Centimeter-level continuous flow reaction device and application of centimeter-level continuous flow reaction device in biological catalysis aerobic reaction

Abstract

The invention discloses a centimeter-level continuous flow reaction device which comprises at least one liquid storage tank, a gas cylinder, a T-shaped mixer and a micro-reactor, inlets of the liquid storage tank and the gas cylinder are communicated with two ends of the T-shaped mixer respectively, the other end of the T-shaped mixer is connected with the micro-reactor, the micro-mixer is arranged between the T-shaped mixer and the micro-reactor, and the T-shaped mixer is connected with the micro-reactor. The micro-mixer is filled at an inlet of the micro-reactor, the micro-mixer is provided with a pore channel communicated with a channel of the micro-reactor, and a gas-liquid mixture generates bubbles after flowing through the micro-mixer. The invention further discloses an application of the device in a gas-liquid two-phase participated reaction in continuous flow, in particular an application of catalyzing cyclohexanol to prepare epsilon-caprolactone by using ADH1-BVMO whole-cell sap. The invention provides a universal, simple and convenient device and method for improving the oxygen transmission efficiency for the biological catalysis aerobic reaction, and provides actual examples and references for the biological catalysis aerobic reaction in continuous flow.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis devices and technologies, and particularly relates to a centimeter-level continuous flow reaction device and its application in biocatalytic aerobic reactions. Background Art

[0002] With the development of biocatalysis towards multi-step reactions, biosynthesis of multifunctional complex molecules has been widely used, but there are still many obstacles that make it difficult to achieve rapid expansion. In the oxidation reaction catalyzed by biological enzymes, the problem of oxygen transfer efficiency caused by the insolubility of oxygen in water is one of the key factors restricting the reaction efficiency. In intermittent reactions, gas-liquid mass transfer is mainly achieved through aeration, stirring and other methods to disperse the bubbles, thereby increasing the dissolution rate and diffusion efficiency of oxygen in the liquid. However, in order to further adapt to the needs of industrial production, when faced with the need to scale up the gas / liquid reaction in the intermittent reactor, simply relying on continuously increasing the size of the reactor to maintain efficient gas / liquid diffusion is obviously not an easy solution to achieve.

[0003] The use of continuous flow mode is an attractive solution to improve mass transfer and productivity. Through the microflow effect and continuous flow reaction, the interfacial surface area can be maximized, the total reaction time can be minimized, and the reaction efficiency can be improved, but the gas-liquid contact area is still very limited. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a continuous flow reactor for improving the oxygen transfer efficiency in view of the shortcomings of the prior art. The present invention can maximize the interface specific surface area, minimize the total reaction time and improve the reaction efficiency through the microflow effect and continuous flow reaction.

[0005] The final problem to be solved by the present invention is to use the ADH1-BVMO whole cell as a biocatalyst to catalyze the reaction of cyclohexanol to produce ε-caprolactone as a model reaction, and apply the above-mentioned continuous flow reactor to improve the conversion rate of the aerobic reaction.

[0006] In order to solve the above technical problems, the present invention discloses a centimeter-level continuous flow reaction device, which is characterized in that it includes at least one liquid storage tank, a gas cylinder, a T-type mixer and a microreactor, the inlets of the liquid storage tank and the gas cylinder are respectively connected to the two ends of the T-type mixer, and the other end of the T-type mixer is connected to the microreactor. Its characteristics are that a micromixer is arranged between the T-type mixer and the microreactor, the micromixer is filled at the inlet of the microreactor, the micromixer has a pore channel connected to the channel of the microreactor, and bubbles are generated after the gas-liquid mixture flows through the micromixer.

[0007] In one embodiment, there are two liquid storage tanks, each of which is connected to a flow pump. The two liquid storage tanks are connected in parallel and then connected to a T-type mixer. Preferably, the two liquid storage tanks are connected using a Y-type mixer and then connected to a T-type mixer.

[0008] In one embodiment, the micro mixer is formed by extruding a diamond wire mesh having a length of 10-50 cm, a width of 5-10 cm, and a pore range of 0.5 mm-1 mm into a structure having a diameter of 1 mm-10 mm and a length of 1 cm-2 cm. In fact, there are many methods for preparing a micro mixer, the purpose of which is to enable the gas-liquid mixture introduced to generate dense bubbles in the micrometer level through a dense pore structure. The method is simple to prepare, and the gas-liquid mixture can generate dense bubbles after flowing through the micro mixer.

[0009] The present application further discloses the application of the continuous flow reaction device in a reaction involving gas and liquid phases in a continuous flow, wherein the liquid is a whole cell solution or a crude enzyme solution. In fact, the system is suitable for aerobic reactions of liquids with a certain viscosity.

[0010] As a specific application, the present application discloses a method for preparing ε-caprolactone using the above-mentioned continuous flow reaction device, comprising the following steps: pumping ADH1-BVMO whole cell fluid and cyclohexanol from a flow synthesizer into a Y-type mixer for mixing, and then connecting the outlet of the Y-type mixer with the air flow meter of the air bottle with a T-shaped mixer, and the gas-liquid mixture flowing out from the other end of the T-type mixer flows into a microreactor for reaction through a micromixer, wherein the ADH1-BVMO whole cell fluid is prepared by the following method ... ADH1 of sp. NCIMB9871 and BVMO derived from Acinetobacter sp. NCIMB9871 were constructed into plasmid pETduet-1, and then introduced into engineered Escherichia coli BL21 (DE3) to obtain ADH1-BVMO bacteria. The bacterial solution was inoculated into LB liquid culture medium, and cultured after adding kanamycin sulfate. The obtained bacterial solution was transferred to TB culture medium containing kanamycin antibiotics for expansion culture, and then IPTG was added to induce protein expression. The bacterial solution after induced expression was centrifuged to obtain the supernatant, the bacterial precipitate was collected, and it was resuspended in PBS buffer to prepare the whole cell solution of the required concentration.

[0011] More specifically, the ADH1-BVMO whole cell solution is prepared by the following method: ADH1 derived from Acinetobacter sp. NCIMB9871 and BVMO derived from Acinetobacter sp. NCIMB9871 are constructed into plasmid pETduet-1, and then introduced into engineered Escherichia coli BL21 (DE3) to obtain ADH1-BVMO bacteria, 50 μL of bacterial solution is inoculated into a 15 mL centrifuge tube containing 5 mL LB liquid culture medium, 5 μL of 50 mg / mL kanamycin sulfate is added, and the mixture is placed in a constant temperature shaker at 37°C and 250 rpm for overnight culture. 3 mL of overnight cultured bacterial solution is transferred to a TB medium (300 mL, 1 L wide-mouthed triangular flask) with a final concentration of 50 μg / mL kanamycin antibiotics, and cultured at 37°C and 250 rpm until OD600 is 0.5-0.7. After cooling the bottle containing the culture in a refrigerator at 4°C for 30 minutes, 300 μL of 0.3 mM IPTG was added and protein expression was induced at 20°C and 250 rpm for 24 hours. The induced bacterial solution was centrifuged at 8500 rpm for 5 minutes at 4°C, the supernatant was discarded, the precipitated bacteria were collected, and the cells were resuspended in PBS buffer to prepare the whole cell solution of the required concentration.

[0012] Preferably, the ADH1-BVMO whole cell solution and cyclohexanol are pumped into a Y-type mixer at a flow rate of 90 μL / min-100 μL / min by a flow synthesizer for mixing.

[0013] The final concentration of the whole cells in the ADH1-BVMO whole cell solution is 0.075 g / mL to 0.2 g / mL, and the final concentration of cyclohexanol is 20 mM to 40 mM.

[0014] Preferably, the flow rate of the air flow meter of the air bottle is 0.5 mL / min to 3 mL / min.

[0015] Preferably, the microreactor is a polytetrafluoroethylene pipe with an inner diameter of 1.2 mm to 10 mm, a residence time of 5 min to 20 min, and a reaction temperature of 20° C. to 30° C.

[0016] Preferably, the water bath temperature is regulated to 20° C. to 30° C. by a magnetic stirrer.

[0017] In order to achieve better gas-liquid mass transfer efficiency, the present invention designs a micro-mixer for continuous flow reaction to greatly increase the gas-liquid contact area, thereby improving the oxygen transfer efficiency, and uses the biocatalytic conversion of cyclohexanol to generate ε-caprolactone as a model reaction, and uses alcohol dehydrogenase and monooxygenase to form a biological hydrogen borrowing system to achieve the circulation of NADPH. In addition, the scale of the reactor is expanded to the centimeter level, which greatly improves the production efficiency and lays a foundation for industrial application. The present invention provides a universal and simple device and method for improving the oxygen transfer efficiency for biocatalytic aerobic reactions, and provides practical examples and references for biocatalytic aerobic reactions in continuous flow.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0019] (1) Compared with the intermittent mode, the present invention has better mass transfer / heat transfer effect, simpler device operation and response setting, and easier batch processing and reaction scale-up by running the reaction in continuous flow;

[0020] (2) The present invention can precisely control the product, such as increasing the gas-liquid contact surface area, sufficient micro-mixing, enhanced mass transfer and online removal of the product, thereby significantly promoting the reaction equilibrium control and avoiding product inhibition and by-product formation. At present, the precise control methods of the product in the continuous flow reaction are mainly divided into the fine regulation of the reaction parameters in the process and the online purification of the crude product;

[0021] (3) The present invention has more significant advantages in terms of green and sustainable development. On the one hand, it can avoid the use of toxic, harmful and difficult to degrade chemical catalysts. On the other hand, since it is no longer limited by the separation of intermediates, the operation time and waste required for the reaction are greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the continuous flow device route;

[0023] Figure 2 Schematic diagram of bubbling of micro mixer in continuous flow reactor;

[0024] Figure 3 Schematic diagram of optimization of air flow rate in a continuous flow reactor;

[0025] Figure 4 Schematic diagram of the effect of tube diameter on conversion rate at different times in a continuous flow reactor. DETAILED DESCRIPTION

[0026] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0027] like Figure 1 As shown, the continuous flow reaction device of the present application includes a flow synthesizer connected by a Y-shaped mixer channel and a T-shaped pipeline and an air flow meter connected to an air bottle, and the gas-liquid mixture flows into the pipeline through the micro mixer for reaction; the pipeline is a polytetrafluoroethylene tube. The micro mixer is prepared by the following method: a diamond wire mesh with a length of 10-50cm, a width of 5-10cm, and a pore range of 0.5mm-1mm is extruded into a nearly cylindrical body with a diameter of 1mm-10mm (matching the inner diameter of the polytetrafluoroethylene tube) and a length of 1cm-2cm.

[0028] In the embodiment of the present invention, it is preferably extruded into a nearly cylindrical body of about 1.5 cm in length. It should be understood by those skilled in the art that the preparation method of the micro mixer is flexible and varied, and the purpose is to enable the gas-liquid mixture introduced to produce dense micron-level bubbles after passing through the micro mixer. In the setting of the present application, the prepared micro mixer can produce bubbles with a diameter of about 100 microns. Those skilled in the art can adjust the compression length of the micro mixer according to the actual reaction situation.

[0029] The ADH1-BVMO whole cell solution described in the following examples was prepared by the following steps:

[0030] (1) ADH1 from Acinetobacter sp. NCIMB9871 and ADH1 from Acinetobacter sp.

[0031] BVMO of NCIMB9871 was constructed into plasmid pETduet-1 and introduced into engineered E. coli BL21 (DE3)

[0032] , and obtained ADH1-BVMO bacteria;

[0033] (2) Take 50 μL of the ADH1-BVMO bacteria constructed in step (1) and inoculate it into a 15 mL centrifuge tube containing 5 mL of LB liquid medium, add 5 μL of 50 mg / mL kanamycin sulfate, and place it at 37°C, 250

[0034] 3 mL of overnight culture was transferred to TB medium (300 mL, 1L wide-mouth Erlenmeyer flask) with a final concentration of 50 μg / mL kanamycin, and cultured at 37°C and 250 rpm until OD600 was 0.5-0.7. After cooling the bottle containing the culture in a refrigerator at 4°C for 30 minutes, 300 μL of 0.3 mM IPTG was added and protein expression was induced at 20°C and 250 rpm for 24 hours.

[0035] (3) The bacterial solution in step (2) was centrifuged at 8500 rpm for 5 min at 4°C, the supernatant was discarded, the precipitated bacteria were collected, and the cells were resuspended in PBS buffer (50 mM, pH 7.4) to prepare a whole cell solution of the required concentration.

[0036] The continuous flow reactor experimental method described in the following examples is as follows: (1) ADH1-BVMO whole cell solution and cyclohexanol are pumped into a Y-type mixer by a flow synthesizer for mixing, and then a T-tube is used to connect the effluent of the Y-type mixer to an air flow meter connected to an air bottle. The gas-liquid mixture flows into a polytetrafluoroethylene (PTFE) pipe through a micro mixer for reaction; (2) the enzyme concentration, substrate concentration and air flow rate in the continuous flow reactor are adjusted, the inner diameter of the continuous flow reaction is expanded, and the substrate conversion rate and product generation rate are analyzed to obtain suitable reaction conditions.

[0037] The effluent from the continuous flow pipeline was extracted with ethyl acetate, and the organic phase extract was filtered through a 0.22 μm organic filter head. The substrate conversion rate and product yield were analyzed by GC.

[0038] The method for analyzing the substrate conversion rate and product formation rate described in the following examples is high performance gas chromatography analysis, chromatographic column: CP-Chirasil (30m x 0.32mm x 0.25μm), using a hydrogen flame (FID) detector. Heating program: initial temperature 100°C, 2°C / min heating to 130°C, 10°C / min heating to 200°C. The retention time of substrate cyclohexanol is 4.603min, the retention time of intermediate product cyclohexanone is 3.611min, and the retention time of product ε-caprolactone is 10.301min.

[0039] Example 1

[0040] The solution containing substrate cyclohexanol and ADH1-BVMO whole cell solution were pumped in at a flow rate of 100 μL / min. After mixing in a Y-type mixer, the final concentration of cyclohexanol was 30 mM and the final concentration of ADH1-BVMO whole cells was 0.1 g / mL. The air flow meter flow rate was adjusted to 1 mL / min, and the reaction solution and air were pumped into a T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 1.2 mm and a micro mixer at the front end, and finally dense bubbles with a diameter of about 100 microns were generated in the reactor pipe. When the reaction temperature was 30°C and the residence time was 10 min, the conversion rate of substrate cyclohexanol was about 35%.

[0041] Example 2

[0042] The solution containing the substrate cyclohexanol and the ADH1-BVMO whole cell solution were pumped in at a flow rate of 100 μL / min. After mixing in the Y-type mixer, the final concentration of cyclohexanol was 30 mM and the final concentration of ADH1-BVMO whole cells was 0.1 g / mL. The air flow meter flow rate was adjusted to 1 mL / min, and the reaction solution and air were pumped into the T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 1.2 mm and a micro mixer at the front end, and finally dense bubbles with a diameter of about 100 microns were generated in the reactor pipe. When the reaction temperature was 30 ° C and the residence time was 20 min, the substrate cyclohexanol was completely converted.

[0043] Example 3

[0044] The solution containing substrate cyclohexanol and ADH1-BVMO whole cell solution were pumped in at a flow rate of 100 μL / min. After mixing in a Y-type mixer, the final concentration of cyclohexanol was 20 mM and the final concentration of ADH1-BVMO whole cells was 0.075 g / mL. The air flow meter flow rate was adjusted to 1 mL / min, and the reaction solution and air were pumped into a T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 1.2 mm and a micro mixer at the front end, and finally dense bubbles with a diameter of about 100 microns were generated in the reactor pipe. When the reaction temperature was 30°C and the residence time was 20 min, the conversion rate of substrate cyclohexanol was about 94%.

[0045] Example 4

[0046] The solution containing substrate cyclohexanol and ADH1-BVMO whole cell solution were pumped in at a flow rate of 100 μL / min. After mixing in a Y-type mixer, the final concentration of cyclohexanol was 40 mM and the final concentration of ADH1-BVMO whole cells was 0.1 g / mL. The air flow meter flow rate was adjusted to 1 mL / min, and the reaction solution and air were pumped into a T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 1.2 mm and a micro mixer at the front end, and finally dense bubbles with a diameter of about 100 microns were generated in the reactor pipe. When the reaction temperature was 30°C and the residence time was 20 min, the conversion rate of substrate cyclohexanol was about 40%.

[0047] Example 5

[0048] The final concentration of cyclohexanol was 20mM, and the final concentration of ADH1-BVMO whole cells was 0.075g / mL. The mixture was pumped into a Y-type mixer at a flow rate of 100μL / min, and the air flow rate was adjusted to 1mL / min. The reaction liquid and air were pumped into a T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 10mm and equipped with a micro mixer at the front end, and dense bubbles with a diameter of about 100 microns were generated in the reactor pipe. When the reaction temperature was 30°C and the residence time was 20min, the conversion rate of the substrate cyclohexanol was about 94%.

[0049] Example 6

[0050] The final concentration of cyclohexanol was 30mM, and the final concentration of ADH1-BVMO whole cells was 0.1g / mL. The mixture was pumped into a Y-type mixer at a flow rate of 100μL / min, and the air flow rate was adjusted to 1mL / min. The reaction liquid and air were pumped into a T-type tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 10mm and equipped with a micro mixer at the front end, and dense bubbles with a diameter of about 100 microns were generated in the reactor pipe. When the reaction temperature was 30°C and the residence time was 20min, the conversion rate of substrate cyclohexanol was about 99%.

[0051] Example 7

[0052] The final concentration of cyclohexanol was 40mM, and the final concentration of ADH1-BVMO whole cells was 0.1g / mL. The mixture was pumped into a Y-type mixer at a flow rate of 100μL / min, and the air flow rate was adjusted to 1mL / min. The reaction liquid and air were pumped into a T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 10mm and equipped with a micro mixer at the front end, generating dense bubbles with a diameter of about 100 microns in the reactor pipe. When the reaction temperature was 30°C and the residence time was 40min, the substrate cyclohexanol was completely converted.

[0053] Example 8

[0054] The final concentration of cyclohexanol was 20mM, and the final concentration of ADH1-BVMO whole cells was 0.125g / mL. The mixture was pumped into a Y-type mixer at a flow rate of 100μL / min, and the air flow rate was adjusted to 1mL / min. The reaction liquid and air were pumped into a T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 10mm and equipped with a micro mixer at the front end, generating dense bubbles with a diameter of about 100 microns in the reactor pipe. When the reaction temperature was 30°C and the residence time was 20min, the substrate cyclohexanol was completely converted.

[0055] Example 9

[0056] The final concentration of cyclohexanol was 20mM, and the final concentration of ADH1-BVMO whole cells was 0.1g / mL. The mixture was pumped into a Y-type mixer at a flow rate of 100μL / min, and the air flow rate was adjusted to 1mL / min. The reaction liquid and air were pumped into a T-type tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 10mm and equipped with a micro mixer at the front end, and dense bubbles with a diameter of about 100 microns were generated in the reactor pipe. When the reaction temperature was 30°C and the residence time was 20min, the conversion rate of substrate cyclohexanol was about 99%.

[0057] Example 10

[0058] The final concentration of cyclohexanol was 40mM, and the final concentration of ADH1-BVMO whole cells was 0.2g / mL. The mixture was pumped into a Y-type mixer at a flow rate of 100μL / min, and the air flow rate was adjusted to 1mL / min. The reaction liquid and air were pumped into a T-tube for mixing, and then passed through a continuous flow polytetrafluoroethylene (PTFE) pipe with an inner diameter of 10mm and equipped with a micro mixer at the front end, generating dense bubbles with a diameter of about 100 microns in the reactor pipe. When the reaction temperature was 30°C and the residence time was 20min, the substrate cyclohexanol was completely converted.

[0059] Comparative Example 1

[0060] In a 25mL stoppered conical flask, add 3mL of 0.2g / mL ADH1-BVMO whole cell solution with a pipette, add 3mL of 45mM cyclohexanol (1% acetonitrile, 99% water), and shake on a shaker at 30°C and 220rpm for 12h. Pipette 1mL of the reaction solution, add 1mL of ethyl acetate, mix thoroughly with a mixer, centrifuge at high speed, and draw the upper ethyl acetate, filter through a 0.22μm organic filter, and analyze the substrate conversion rate with GC, which is 62%.

[0061] Comparative Example 2

[0062] In a 25mL blue thread bottle, add 6mL 0.2g / mL ADH1-BVMO whole cell solution with a pipette, add 6mL 60mM cyclohexanol (1% acetonitrile, 99% water), and shake in a constant temperature shaker at 30°C and 220rpm. Pipette 1mL of the reaction solution, add 1mL ethyl acetate, mix thoroughly with a mixer, centrifuge at high speed, and draw the upper ethyl acetate, filter through a 0.22μm organic filter, and analyze the substrate conversion rate with GC, which is 55%.

[0063] In summary, the present invention uses ADH1-BVMO whole cells as a biocatalyst to catalyze the reaction of cyclohexanol to generate ε-caprolactone as a model reaction, and designs a micro-mixer in a continuous flow. Compared with the plunger flow generated when the gas-liquid is in contact with the ordinary gas, the present design can form micron-sized small bubbles at the moment of gas-liquid contact, which greatly improves the gas-liquid contact specific surface area in the continuous flow reactor, thereby improving the oxygen transfer efficiency. As the inner diameter of the pipeline increases, the reaction still maintains a high reaction rate, which may be because the apparent size of the tiny bubbles bulging out of the micro-mixer affects the reaction rate. To some extent, such a process design breaks through the amplification effect of the continuous flow pipeline and provides more possibilities for the industrial application of this system.

[0064] The present invention provides a method and idea for increasing the oxygen transfer efficiency of an aerobic enzyme-catalyzed reaction system. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A centimeter-level continuous flow reaction device, characterized in that: The invention comprises at least one liquid storage tank, a gas cylinder, a T-type mixer and a microreactor, wherein the inlets of the liquid storage tank and the gas cylinder are respectively communicated with the two ends of the T-type mixer, and the other end of the T-type mixer is connected with the microreactor. The invention is characterized in that a micromixer is arranged between the T-type mixer and the microreactor, the micromixer is filled at the inlet of the microreactor, the micromixer has a pore channel communicated with the channel of the microreactor, and bubbles are generated after the gas-liquid mixture flows through the micromixer.

2. The continuous flow reaction device according to claim 1, characterized in that: There are two liquid storage tanks, each of which is connected to a flow pump. The two liquid storage tanks are connected in parallel and then connected to a T-type mixer. Preferably, the two liquid storage tanks are connected by a Y-type mixer and then connected to a T-type mixer.

3. The continuous flow reaction device according to claim 1, characterized in that: The micro mixer is prepared by extruding a diamond wire mesh having a length of 10-50 cm, a width of 5-10 cm, and a pore range of 0.5 mm-1 mm into a structure having a diameter of 1 mm-10 mm and a length of 1 cm-2 cm.

4. Use of the continuous flow reaction device according to any one of claims 1 to 3 in a reaction involving gas and liquid phases in a continuous flow, wherein: The liquid is whole cell liquid or crude enzyme liquid.

5. A method for preparing ε-caprolactone using the continuous flow reaction device according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: pumping ADH1-BVMO whole cell solution and cyclohexanol into a Y-type mixer through a flow synthesizer for mixing, and then connecting the outlet of the Y-type mixer to the air flow meter of an air bottle through a T-type mixer, and the gas-liquid mixed liquid flowing out from the other end of the T-type mixer flows into a microreactor through a micromixer for reaction, wherein the ADH1-BVMO whole cell solution is prepared by the following method: ADH1 derived from Acinetobacter sp. NCIMB9871 and cyclohexanol derived from Acinetobacter sp. The BVMO of NCIMB9871 was constructed into the plasmid pETduet-1, and then introduced into the engineered Escherichia coli BL21 (DE3) to obtain ADH1-BVMO bacteria. The bacterial solution was inoculated into LB liquid culture medium, and cultured after adding kanamycin sulfate. The obtained bacterial solution was transferred to TB medium containing kanamycin antibiotics for expansion culture, and then IPTG was added to induce protein expression. The bacterial solution after induced expression was centrifuged to obtain the supernatant, and the bacterial precipitate was collected and resuspended in PBS buffer to prepare the whole cell solution of the required concentration.

6. The method according to claim 5, characterized in that ADH1-BVMO whole cell solution and cyclohexanol were pumped into the Y-type mixer at a flow rate of 90 µL / min-100 µL / min by the flow synthesizer for mixing.

7. The method according to claim 5, characterized in that The final concentration of the whole cells in the ADH1-BVMO whole cell solution is 0.075 g / mL to 0.2 g / mL, and the final concentration of cyclohexanol is 20 mM to 40 mM.

8. The method according to claim 5, characterized in that The flow rate of the air flow meter of the air bottle is 0.5 mL / min~3 mL / min.

9. The method according to claim 5, characterized in that The microreactor is a polytetrafluoroethylene pipe with an inner diameter of 1.2 mm to 10 mm, a residence time of 5 min to 20 min, and a reaction temperature of 20° C. to 30° C.

10. The method according to claim 9, characterized in that The water bath temperature was regulated at 20°C~30°C by a magnetic stirrer.

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