidation of chiral 3-hydroxytetrahydrofuran into 3-ketotetrahydrofuran in whole cell and pure enzyme, and has the advantages of mild catalytic conditions, environmental friendliness, low cost, and effective solution to the problem of large amount of "three wastes" in the existing synthesis method, and has high practical application value.

[0071] Example 10: Whole cell catalysis of 3-hydroxytetrahydrofuran to synthesize 3-ketotetrahydrofuran

[0072] S-3-hydroxytetrahydrofuran (concentration of 30 g / L), a co-substrate acetone (concentration of 60%), whole cell catalyst (0.5 g), NADP + (0.1 Mm) were added into the reaction system, and 0.1 M phosphate buffer was added to 5 mL. The reaction was carried out at 30℃ and 200 rpm for 48 h, centrifuged (8000 rpm, 10 min), and 100 μL supernatant was diluted with methanol and determined by gas phase to have a reaction conversion rate of 5.3% and a yield of 0.74%.

[0073] It should be understood that the use of these examples is only for the purpose of illustrating the present application and is not intended to limit the protection scope of the present application. In addition, it should also be understood that after reading the technical content of the present application, those skilled in the art can make various modifications, modifications and / or variations to the present application, and all these equivalent forms also fall within the protection scope defined by the appended claims of the present application.

Claims

1. The application of a mutant of alcohol dehydrogenase with the amino acid sequence shown in SEQ ID NO.3 or thermophilic anaerobic bacteria containing the gene shown in SEQ ID NO.2 in the catalytic synthesis of 3-ketotetrahydrofuran, wherein the reaction substrate for the catalytic synthesis is 5-50 g / L of (R)-(-)-3-hydroxytetrahydrofuran, 60% acetone is used as a co-substrate, and 0.1-1 mM of coenzyme NADP is added. + The amount of the alcohol dehydrogenase mutant added is 40 mg / mL or the amount of the thermophilic anaerobic bacteria in whole cells is 0.05-0.25 g / mL.

2. The application according to claim 1, characterized in that, The alcohol dehydrogenase mutant was obtained by mutating isoleucine at position 86 of the amino acid sequence shown in SEQ ID NO.1 to asparagine, cysteine ​​at position 295 to asparagine, and serine at position 39 to threonine.

3. A method for preparing 3-ketotetrahydrofuran, characterized in that, Using (R)-(-)-3-hydroxytetrahydrofuran as a substrate, 3-ketotetrahydrofuran was obtained by catalysis with an alcohol dehydrogenase mutant containing the amino acid sequence shown in SEQ ID NO.3 or thermophilic anaerobic bacteria containing the gene sequence shown in SEQ ID NO.

2. The substrate for the catalytic reaction was 5-50 g / L of (R)-(-)-3-hydroxytetrahydrofuran, 60% acetone as a co-substrate, and 0.1-1 mM of coenzyme NADP added. + The reaction was carried out in phosphate buffer at pH 7.2-7.4 at 30°C for 1-72 hours, wherein the amount of the alcohol dehydrogenase mutant added was 40 mg / mL or the amount of the whole cells of the thermophilic anaerobic bacillus was 0.05-0.25 g / mL.

4. A method for improving the yield, conversion, and selectivity of 3-ketotetrahydrofuran, characterized in that, Using (R)-(-)-3-hydroxytetrahydrofuran (5-50 g / L) as the substrate and 60% acetone as the co-substrate, 0.1-1 mM coenzyme NADP was added. + The yield, conversion and selectivity of 3-ketotetrahydrofuran were improved by using an alcohol dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO.3 at a concentration of 40 mg / mL.

5. The application of thermophilic anaerobic bacteria containing an alcohol dehydrogenase mutant with the amino acid sequence shown in SEQ ID NO.3 and a gene with the nucleotide sequence shown in SEQ ID NO.2 in improving the yield, conversion rate, and selectivity of 3-ketotetrahydrofuran; the application is based on 5-50 g / L of (R)-(-)-3-hydroxytetrahydrofuran as a substrate, 60% acetone as a co-substrate, and the addition of 0.1-1 mM coenzyme NADP. + The yield, conversion rate, and selectivity of 3-ketotetrahydrofuran were improved by using an alcohol dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO.3 or by thermophilic anaerobic bacteria containing the gene shown in SEQ ID NO.

2. The amount of the alcohol dehydrogenase mutant added is 40 mg / mL or the total amount of the thermophilic anaerobic bacteria is 0.05-0.25 g / mL.

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