Preparation method of 3-ketotetrahydrofuran

Abstract

The invention discloses a preparation method of 3-ketotetrahydrofuran, and belongs to the technical field of biological catalysis and pharmaceutical engineering. The invention aims to provide an enzymatic oxidation reaction method for obtaining 3-ketotetrahydrofuran with high conversion rate and high yield. According to the present invention, the alcohol dehydrogenase mutant is adopted to perform whole-cell and pure enzyme catalysis chiral 3-hydroxytetrahydrofuran oxidation so as to achieve the biosynthesis of 3-ketotetrahydrofuran, the highest yield is 29.73%, the selectivity is 95.3%, the method uses water as the solvent and the biological enzyme as the catalyst, the reaction is performed at the room temperature, and the advantages of mild condition, environmental protection and low cost are provided; the application potential is higher.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis and pharmaceutical engineering, and specifically relates to a method for preparing 3-ketotetrahydrofuran. Background Art

[0002] 3-Ketotetrahydrofuran is an important pharmaceutical intermediate, mainly involved in the synthesis of drugs for the treatment of diseases such as cancer, rheumatoid arthritis, and AIDS. At present, there are many reports on the chemical synthesis preparation of 3-ketotetrahydrofuran, and there are problems such as the need to use strong oxidants, potential safety hazards, and large amounts of "three wastes". Its synthetic route mostly uses 3-hydroxytetrahydrofuran as raw materials, and the products are directly oxidized by strong oxidants such as PCC and PDC. The reagents are toxic and cause serious environmental pollution. Therefore, the development of a green method for preparing 3-ketotetrahydrofuran with mild reaction conditions is an urgent problem to be solved.

[0003] Patent CN102321054A uses 1,4-butynediol as raw material and Ce(SO4)2 as catalyst to dehydrate and hydrate it to synthesize 3-ketotetrahydrofuran. The advantage of the synthetic route is that after the product is distilled and refined, the water layer containing H2SO4 and Ce(SO4)2 can be reused, saving costs. However, the production needs to be carried out in a reactor, and cerium sulfate and sulfuric acid aqueous solution will not only corrode the equipment, but also cause serious pollution to the environment.

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

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

[0006] Patent CN113185481A uses NaH to catalyze the condensation of methyl glycolate and methyl acrylate to obtain 4-formate-tetrahydrofuran-3-one, which can be refluxed and decarboxylated under HCl to obtain 3-ketotetrahydrofuran. The advantage of the synthetic route is that the total yield is relatively high, about 81%, but the disadvantage is that the raw material methyl acrylate is highly toxic, and the strong base sodium hydride is flammable, which poses a great safety hazard.

[0007] The literature (Advanced Synthesis & Catalysis 346 (2004): 268-274) uses N2O as a catalyst to oxidize the olefin bond of 2,5-dihydrofuran to synthesize 3-ketotetrahydrofuran at 10atm and 220°C. After 12 hours of reaction, the raw material conversion rate is only 16%. The reaction process requires high temperature and continuous maintenance of N2O pressure. These factors make the synthetic route difficult to apply to actual production. Summary of the invention

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

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

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

[0011] The invention provides a method for preparing 3-ketotetrahydrofuran. (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran is used as a substrate and 3-ketotetrahydrofuran is obtained by catalysis using an alcohol dehydrogenase mutant having 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.

[0012] It is further defined that the concentration of (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran in the reaction system is 5-50 g / L.

[0013] Further defined, the conversion reaction is carried out in a phosphate buffer at pH 7.2-7.4 at 30°C for 1-72 hours.

[0014] It is further defined that the reaction system contains the coenzyme NADP+ at a concentration of 0.1-1 mM.

[0015] It is further defined that the reaction system contains a co-substrate with a concentration of 10-100%: any one of acetone, 2-pentanone, methyl isopropyl ketone, cyclopropyl methyl ketone, 2-hexanone, methyl isobutyl ketone, tetrahydrothiophene-3-one, tert-butyl 3-oxopyrrolidine-1-carboxylate, N-tert-butoxycarbonyl-3-piperidone and 1,3-dihydroxyacetone co-substrate.

[0016] It is further defined that the amount of the alcohol dehydrogenase mutant having an amino acid sequence as shown in SEQ ID NO. 3 is 1.5-2.0 mg / mL; the amount of the whole cells of the thermophilic anaerobic bacteria is 0.05-0.25 g / mL.

[0017] The invention provides a method for improving the yield, conversion rate and selectivity of 3-ketotetrahydrofuran. The method comprises the following steps: using (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran as a substrate, and any one of acetone, 2-pentanone, methyl isopropyl ketone, cyclopropyl methyl ketone, 2-hexanone, methyl isobutyl ketone, tetrahydrothiophene-3-one, tert-butyl 3-oxopyrrolidine-1-carboxylate, N-tert-butyloxycarbonyl-3-piperidone and 1,3-dihydroxyacetone as a cosubstrate, adding coenzyme NADP+, and utilizing an alcohol dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO.3 for catalysis to improve the yield of 3-ketotetrahydrofuran.

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

[0019] Beneficial effects: The 3-keto-tetrahydrofuran biological preparation method adopted by the present invention has the following advantages over the chemical synthesis method: (1) The reversibility of the catalytic activity of the alcohol dehydrogenase (TbSADH) of the biocatalyst Thermoanaerobacterium anaerobicum is utilized to realize the synthesis of high value-added 3-keto-tetrahydrofuran from 3-hydroxytetrahydrofuran. (2) The biocatalyst catalyzes the oxidation of hydroxyl groups to synthesize 3-keto-tetrahydrofuran, which has the advantages of being easily available, safe, and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The graph shows the yield and conversion rate of 3-keto-tetrahydrofuran catalyzed by whole cells at different substrate concentrations;

[0021] Figure 2 The mass spectrometry results of the product 3-ketotetrahydrofuran standard and the prepared product; A is the result of the 3-ketotetrahydrofuran standard; B is the result of the whole cell catalysis product;

[0022] Figure 3The results of the yield and conversion rate of 3-keto-tetrahydrofuran catalyzed by whole cells at different times are shown in the figure;

[0023] Figure 4 The results of 3-ketotetrahydrofuran yield and conversion rate at different whole cell dosages are shown;

[0024] Figure 5 For different NADP + The effect of dosage on the yield and conversion rate of 3-ketotetrahydrofuran synthesized by whole cell catalysis;

[0025] Figure 6 The graphs show the effects of different acetone concentrations on the yield and conversion rate of 3-ketotetrahydrofuran catalyzed by whole cells; A is the result of a 6-hour reaction; B is the result of a 48-hour reaction. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific examples, but the use and purpose of these exemplary implementations are only used to illustrate the present invention and do not constitute any form of limitation on the actual protection scope of the present invention, nor do they limit the protection scope of the present invention to these.

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

[0028] Example 1: Preparation of whole cell catalyst

[0029] Pick a single clone and place it in 40mL LB medium containing 80mg / mL ampicillin sodium, and culture it at 37℃, 200rpm for 4h. Take 2mL of culture solution and transfer it to 200mL fresh LB medium containing the same antibiotics for secondary culture (37℃, 200rpm). When the OD600 of the culture solution reaches about 0.6, add IPTG with a final concentration of 0.2mM, and culture it at 30℃, 200rpm overnight. Centrifuge the fermentation broth (8000rpm, 4℃, 10min), remove the supernatant, collect the bacterial cells (containing the gene shown in SEQ IDNO.3), wash and suspend the cells with phosphate buffer (100mM pH7.2-7.4), centrifuge again to discard the supernatant and repeat the washing and store in a -20℃ refrigerator for use.

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

[0031] Add chiral R-3-hydroxytetrahydrofuran (concentration of 5-50 g / L), co-substrate acetone (concentration of 10%), whole cell catalyst (0.5 g), NADP to the reaction system.+ (concentration of 0.1 Mm), and dilute to 5 mL with 0.1 M phosphate buffer. React at 30°C, 200 rpm for 6 h, centrifuge (8000 rpm, 10 min), take 100 μL of supernatant, dilute with methanol, and use gas chromatography to determine the reaction conversion rate and yield, and use gas chromatography-mass spectrometry to determine the molecular weight of the product 3-ketotetrahydrofuran.

[0032] The experimental results are as follows Figure 1 As shown in the figure, as the substrate concentration of the system increases, the conversion rate and yield generally show a downward trend. Considering that when the substrate concentration is much less than 100mM, the actual production effect is extremely limited. Therefore, in subsequent experiments, the substrate concentration is continued to be 30g / L (348.47mM), hoping to improve the catalytic performance of the whole cell to the substrate by optimizing the catalytic conditions such as reaction time and co-substrate concentration. The mass spectrometry detection results of the product are shown in 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 86 of the standard 3-ketotetrahydrofuran.

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

[0034] R-3-hydroxytetrahydrofuran (concentration of 30 g / L), co-substrate acetone (concentration of 10%), whole cell catalyst (0.5 g), NADP + (concentration of 0.1 Mm), and dilute to 5 mL with 0.1 M phosphate buffer. React at 30°C, 200 rpm for 1-48 h, centrifuge (8000 rpm, 10 min), take 100 μL of supernatant, dilute with methanol, and use gas phase to determine reaction conversion rate and yield.

[0035] The experimental results are as follows Figure 3 As shown, as the reaction time increases, the yield increases first and then slowly decreases. When the reaction is carried out for 6 hours, the conversion rate is 15.39%, the yield is 9.2%, and the selectivity is 59.7%. Continuing to extend the reaction time, the yield does not increase significantly.

[0036] Example 4: Synthesis of 3-ketotetrahydrofuran catalyzed by whole cells at different dosages

[0037] Add R-3-hydroxytetrahydrofuran (concentration of 30 g / L), co-substrate acetone (concentration of 10%), whole cell catalyst (0.25-1.25 g), NADP to the reaction system. +(concentration of 0.1 Mm) and dilute to 5 mL with 0.1 M phosphate buffer. React at 30°C, 200 rpm for 6 h, centrifuge (8000 rpm, 10 min), take 100 μL of supernatant, dilute with methanol and use gas phase to determine reaction conversion rate and yield.

[0038] The experimental results are as follows Figure 4 As shown, too much whole cells will increase the difficulty of mass transfer, limiting the substrate from crossing the cell membrane and binding to the active pocket of TbSADH. At the same time, the generated product is difficult to cross the cell membrane in time, resulting in a decrease in yield. Therefore, it is more appropriate to select a whole cell addition amount of 0.5g.

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

[0040] R-3-hydroxytetrahydrofuran (concentration of 30 g / L), co-substrate acetone (concentration of 10%), whole cell catalyst (0.5 g), NADP + (concentration of 0.1-1Mm), and dilute to 5mL with 0.1M phosphate buffer. React at 30°C, 200rpm for 6h, centrifuge (8000rpm, 10min), take 100μL of supernatant, dilute with methanol and use gas phase to determine reaction conversion rate and yield.

[0041] The experimental results are as follows Figure 5 As shown, with the increase of coenzyme concentration, the yield of 3-ketotetrahydrofuran showed a trend of first increasing and then decreasing, but the overall change was not obvious. When the coenzyme concentration was 0.4 mM compared with the concentration of 0.1 mM, the yield was only increased by 0.31%.

[0042] Example 6: Whole-cell catalytic synthesis of 3-ketotetrahydrofuran under different types of cosubstrates

[0043] R-3-hydroxytetrahydrofuran (concentration of 30 g / L), different types of co-substrates including but not limited to acetone, 2-pentanone, methyl isopropyl ketone, etc. (concentration of 10%), whole cell catalyst (0.5 g), NADP + (concentration of 0.1 Mm) and dilute to 5 mL with 0.1 M phosphate buffer. React at 30°C, 200 rpm for 6 h, centrifuge (8000 rpm, 10 min), take 100 μL of supernatant, dilute with methanol and use gas phase to determine reaction conversion rate and yield.

[0044] The experimental results are shown in Table 1. Acetone as a cosubstrate has the best effect on the whole-cell catalytic substrate, with a conversion rate of 20.8%, a yield of 8.9%, and a selectivity of 42.8%; followed by methyl isopropyl ketone as a cosubstrate with a conversion rate of 12.7%, a yield of 4.36%, and a selectivity of 21.0%, while N-tert-butyloxycarbonyl-3-piperidone as a cosubstrate has a yield of only 1.05%.

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

[0046]

[0047] Yield formula: actual mass of product generated / theoretically generated mass of product;

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

[0049] Formula for selectivity: yield / conversion.

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

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

[0052] The experimental results are as follows Figure 6 As shown in the figure, with the increase of acetone concentration, the conversion rate and yield of both reactions showed a trend of increasing first and then decreasing. When the reaction time was 6h and the acetone concentration was 20%, the conversion rate was 19.05%, and the yield reached a maximum value of 11.10%. When the reaction time was extended to 48h and the acetone concentration was 60%, the conversion rate and yield were significantly improved. At this time, the conversion rate was 39.15%, the yield reached a maximum value of 23.74%, and the selectivity was 60.6%.

[0053] Example 8: Preparation of pure enzyme catalyst

[0054] After the whole cells cultured in Example 1 were collected, they were resuspended in a pre-cooled 50 mL cell lysis solution and the cells were broken using a high-pressure cell disruptor. The broken bacterial solution was collected in a 50 mL centrifuge tube, centrifuged at 4 ° C, 13000 rpm for 1 hour, and the supernatant was collected and filtered with a 0.22 μm microporous filter membrane. 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 (cutoff 30 KDa), and the protein solution was stored at -80 ° C for standby use.

[0055] I86N / C295N / S39T was obtained by mutating the isoleucine at position 86 to asparagine, the cysteine ​​at position 295 to asparagine, and the serine at position 39 to threonine in the amino acid sequence shown in SEQ ID NO.1;

[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] MKGFAMLSIGKVGWIEKEKPAPGPFDAIVRPLAVAPCTTDIHTVFEGAIGERHNMILGHEAVGEVVEVGSEVKDFKPGDRVVVPANTPDWRTSEVQRGYHQHSGGMLAGWKFSNVKDGVFGEFFHVNDADMNLAHLPKEIPLEAAVMIPDMMTTGFHGAELADIELGATVAVLGIG PVGLMAVAGAKLRGAGRIIAVGSRPVCVDAAKYYGATDIVNYKDGPIESQIMNLTEGKGVDAAIIAGGNADIMATAVKIVKPGGTIANVNYFGEGEVLPVPRLEWGCGMAHKTIKGGLNPGGRLRMERLIDLVFYKRVDPSKLVTHVFRGFDNIEKAFMLMKDKPKDLIKPVVILA.

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

[0066] R-3-hydroxytetrahydrofuran (concentration of 30 g / L), co-substrate acetone (concentration of 10 or 60%), 43 μL of I86N / C295N / S39T pure enzyme (concentration of 40 mg / mL), NADP + (concentration of 1 Mm), and dilute to 1 mL with 0.1 M phosphate buffer. React at 30°C, 200 rpm for 6 h or 48 h, take 100 μL of the supernatant, dilute with methanol, and use gas phase to determine the reaction conversion rate and yield.

[0067] The experimental results are shown in Table 2. When the acetone concentration is 60%, the conversion rate and yield are both increased by about 19% compared with the acetone concentration of 10%. When the acetone concentration of the system is 60% and the reaction time is 48h, the conversion rate and yield can reach the maximum value of 31.21% and 29.73% respectively, and the selectivity is 95.3%.

[0068] Table 2: Whole-cell catalysis results under different types of cosubstrates

[0069]

[0070] In summary, the present invention provides a method for preparing 3-ketotetrahydrofuran, wherein alcohol dehydrogenase (TbSADH) is used to catalyze chiral 3-hydroxytetrahydrofuran to 3-ketotetrahydrofuran using whole-cell and pure enzyme. The method has mild catalytic conditions, is environmentally friendly, and has low cost. The problem of large amounts of "three wastes" in existing synthesis methods can be effectively solved, and the method has high practical application value.

[0071] Example 10: Whole cell catalysis - synthesis of 3-hydroxytetrahydrofuran into 3-ketotetrahydrofuran

[0072] S-3-hydroxytetrahydrofuran (concentration of 30 g / L), co-substrate acetone (concentration of 60%), whole cell catalyst (0.5 g), NADP + (concentration of 0.1 Mm), and dilute to 5 mL with 0.1 M phosphate buffer. React at 30°C, 200 rpm for 48 h, centrifuge (8000 rpm, 10 min), take 100 μL of supernatant, dilute with methanol, and measure the reaction conversion rate by gas phase, 5.3%, and yield 0.74%.

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

Claims

1. Use of an alcohol dehydrogenase mutant having an amino acid sequence as shown in SEQ ID NO.3 or a thermophilic anaerobic bacillus containing the gene as shown in SEQ ID NO.2 in catalyzing the synthesis of 3-ketotetrahydrofuran.

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

1.

3. A method for preparing 3-ketotetrahydrofuran, characterized in that: Using (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran as a substrate, 3-ketotetrahydrofuran is obtained by catalysis using 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.

4. The method according to claim 3, characterized in that The concentration of (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran in the reaction system is 5-50 g / L.

5. The method according to claim 3, characterized in that: The conversion reaction was carried out at 30°C for 1-72 hours in phosphate buffer at pH 7.2-7.

4.

6. The method according to claim 3, characterized in that The reaction system contains 0.1-1mM coenzyme NADP + .

7. The method according to claim 3, characterized in that: The reaction system contains a co-substrate with a concentration of 10-100%, which is any one of acetone, 2-pentanone, methyl isopropyl ketone, cyclopropyl methyl ketone, 2-hexanone, methyl isobutyl ketone, tetrahydrothiophene-3-one, tert-butyl 3-oxopyrrolidine-1-carboxylate, N-tert-butyloxycarbonyl-3-piperidone and 1,3-dihydroxyacetone co-substrate.

8. The method according to claim 3, characterized in that The amount of the alcohol dehydrogenase mutant with the amino acid sequence shown in SEQ ID NO.3 added is 1.5-2.0 mg / mL; the amount of the whole cells of the thermophilic anaerobic bacteria used is 0.05-0.25 g / mL.

9. A method for improving the yield, conversion rate and selectivity of 3-ketotetrahydrofuran, characterized in that: With (R)-(-)-3-hydroxytetrahydrofuran or (S)-(+)-3-hydroxytetrahydrofuran as substrate, any one of acetone, 2-pentanone, methyl isopropyl ketone, cyclopropyl methyl ketone, 2-hexanone, methyl isobutyl ketone, tetrahydrothiophene-3-one, tert-butyl 3-oxopyrrolidine-1-carboxylate, N-tert-butyloxycarbonyl-3-piperidone and 1,3-dihydroxyacetone as cosubstrate, and adding coenzyme NADP + , using the alcohol dehydrogenase mutant with the amino acid sequence shown in SEQ ID NO.3 to catalyze and improve the yield of 3-ketotetrahydrofuran.

10. Use of a thermophilic anaerobic bacillus having an alcohol dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO.2 and a gene with a nucleotide sequence as shown in SEQ ID NO.3 in improving the yield, conversion rate and selectivity of 3-ketotetrahydrofuran.

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