Method for immobilizing halohydrin dehalogenase based on single-layer MnO2 nanosheet and application of halohydrin dehalogenase
By using a single -layer MNO2 nanofatter fixed halogenic halide enzyme, the problem of reduced catalytic efficiency during enzyme fixation is solved, the efficient load and reuse of enzyme are achieved, and it is successfully applied to a specific process.
Patent Information
- Application Number
- CN202411631632.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-15
- Publication Date
- 2025-01-28
AI Technical Summary
The prior art can easily lead to a reduction in the catalytic efficiency of the enzyme when immobilizing the enzyme on a single layer two-dimensional nanomaterial.
Using a single -layer MNO2 nanos tablet as a fixed carrier of the enzyme, through multiple forces such as static electricity interaction, Van Dehua and hydrogen bonds, fix the halogen enzymes, and optimize the fixed conditions to improve the load and reuse of enzymes and reused utilization sex.
It significantly improves the load and reusability of enzymes, maintains the activity and structural stability of the enzyme, and is successfully applied to the process of catalyzing 1,3-dichloropllori-2-propionine synthetic acromatine.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a method and application of immobilizing halohydrin dehalogenase based on a single-layer MnO2 nanosheet. Background Art
[0002] In the modern biochemical industry, enzymes, as a biocatalyst, are widely used in various complex chemical reactions due to their high efficiency and strong specificity. However, due to the poor stability and difficulty in recycling of free enzymes, their large-scale application in industrial production is limited. Compared with free enzymes, immobilized enzymes have the advantages of high stability and reusability, and have stronger economic benefits in industrial production. Enzyme immobilization technology based on adsorption is a simple immobilization method that requires fewer materials and steps, is cost-effective and easy to implement. In large-scale industrial production, adsorption technology is often the preferred method for enzyme immobilization.
[0003] Ultrathin two-dimensional nanomaterials are a type of lamellar nanomaterials with a lateral dimension of more than 100 nanometers and a thickness of less than 5 nanometers. They have been widely used in many fields such as biomedicine, targeted therapy, biosensors, environmental remediation, and enzyme immobilization. It can be imagined that single-layer nanosheets can show the most active sites for the binding of biomolecules and are an ideal enzyme immobilization carrier. So far, graphene oxide and its derivatives have been the most widely studied for enzyme immobilization among single-layer two-dimensional nanomaterials. However, due to the presence of oxygen-containing functional groups (such as carbonyl, carboxylic acid, hydroxyl, and alkoxy) on the surface of graphene oxide, the enzyme activity is often reduced after immobilization. Therefore, it is of great significance to develop a new single-layer two-dimensional nanomaterial for enzyme immobilization.
[0004] MnO2 is a common transition metal oxide with unique properties, high abundance, low cost and environmental friendliness. Studies have shown that MnO2 nanosheets can be used as carriers for nucleic acids, drugs and nanoprobes. Single-layer MnO2 nanosheets have the largest surface area and the smallest thickness, and are expected to be effective carriers for enzyme immobilization. Although other forms of MnO2 nanostructures, such as mesoporous or hollow sea urchin-like structures, have been used for enzyme immobilization, they have defects such as low enzyme loading and low enzyme catalytic efficiency. Summary of the invention
[0005] The present invention aims to overcome the defect that the catalytic efficiency of the enzyme is easily reduced when the enzyme is fixed on the existing monolayer two-dimensional nanomaterial. A method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet is provided, and the method is applied to the process of catalyzing the synthesis of chiral epichlorohydrin from 1,3-dichloro-2-propanol.
[0006] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet comprises the following steps: (S.1) mixing sodium dodecyl sulfate solution and sulfuric acid solution in ultrapure water and preheating in an oil bath, then adding potassium permanganate solution, reacting fully until a colloidal solution is obtained, and continuing to heat to obtain a precipitate; taking the precipitate and letting it stand overnight, washing it, and redispersing it in ultrapure water to obtain a colloidal suspension of MnO2 nanosheets; (S.2) The colloidal suspension of MnO2 nanosheets obtained in step (S.1) is mixed with the purified halohydrin dehalogenase solution and the pH of the mixed solution is adjusted. The temperature is controlled and oscillated. After the reaction is complete, the solution is centrifuged and washed to obtain halohydrin dehalogenase immobilized on a monolayer of MnO2 nanosheets.
[0007] Single-layer MnO2 nanosheets have become a promising enzyme immobilization carrier due to their good chemical stability, large specific surface area and good biocompatibility. The surface of MnO2 nanosheets can be easily functionalized by physical or chemical methods, and can effectively bind to enzyme molecules through electrostatic interactions, van der Waals forces and hydrogen bonds, and their wide specific surface area provides numerous binding sites for enzyme immobilization. In addition, the layered structure of this material helps to increase the loading capacity of immobilized enzymes and provide a stable microenvironment for the enzymes, thereby maintaining their activity and structural stability.
[0008] Therefore, the present invention provides an enzyme immobilization method based on monolayer manganese dioxide nanosheets, using soil radiobacterium Agrobacterium radiobacter AD1's halohydrin dehalogenase (HheC) is a model enzyme. Through multiple forces such as electrostatic interaction, van der Waals force and hydrogen bond between the monolayer MnO2 nanosheets and the enzyme, efficient immobilization of the halohydrin dehalogenase is achieved, which significantly improves the enzyme loading capacity and reusability, which is of great significance to the industrial application of halohydrin dehalogenase.
[0009] Preferably, the concentration of the sodium dodecyl sulfate solution added in step (S.1) is 50-200 mM; the concentration of the sulfuric acid solution is 50-200 mM.
[0010] As further preferred, the concentration of the sodium dodecyl sulfate solution added in step (S.1) is 100 mM; the concentration of the sulfuric acid solution is 100 mM.
[0011] Preferably, the volume of ultrapure water added in step (S.1) is 250-300 mL.
[0012] As a further preference, the volume of ultrapure water added in step (S.1) is 283.2 mL.
[0013] Preferably, the oil bath temperature during the preheating process in the oil bath in step (S.1) is 90-100°C, and the preheating time is 10-30 min.
[0014] As a further preference, in the step (S.1), the oil bath temperature of the preheating process in the oil bath is 95° C., and the preheating time is 20 min.
[0015] Preferably, the concentration of the potassium permanganate solution added in step (S.1) is 10-100 mM.
[0016] As further preferred, the concentration of the potassium permanganate solution added in step (S.1) is 50 mM.
[0017] Preferably, the mass concentration of MnO2 nanosheets contained in the MnO2 nanosheet colloidal suspension added in step (S.2) is 0.1-2 mg / mL.
[0018] As a further preference, the mass of MnO2 nanosheets contained in the 1.5 mL MnO2 nanosheet colloidal suspension added in step (S.2) is 1.5 mg.
[0019] Preferably, the concentration of the purified halohydrin dehalogenase solution added in step (S.2) is 1-5 mg / mL.
[0020] As further preferred, the concentration of the purified halohydrin dehalogenase solution added in step (S.2) is 2 mg / mL.
[0021] Preferably, in step (S.2), the pH of the mixed solution is adjusted to 4-8.
[0022] As a further preference, in step (S.2), the pH of the mixed solution is adjusted to 6.
[0023] Preferably, the reaction temperature of the temperature control and oscillation process in step (S.2) is 0-10°C, the oscillation speed is 500-1000 rpm, and the reaction time is 20-90 min.
[0024] As a further preference, in the temperature control and oscillation process in step (S.2), the reaction temperature is 4°C, the oscillation speed is 800 rpm, and the reaction time is 40 min.
[0025] The application of the monolayer MnO2 nanosheet-immobilized halohydrin dehalogenase prepared by the method of immobilizing halohydrin dehalogenase on monolayer MnO2 nanosheets as described above in catalyzing the synthesis of chiral epichlorohydrin from 1,3-dichloro-2-propanol.
[0026] The present invention uses single-layer manganese dioxide nanosheets (Single-layer MnO2Nanosheets) and halohydrin dehalogenase (HheC) as research objects, immobilizes HheC on single-layer MnO2 nanosheets, and prepares single-layer MnO2 nanosheet-immobilized halohydrin dehalogenase. Subsequently, the immobilized enzyme is applied to the reaction of catalyzing 1,3-dichloro-2-propanol (1,3-DCP) to synthesize chiral epichlorohydrin ((S)-ECH).
[0027] Therefore, the present invention has the following beneficial effects: (1) The present invention prepares a single-layer MnO2 nanosheet, and uses the characteristics of the single-layer MnO2 nanosheet with large specific surface area and good compatibility for the first time to immobilize the single-layer MnO2 nanosheet, thus developing a new method for immobilizing enzymes using the single-layer MnO2 nanosheet; (2) The present invention optimizes the immobilization temperature, time and pH conditions of the monolayer MnO2 nanosheet-immobilized halohydrin dehalogenase, and obtains the monolayer MnO2 nanosheet-immobilized halohydrin dehalogenase with good performance; (3) The halohydrin dehalogenase immobilized on the single-layer MnO2 nanosheet prepared by the present invention has strong operability, high loading rate and good reusability, and can effectively catalyze the synthesis of chiral epichlorohydrin from 1,3-dichloro-2-propanol. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Schematic diagram of the immobilization of halohydrin dehalogenase on the surface of monolayer manganese dioxide nanosheets.
[0029] Figure 2 Comparison of the relative enzyme activities of halohydrin dehalogenase immobilized on monolayer manganese dioxide nanosheets at different immobilization times.
[0030] Figure 3 Figure a) shows the relative enzyme activity comparison of halohydrin dehalogenase immobilized on monolayer manganese dioxide nanosheets at different immobilization pHs; Figure 3 (b) is a comparison of the loading rates of halohydrin dehalogenase immobilized on monolayer manganese dioxide nanosheets at different immobilization pHs.
[0031] Figure 4 The enzyme activity recovery rate and product yield in the reaction process of free halohydrin dehalogenase and halohydrin dehalogenase immobilized on monolayer MnO2 nanosheets catalyzing 200 mM 1,3-DCP to (S)-ECH ee Value comparison chart.
[0032] Figure 5 Cyclic batch performance diagram of halohydrin dehalogenase immobilized on monolayer manganese dioxide nanosheets. DETAILED DESCRIPTION
[0033] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.
[0034]
Enzyme expression and protein purification
[0035] Example 1 This embodiment provides a method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet, comprising the following steps: (S.1) Add 32 mL (100 mM) of sodium dodecyl sulfate solution (SDS) and 1.6 mL (100 mM) of H2SO4 solution to 283.2 mL of ultrapure water, mix well, and preheat in an oil bath at 95 °C for 20 min. Subsequently, 3.2 mL (50 mM) of KMnO4 solution is quickly added to start the chemical reaction. After about 2 h, a uniform dark brown colloidal solution can be obtained. Continue to heat for a long time, MnO2 nanosheets are formed and aggregated to obtain black precipitates. The precipitate is allowed to stand overnight and the black precipitate is collected by filtration, and then repeatedly washed with ultrapure water and ethanol for purification. Finally, the purified black precipitate (i.e., MnO2 nanosheet precipitate) is redispersed in ultrapure water to form a MnO2 nanosheet colloidal suspension; (S.2) The MnO2 nanosheet colloidal suspension (1.5 mL) containing 1.5 mg of monolayer MnO2 nanosheets obtained in step (S.1) was mixed with 0.5 mL of the purified halohydrin dehalogenase solution (2 mg mL -1 ) and the pH of the mixed solution was adjusted to 6. The mixed solution was shaken at 4 °C and 800 rpm for 40 min. After the reaction was complete, the mixed solution was centrifuged at 12000 rpm for 20 min and washed with ultrapure water to obtain the halohydrin dehalogenase immobilized on the surface of the monolayer MnO2 nanosheet. The schematic diagram of the immobilization of halohydrin dehalogenase on the surface of the monolayer manganese dioxide nanosheet is shown in Figure 1 As shown in Figure 2, since the surface of MnO2 nanosheets is negatively charged, the positively charged regions on the surface of halohydrin dehalogenase will adsorb to it through electrostatic interaction, and electrostatic adsorption enables the halohydrin dehalogenase to be successfully immobilized on the monolayer manganese dioxide nanosheets.
[0036] Example 2 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet, wherein the mixed solution in step (S.2) is subjected to an oscillation reaction at 4°C and 800 rpm for 20 min. The rest is the same as in Example 1.
[0037] Example 3 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet, wherein the mixed solution in step (S.2) is subjected to an oscillation reaction at 4°C and 800 rpm for 60 min. The rest is the same as in Example 1.
[0038] Example 4 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet, wherein the mixed solution in step (S.2) is subjected to an oscillation reaction at 4°C and 800 rpm for 90 min. The rest is the same as in Example 1.
[0039] Example 5 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein the pH of the mixed solution is adjusted to 4 in step (S.2). The rest is the same as in Example 1.
[0040] Example 6 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein the pH of the mixed solution is adjusted to 5 in step (S.2). Others are the same as in Example 1.
[0041] Example 7 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein the pH of the mixed solution is adjusted to 7 in step (S.2). Other steps are the same as those in Example 1.
[0042] Example 8 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein the pH of the mixed solution is adjusted to 8 in step (S.2). The rest is the same as in Example 1.
[0043] Example 9 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein in step (S.1), 10 mL (50 mM) of sodium dodecyl sulfate solution (SDS) and 1 mL (50 mM) of H2SO4 solution are added to 250 mL of ultrapure water and mixed evenly, and then preheated in an oil bath at 90 °C for 10 min. Subsequently, 1 mL (10 mM) of KMnO4 solution is quickly added to start the chemical reaction; in step (S.2), the MnO2 nanosheet colloidal suspension (1 mL) containing 1 mg of monolayer MnO2 nanosheets obtained in step (S.1) is mixed with 0.1 mL of the purified halohydrin dehalogenase solution (1 mg mL -1 ) and the pH of the mixed solution was adjusted to 6, and the mixed solution was shaken at 0°C and 500 rpm for 40 min. Others were the same as in Example 1.
[0044] Example 10 The difference between this embodiment and embodiment 1 is that: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein in step (S.1), 50 mL (200 mM) of sodium dodecyl sulfate solution (SDS) and 2 mL (200 mM) of H2SO4 solution are added to 300 mL of ultrapure water and mixed evenly, and then preheated in an oil bath at 100 °C for 30 min. Subsequently, 5 mL (100 mM) of KMnO4 solution is quickly added to start the chemical reaction; in step (S.2), the MnO2 nanosheet colloidal suspension (2 mL) containing 2 mg of monolayer MnO2 nanosheets obtained in step (S.1) is mixed with 1 mL of the purified halohydrin dehalogenase solution (5 mg mL -1 ) and the pH of the mixed solution was adjusted to 6, and the mixed solution was shaken at 10° C. and 1000 rpm for 40 min. Others were the same as in Example 1.
[0045] Comparative Example 1 The difference between this comparative example and Example 1 is: A method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet, wherein the mixed solution in step (S.2) is subjected to an oscillation reaction at 4°C and 800 rpm for 10 min. The rest is the same as in Example 1.
[0046] Comparative Example 2 The difference between this comparative example and Example 1 is: A method for immobilizing halohydrin dehalogenase based on a monolayer MnO2 nanosheet, wherein the mixed solution in step (S.2) is subjected to an oscillation reaction at 4°C and 800 rpm for 120 min. The rest is the same as in Example 1.
[0047] Comparative Example 3 The difference between this comparative example and Example 1 is: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein the pH of the mixed solution is adjusted to 3 in step (S.2). Others are the same as in Example 1.
[0048] Comparative Example 4 The difference between this comparative example and Example 1 is: A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, wherein the pH of the mixed solution is adjusted to 9 in step (S.2). Others are the same as in Example 1.
[0049]
Performance testing and analysis
Calculation of the enzyme activity and enzyme activity recovery rate of halohydrin dehalogenase immobilized on monolayer MnO2 nanosheets
[0050] Where: U1 represents the activity of immobilized enzyme; U2 represents the activity of free enzyme.
[0051] The monolayer MnO2 nanosheet-immobilized halohydrin dehalogenase was prepared according to the methods in Examples 1 to 4 and Comparative Examples 1 to 2, and its enzyme activity was determined by gas chromatography. The relative enzyme activity comparison of the monolayer MnO2 nanosheet-immobilized halohydrin dehalogenase at different immobilization times is shown in the figure below: Figure 2 shown.
[0052] Depend on Figure 2 Analysis shows that when the immobilization time is 40 min, the enzyme activity of halohydrin dehalogenase immobilized on monolayer manganese dioxide nanosheets is the highest and the immobilization effect is the best.
[0053]
Determination of enzyme amount and loading rate of halohydrin dehalogenase immobilized on monolayer MnO2 nanosheets
[0054] After the halohydrin dehalogenase was fixed under the above optimal operating conditions, the supernatant was collected by centrifugation and the protein content in the supernatant was determined using a BCA kit; the calculation formula for the loading rate was as follows: Where: M represents the amount of enzyme added; m is the amount of enzyme in the supernatant.
[0055] The halohydrin dehalogenase immobilized on the monolayer MnO2 nanosheets was prepared according to the methods in Example 1, Examples 5-8 and Comparative Examples 3-4, and the enzyme activity and the loading rate of the halohydrin dehalogenase immobilized on the monolayer manganese dioxide nanosheets were determined by gas chromatography. Figure 3 Figure a) shows the relative enzyme activity comparison of halohydrin dehalogenase immobilized on monolayer manganese dioxide nanosheets at different immobilization pH values. Figure 3Figure b) shows the comparison of the loading rates of halohydrin dehalogenase immobilized on monolayer manganese dioxide nanosheets at different immobilization pHs.
[0056] from Figure 3 From the data analysis, it can be seen that the halohydrin dehalogenase immobilized on the monolayer MnO2 nanosheets exhibited the highest enzymatic activity under the condition of pH=6; however, as the pH value decreased, the loading rate of the halohydrin dehalogenase showed an increasing trend. For example, the loading rate reached 55.3% under pH=3, while the loading rate dropped to 19.8% at pH=9. Considering the enzyme activity and loading rate, pH=6 was determined to be the optimal pH condition for the immobilization process. Under this pH value condition, the halohydrin dehalogenase immobilized on the monolayer MnO2 nanosheets can not only maintain a high activity, but also achieve a relatively high loading efficiency.
[0057]
Synthesis of (S)-ECH catalyzed by halohydrin dehalogenase immobilized on monolayer MnO2 nanosheets
[0058] from Figure 4 The analysis showed that the enzyme activity recovery rate of the halohydrin dehalogenase immobilized on the monolayer MnO2 nanosheets was 80.9%, and the (S)-ECH of the free halohydrin dehalogenase and the halohydrin dehalogenase immobilized on the monolayer MnO2 nanosheets reached 82.6% and 79.1% respectively after 2 h of reaction. ee The values reached 91.6% and 91.7% respectively. This proves that the halohydrin dehalogenase immobilized on the monolayer MnO2 nanosheets has good application.
[0059] Batch stability of halohydrin dehalogenase immobilized on monolayer MnO2 nanosheets The number of batches of halohydrin dehalogenase immobilized on a single-layer MnO2 nanosheet is also an important indicator for judging its performance. After verifying the catalytic performance of halohydrin dehalogenase immobilized on a single-layer MnO2 nanosheet, the halohydrin dehalogenase immobilized on a single-layer MnO2 nanosheet (i.e., prepared according to the method in Example 1 above) was recovered by centrifugation and its recycling performance was tested. The recycling batch performance of halohydrin dehalogenase immobilized on a single-layer MnO2 nanosheet is shown in FIG. Figure 5 shown.
[0060] from Figure 5 The analysis showed that after repeated use of 5 batches, 85% of the enzyme activity was still retained compared to the first batch, indicating that the halohydrin dehalogenase immobilized on monolayer MnO2 nanosheets has good batch stability.
[0061] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.
Claims
1. A method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets, characterized in that: The following steps are involved: (S.1) mixing sodium dodecyl sulfate solution and sulfuric acid solution in ultrapure water and preheating in an oil bath, then adding potassium permanganate solution, reacting fully until a colloidal solution is obtained, and continuing to heat to obtain a precipitate; taking the precipitate and letting it stand overnight, washing it, and redispersing it in ultrapure water to obtain a colloidal suspension of MnO2 nanosheets; (S.2) The colloidal suspension of MnO2 nanosheets obtained in step (S.1) is mixed with the purified halohydrin dehalogenase solution and the pH of the mixed solution is adjusted. The temperature is controlled and oscillated. After the reaction is complete, the solution is centrifuged and washed to obtain halohydrin dehalogenase immobilized on a monolayer of MnO2 nanosheets.
2. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: The concentration of the sodium dodecyl sulfate solution added in step (S.1) is 50-200 mM; the concentration of the sulfuric acid solution is 50-200 mM.
3. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: The volume of ultrapure water added in step (S.1) is 250~300 mL.
4. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: In step (S.1), the oil bath temperature during the preheating process in the oil bath is 90-100°C, and the preheating time is 10-30 min.
5. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: The concentration of the potassium permanganate solution added in step (S.1) is 10-100 mM.
6. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: The mass concentration of MnO2 nanosheets contained in the MnO2 nanosheet colloidal suspension added in step (S.2) is 0.1~2 mg / mL.
7. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: The concentration of the purified halohydrin dehalogenase solution added in step (S.2) is 1-5 mg / mL.
8. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: In step (S.2), the pH of the mixed solution is adjusted to 4-8.
9. The method for immobilizing halohydrin dehalogenase based on monolayer MnO2 nanosheets according to claim 1, characterized in that: The reaction temperature of the temperature control and oscillation process in step (S.2) is 0-10°C, the oscillation speed is 500-1000 rpm, and the reaction time is 20-90 min.
10. Use of the halohydrin dehalogenase immobilized on a single-layer MnO2 nanosheet prepared by the method for immobilizing halohydrin dehalogenase on a single-layer MnO2 nanosheet as claimed in any one of claims 1 to 9 in catalyzing the synthesis of chiral epichlorohydrin from 1,3-dichloro-2-propanol.