A catalytic system and method for the enzymatic hydrolysis of halogenated organics at the oil-water interface

Abstract

The application discloses a catalytic system and method for hydrolysis of halogenated organic matter catalyzed by enzyme at an oil-water interface. The catalytic system comprises an organic solvent, an aqueous solution and an enzyme-polymer conjugate enzyme; the enzyme-polymer conjugate enzyme is a conjugate of halogenated alkane dehalogenase and amphiphilic block copolymer covalently combined; in the enzyme-polymer conjugate enzyme, 2-3 molecules of amphiphilic block copolymer are connected to each molecule of enzyme. The application enriches the enzyme at the oil-water interface by constructing an oil-water two-phase reaction system and a conjugate synthesized by the amphiphilic block polymer and the enzyme; the accessibility of the substrate in the organic solvent is improved, meanwhile, the enzyme inactivation caused by the organic solvent is avoided, and the hydrolysis activity of the enzyme on the halogenated organic matter is improved; the enzyme catalytic reaction product hydrogen halide can be dissolved in the aqueous solution by constructing the oil-water two-phase reaction system, and the reduction of the enzyme catalytic activity caused by the retention of halogen ions in the enzyme product pocket is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to a catalytic system and method for enzymatic catalysis of hydrolysis of halogenated organic matter at the oil-water interface, belonging to the field of biological catalysis. BACKGROUND

[0002] Halogenated organic matter is widely used in chemical industry as raw materials, intermediates, solvents, etc., which also makes many halogenated organic matter inevitably discharged into the environment, becoming a common industrial pollutant. Halogenated organic pollutants are persistent and difficult to biodegrade, causing serious harm to the environment and human health.

[0003] At present, the treatment of halogenated organic pollutants mainly includes incineration, catalytic oxidation, catalytic reduction, etc., and the incineration method is more widely used. Incineration method may produce dioxin / furan at high temperature, which enhances the toxicity of organic matter. Although high temperature and high pressure incineration can inhibit the generation of dioxin / furan, the technology has high cost and serious equipment corrosion. Catalytic oxidation and chemical catalytic reduction usually require noble metal catalysts, and also need oxygen or hydrogen and other oxidizing or reducing substances.

[0004] Dehalogenation is the main way to reduce the toxicity of halogenated organic pollutants. Hydrolytic dehalogenation catalyzed by halogenated alkane dehalogenase is a simple and effective way to reduce the toxicity of halogenated organic pollutants, which is carried out at room temperature and pressure without oxygen or hydrogen, and does not require additional coenzyme / cofactor.

[0005] The process of enzymatic catalysis of hydrolytic dehalogenation needs to be carried out in water environment, however, most halogenated organic matter is water-insoluble. If catalysis is carried out in water phase, the accessibility of substrate is poor, and the reaction rate is very low; if catalysis is carried out in organic solvent, the basic water of enzyme is removed, and the activity of enzyme is very low. In addition, hydrogen halide produced by hydrolysis of halogenated organic matter is also difficult to dissolve in organic solvent, causing product inhibition. The most ideal case of enzymatic catalysis of hydrolytic dehalogenation is to carry out at the interface of oil-water two phases.

[0006] Common oil-water two-phase enzyme catalysis techniques include the use of free enzymes and immobilized enzymes. When using free enzymes, an oil-water dispersion is typically prepared, and the enzyme is then dissolved in the aqueous phase for catalysis. For example, patent CN201610529540.8 discloses a technique for catalyzing transesterification reactions using free lipases in an oil-water two-phase system; patent CN202311447751.3 discloses a technique for catalyzing the debenzylation synthesis of a key intermediate of sitafloxacin using free laccase in an oil-water two-phase system. Using immobilized enzymes can reduce the influence of organic solvents on the enzyme to some extent, and also facilitates enzyme recovery and reuse. For instance, patent CN202211616988.5 reports a two-phase catalysis method using immobilized enzymes in a packed column. This technique loads the enzyme onto porous particles and immobilizes the porous particles on the packing material, while the oil-water two-phase system is operated in a packed extraction column manner to achieve two-phase dispersion and contact with the enzyme. The above techniques cannot avoid the reduction in enzyme activity caused by direct contact between organic solvents and enzymes. At the same time, enzymes in the aqueous phase cannot fully contact the substrates in the organic phase, resulting in low enzyme activity.

[0007] To achieve more efficient catalysis at the oil-water interface, some novel technologies for constructing oil-water two-phase systems and immobilizing enzymes have been developed. For example, the Pickering emulsion reaction system immobilizes enzymes on solid particles that serve as Pickering emulsion stabilizers, thereby enabling enzyme catalysis at the oil-water interface. For instance, the literature [Acs Sustainable Chemistry & Engineering 2019, 7(8): 7619-7629] reported the preparation of Pickering emulsions by adsorbing and immobilizing lipases on mesoporous carbon spheres, which were applied to the esterification reaction of phytosterols and α-linolenic acid. Literature and invention patent CN202210057297.X reported the technology of using amphiphilic polymer-enzyme-inorganic hybrid nanoflowers as immobilized enzyme preparations to catalyze reactions at the oil-water interface. Although these methods can achieve high enzyme activity and catalytic efficiency, they require very complex immobilized enzyme preparation techniques, making large-scale preparation very difficult, and the stability of these immobilized supports still faces challenges.

[0008] In summary, the technology to enrich enzymes at the oil-water interface, maintain high enzyme activity, and achieve high enzyme catalytic efficiency without the use of additional emulsifiers or auxiliaries, and by adopting a relatively simple, reliable, and scalable immobilized enzyme method, still needs further development. Summary of the Invention

[0009] The purpose of this invention is to provide a catalytic system and method for the enzymatic hydrolysis of halogenated organic compounds at the oil-water interface, which can significantly improve the activity of enzyme-catalyzed dehalogenation reactions of halogenated hydrocarbons.

[0010] To construct an oil-water interface enzyme catalytic system, this invention first provides an enzyme-polymer conjugate that can be enriched at the oil-water interface, which is a conjugate of an enzyme and an amphiphilic block copolymer covalently linked together.

[0011] The amphiphilic block copolymer is a polyoxyethylene-polyoxypropylene block copolymer, preferably a polyoxyethylene-polyoxypropylene block copolymer, Pluronic F127.

[0012] The amphiphilic block copolymer may specifically be a polyoxyethylene-polyoxypropylene block copolymer.

[0013] The enzyme is a haloalkane dehalogenase, used to catalyze the hydrolysis reaction of haloalkane at the oil-water interface.

[0014] Preferably, the haloalkane dehalogenase is LinB, a hydrolytic dehalogenase from Sphingosine monocytogenes.

[0015] In the enzyme-polymer coupled enzyme, each molecule of the enzyme is linked to 2 to 3 molecules of the amphiphilic block copolymer.

[0016] The enzyme-polymer coupled enzyme can be prepared according to the following method:

[0017] The amphiphilic block copolymer is modified with terminal aldehyde groups; the resulting terminally aldehyde-modified amphiphilic block copolymer is covalently linked to the free amino groups on the surface of the enzyme in solution.

[0018] Preferably, the terminal aldehyde group is modified using a Desmond-Martin oxidant;

[0019] The covalent linkage steps are as follows: dissolve the enzyme in a phosphate buffer solution, then add it to an organic solution containing the amphiphilic block copolymer with terminal aldehyde modification, stir the reaction, and then add a reducing agent (such as sodium cyanoborohydride) to continue the reaction to obtain the final product.

[0020] Based on the enzyme-polymer conjugate, the present invention further provides a catalytic system for the hydrolysis of halogenated organic compounds by enzyme at the oil-water interface, comprising an organic solvent, an aqueous solution and the enzyme-polymer conjugate.

[0021] Preferably, the organic solvent is a high-boiling-point organic solvent that is partially miscible with water and has high solubility for the reactants, selected from n-dodecane or paraffin oil; more preferably n-dodecane.

[0022] The aqueous solution is pure water, glycine-NaOH buffer solution, or phosphate buffer solution;

[0023] The volume ratio of the organic solvent to the aqueous solution is 1:20 to 1:5.

[0024] The catalytic system provided by this invention can catalyze the hydrolysis of halogenated organic compounds, and can be carried out according to the following steps:

[0025] S1. Dissolve the enzyme-polymer coupled enzyme in the aqueous solution of the catalytic system to form the aqueous phase;

[0026] S2. Dissolve the halogenated organic compound in the organic solvent of the catalytic system to form the oil phase;

[0027] S3. The oil phase and the aqueous phase are mixed to form an oil-water dispersion system, and the halogenated organic compound is hydrolyzed by enzyme catalysis at the oil-water interface.

[0028] Preferably, the halogenated organic compound is a halogenated alkane, including chain halogenated hydrocarbons, cyclic halogenated hydrocarbons, halogenated esters, halogenated nitriles, and halogenated amides containing a benzene ring structure.

[0029] Preferably, the mass concentration ratio of the enzyme-polymer coupled enzyme (based on the mass of the enzyme contained therein) to the mass concentration ratio of the halogenated organic compound is 1:200 to 1:1000, and this ratio affects the reaction rate.

[0030] The enzyme-catalyzed reaction at the oil-water interface involved in this invention has the following characteristics: the substrate (reactant) of the enzyme-catalyzed reaction is a substance that is poorly soluble in water and is dissolved in an organic solvent to carry out the reaction; the product of the enzyme-catalyzed reaction is a substance that is poorly soluble in organic solvents and needs to be dissolved in an aqueous solution; the catalytic reaction occurs at the oil-water interface.

[0031] There are several ways to prepare the oil-water two-phase system of the catalytic system: an emulsion in which the organic solvent (aqueous solution) is suspended in the aqueous solution (organic solvent) in the form of droplets; or an oil-water dispersion system in which the aqueous solution (organic solvent) forms a flowing liquid film along the surface of the packing material and the organic solvent is attached to the surface of the aqueous solution liquid film.

[0032] The oil-water interface enzyme-catalyzed reaction involved in this invention has the following advantages:

[0033] 1) By constructing an oil-water two-phase reaction system and using a coupling compound synthesized from amphiphilic block polymers and enzymes, the enzyme is enriched at the oil-water interface, which improves the accessibility of substrates in organic solvents and avoids enzyme inactivation caused by organic solvents, thereby improving the enzyme's hydrolytic activity on halogenated organic compounds.

[0034] 2) By constructing an oil-water two-phase reaction system, the hydrogen halide product of the enzyme catalytic reaction is dissolved in the aqueous solution, thus avoiding the reduction in enzyme catalytic activity caused by the retention of halide ions in the enzyme active pocket.

[0035] 3) The conjugate of amphiphilic block polymer and enzyme synthesis also acts as a surfactant to stabilize the oil-water interface, eliminating the need for additional surfactants to stabilize the oil-water dispersion system.

[0036] The hydrolysis reaction equation of haloalkanes (RX, where R represents a hydrocarbon chain and X represents a halogen) catalyzed by the dehalogenase of haloalkanes involved in this invention is: RX + H₂O = R-OH + HX. Here, the reactant haloalkanes (RX) are typically poorly soluble in water and need to be dissolved in an organic solvent for the reaction, which in turn requires water. Simultaneously, the product HX is also poorly soluble in an organic solvent and needs to be dissolved in an aqueous solution. Therefore, this reaction requires a two-phase dispersion system of oil and water. Typically, in such systems, the free enzyme is uniformly dispersed in the aqueous phase, resulting in poor accessibility to the organic substrate RX and very low apparent catalytic activity. This invention combines the enzyme with an amphiphilic block copolymer to construct an enzyme-copolymer conjugate that can be distributed at the oil-water interface (but mainly on the aqueous side), greatly improving the enzyme's accessibility to the substrate RX in the organic phase. Simultaneously, the amphiphilic enzyme-copolymer conjugate acts as a surfactant and can also stabilize the oil-water interface.

[0037] In this invention, by specifically selecting the polymer and organic solvent, the protein bulk of the coupling enzyme molecule is located in the aqueous environment, rather than in the organic solvent, at the oil-water interface, thus ensuring the enzyme's structural flexibility and activity. Therefore, for constructing the amphiphilic copolymer of the enzyme-copolymer coupling, this invention selects the slightly more hydrophilic amphiphilic polymer Pluronic F127 (hydrophilic-lipophilic balance coefficient HLB of 22). Simultaneously, the preferred organic solvent, besides its good solubility for haloalkanes, also possesses the following characteristics: 1) minimal impact on enzyme activity; 2) suitable viscosity and density, facilitating the construction of a relatively stable oil-water dispersion system; 3) suitable solubility for the copolymer linked to the enzyme, thereby ensuring that the coupling enzyme is mainly concentrated at the oil-water interface, primarily on the aqueous phase side. This invention, through molecular simulation of the distribution of various types of polyoxyethylene-polyoxypropylene block copolymers at the oil-water interface, found that by using Pluronic F127, which has poor solubility in the organic phase n-dodecane, the main body of the coupling enzyme molecule can be distributed in the aqueous phase, thus ensuring the necessary water environment for the enzyme. Figure 1 The laser confocal microscopy image in Figure a shows that the fluorescently labeled coupling enzyme is distributed at the interface between the oil and water phases, mainly on the aqueous phase side.

[0038] In addition, the product HX generated by the dehalogenation reaction of haloalkanes remains in the active pocket of the enzyme, resulting in significant product inhibition; placing the main body of the coupling enzyme molecule in an aqueous solution facilitates the removal of HX and reduces product inhibition. Attached Figure Description

[0039] Figure 1These are confocal micrographs showing the distribution of enzymes in oil-water two-phase systems; in figure a, Rhodamine B-labeled LinB-Pluronic F127 is distributed in n-dodecane-glycine buffer, and in figure b, FITC-labeled LinB-Pluronic F127 is distributed in toluene-glycine buffer.

[0040] Figure 2 The catalytic activity of the coupling enzyme in the oil-water two-phase reaction system of dodecane-glycine buffer is determined.

[0041] Figure 3 The catalytic activity of the coupling enzyme in the toluene-glycine buffer oil-water two-phase reaction system was determined.

[0042] Figure 4 The hydrolysis rate of 1-bromobutane catalyzed by the coupling enzyme in the oil-water two-phase reaction system is given. Detailed Implementation

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] In the examples below, the dehalogenases for haloalkanes were all hydrolytic dehalogenase B from Sphingomonas hypokinesi (LinB, commercially available), and all other chemicals were commercially available.

[0046] Example 1: Synthesis of LinB-Pluronic F127 Coupling

[0047] First, the aldehyde-terminated polymer Pluronic was modified: 10 g of Pluronic F127 was dissolved in 500 mL of dichloromethane, and 5 times the amount of Dysmart oxidant (1.7 g) was added. The mixture was stirred at room temperature for 24 hours. Most of the organic solvent was removed using a rotary evaporator, resulting in a white turbid liquid. Cold diethyl ether was added to form a precipitate, which was collected by filtration and washed repeatedly with cold diethyl ether to remove excess Dysmart oxidant, yielding aldehyde-terminated Pluronic F12. The aldehyde modification rate was determined using the Purpald method, and the modification rate was approximately 30%.

[0048] The haloalkane dehalogenase LinB was dissolved in 10 mmol / L phosphate buffer (pH = 8.0) to obtain a solution with an enzyme concentration of 2 mg / L. Terminally aldehyde-terminated Pluronic F127 solid powder was dissolved in the same phosphate buffer, ensuring a molar ratio of 1.5–2 between the terminal aldehyde group of Pluronic F127 and the amino group on the LinB surface. The mixture was then stirred at room temperature for 2 h, followed by the addition of sodium cyanoborohydride (10% by weight of the Pluronic F127 feed), and stirring continued at room temperature for another 12 h. The product was purified using a protein purification system to obtain the LinB-Pluronic F127 conjugate.

[0049] The grafting rate of LinB-Pluronic conjugates was determined and calculated using the TNBS method, with an average of 2 Pluronic F127 molecules grafted onto each LinB enzyme molecule.

[0050] Example 2: Construction and catalytic properties of the LinB-Pluronic F127 coupling oil-water reaction system.

[0051] The distribution of LinB-Pluronic F127 at the oil-water interface was observed using laser confocal microscopy.

[0052] LinB-Pluronic F127 was labeled with the fluorescent dye Rhodamine B. The Rhodamine B-labeled LinB-Pluronic F127 was dissolved in 0.1 mol / L glycine buffer (50 mmol / L, pH = 9.8) to obtain an enzyme-containing solution with a concentration of 0.05 mg / mL. 200 μL of this enzyme-containing solution was added dropwise to an optically precise 96-well plate, followed by the addition of 200 μL of n-dodecane (without reactants) to construct an oil-water two-phase system. The sample in the well plate was horizontally scanned along the oil-water interface using a laser confocal microscope in z-stack mode, with an excitation wavelength of 595 nm and a detection excitation wavelength of 561 nm. The obtained laser confocal images are shown below. Figure 1 As shown in Figure a, the red fluorescent dots represent Rhodamine B-labeled LinB-Pluronic F127, and these dots are focused at the oil-water interface. In contrast, a similar oil-water two-phase system was constructed using the same mass concentration of free LinB enzyme solution, and the free LinB enzyme was found to be evenly distributed in the glycine buffer.

[0053] In two-phase reaction systems with different oil-to-water ratios of n-dodecane-glycine buffer, using 1-bromobutane as a substrate, the relative activity (relative to the activity of the free enzyme in the aqueous phase) of the coupling enzyme was determined. The reaction rate was determined by the mercuric thiocyanate-ferric ammonium sulfate colorimetric method (the reaction rate was measured in the initial 30 min). Results are as follows: Figure 2As shown, constructing the LinB-Pluronic F127 conjugate significantly enhances the relative enzyme activity of LinB in the oil-water two-phase reaction system. The kinetic constant K of the enzyme-catalyzed reaction was determined. m and k cat The results are shown in Table 1 (oil-to-water ratio 1:10). It can be seen that compared to the free enzyme, the K of the LinB-Pluronic F127 conjugate... m The significant decrease indicates a significant improvement in substrate accessibility; meanwhile, k cat With free enzyme in aqueous phase k cat Similar results indicate a significant decrease in enzyme activity.

[0054] Table 1 Kinetic constants of enzyme-catalyzed reactions

[0055] Enzyme / reaction system K m (mmol / L) k cat (s -1 )]]> Free enzyme / glycine buffer 0.59 6.26 Free enzyme / n-dodecane-glycine buffer 22.14 4.70 Coupled enzyme / n-dodecane-glycine buffer 3.21 7.39

[0056] Using the same method described above, an oil-water two-phase system (oil-water ratio 1:10) was constructed using paraffin oil as the solvent, and the LinB-Pluronic F127 conjugate was also enriched in the aqueous phase. Enzyme activity was measured using the same method, and the relative enzyme activities (with the free enzyme activity in the pure aqueous phase as 100%) of the conjugate and free enzyme in the oil-water two-phase system were 114% and 72%, respectively. This indicates that the catalytic activity is significantly enhanced in the paraffin oil-water system.

[0057] Comparative Example 1: Effect of Organic Solvents

[0058] An oil-water two-phase reaction system was constructed using toluene, which has high solubility in Pluronic F127, as the organic solvent. The composition of the aqueous phase and the ratio of the oil and water phases were the same as in Example 2.

[0059] LinB-Pluronic F127 was labeled with the fluorescent dye FTIC, and its distribution in the oil-water two-phase mixture was observed using laser confocal microscopy as described in Example 2. Figure 1 As shown in Figure b, the LinB-Pluronic F127 coupling enzyme is abundantly distributed in the organic solvent and not concentrated at the oil-water interface. Enzyme activity was measured using the same method as in Example 2, and the results were... Figure 3 The results showed that the catalytic activity of the coupled enzyme in the oil-water two-phase system was almost the same as that of the free enzyme, but significantly lower than that in Example 2 when n-dodecane was used as the oil phase.

[0060] The above results indicate that the solubility of the organic solvent in the oil-water two-phase system for the copolymer of the constructed enzyme is an important factor affecting the distribution of the coupling enzyme in the reaction system. It is necessary to select an organic solvent with low solubility for the selected copolymer to ensure that the coupling enzyme is enriched at the oil-water interface and its main body exists in the aqueous phase in the constructed oil-water two-phase reaction system.

[0061] Comparative Example 2: Effects on Polymers

[0062] Polymer-enzyme conjugates were prepared using the more lipophilic polyoxyethylene-polyoxypropylene block copolymer Pluronic P123 (Pluronic P123 has a hydrophilic-lipophilic balance coefficient of 8, while Pluronic F127 has an HLB of 22) and the non-amphiphilic polyethylene glycol PEG 10K (relative molecular mass 10000, close to that of Pluronic F127), following the method described in Example 1. An oil-water two-phase reaction system was constructed according to the method described in Example 2, with an oil-water ratio of 1:10. Enzyme activity and the kinetic constant K were determined using the same method as in Example 2. m and k cat The results are shown in Table 2. It can be seen that choosing a polymer with strong lipophilicity significantly reduces the relative enzyme activity in the oil-water two-phase catalytic system because the enzyme-polymer bulk dissolves in the oil phase. Similarly, using the non-amphiphilic polymer PEG10K to construct polymer-enzyme conjugates results in poor substrate accessibility and significantly reduced relative enzyme activity in the oil-water two-phase catalytic system (even lower than that of the free enzyme).

[0063] Table 2 Comparison of enzyme activities of enzyme-polymer conjugates prepared using different polymers.

[0064] Polymer HLB Relative enzyme activity (%) K m (mmol / L) k cat (s -1 )]]> Pluronic P123 8 89 6.33 5.88 Pluronic F127 22 121 3.21 7.39 PEG 10K — 47 18.32 3.47

[0065] Example 3: Construction of the LinB-Pluronic F127 coupling oil-water reaction system and its catalytic hydrolysis of 1-bromobutane

[0066] A 50 mmol / L glycine buffer (pH = 9.75) was used as the aqueous phase, and n-dodecane was used as the oil phase. The volume ratio of the oil to water phases was 1:10. The LinB-Pluronic F127 conjugate was dissolved in the glycine buffer at a concentration (based on the mass of LinB) of 0.03 mg / L, and 1-bromobutane was dissolved in n-dodecane at a concentration of 15 mg / mL. The n-dodecane was added dropwise to the glycine buffer while slowly stirring to form an oil-water dispersion system in which oil droplets were dispersed in the glycine buffer. The hydrolysis rate of 1-bromobutane was determined, and the results are shown in the attached figure. Figure 4 As shown.

[0067] In contrast, the LinB-Pluronic F127 conjugate was replaced with the same amount of free LinB, while other conditions remained the same. It is evident that by constructing an oil-water reaction system and simultaneously using the LinB-Pluronic F127 conjugate for interfacial catalysis, the hydrolysis rates at 60 min and 90 min were improved by 21% and 17% respectively compared to the hydrolysis rate catalyzed by free LinB enzyme in the aqueous phase.

[0068] Example 4: Catalytic hydrolysis of 4-chloro-1-butene in an oil-water reaction system using LinB-Pluronic F127 coupling enzyme

[0069] The oil-water two-phase reaction system and its construction method are the same as in Example 3.

[0070] The organic phases contained 10 mg / mL, 15 mg / mL, and 25 mg / mL of the substrate 4-chloro-1-butene, respectively, while the aqueous phase contained 0.03 mg / L of LinB-Pluronic F127 (based on the mass of LinB). For comparison, the same amount of free LinB was used to catalyze the hydrolysis of 4-chloro-1-butene in glycine buffer under identical conditions. The hydrolysis rate was measured after 90 min of hydrolysis, and the results are shown in Table 3. It can be seen that for different substrate concentrations, the conversion rate of the oil-water phase reaction of the coupled enzyme was higher than that of the aqueous phase reaction of the free enzyme, and the difference was more pronounced at lower substrate concentrations.

[0071] Table 3. Hydrolysis rate (%) of 4-chloro-1-butene

[0072] Substrate content (mg / mL) Coupled enzyme oil-water two-phase system Free enzyme aqueous phase reaction system 10 28 17 15 29 21 25 19 16

[0073] Example 5: Effect of oil-water volume ratio in the LinB-Pluronic F127 coupling oil-water reaction system

[0074] The oil-water reaction system is the same as in Example 2, wherein the volume ratio of n-dodecane solvent to carbonate buffer is 1:20, 1:10, 3:20 and 1:5 respectively.

[0075] The catalytic activity of LinB-Pluronic F127 was determined according to the method in Example 2, and the results are shown in Table 4. It can be seen that the catalytic activity decreased significantly when the oil-water ratio was greater than 15%.

[0076] Table 4 Effect of oil-water volume ratio on catalytic activity

[0077] Oil-water volume ratio Specific activity K m (mmol / L) k cat (s -1 )]]> 1:20 126％ 1.25 7.83 1:10 119％ 3.21 7.39 3:20 112％ 5.05 6.89 1:5 93％ 10.01 6.63

Claims

1. A catalytic system for the enzymatic hydrolysis of halogenated organic compounds at the oil-water interface, comprising an organic solvent, an aqueous solution, and an enzyme-polymer coupled enzyme; The enzyme-polymer coupling enzyme is a coupling product of a haloalkane dehalogenase and an amphiphilic block copolymer covalently linked; The haloalkane dehalogenase is LinB, a hydrolytic dehalogenase from Sphingosine monocytogenes. The amphiphilic block copolymer is a polyoxyethylene-polyoxypropylene block copolymer, Pluronic F127. In the enzyme-polymer coupled enzyme, each enzyme molecule is linked to 2 to 3 molecules of the amphiphilic block copolymer; In the catalytic system: 1) The organic solvent and the aqueous solution constitute an oil-water dispersion system; The aqueous solution is pure water, glycine buffer solution, or phosphate buffer solution; 2) The enzyme-polymer coupled enzyme is dispersed at the oil-water interface of the oil-water dispersion system, and the bulk of the enzyme-polymer coupled enzyme molecules is dispersed in the aqueous phase. 3) The reactant halogenated organic compound is dissolved in the organic solvent, and at least one product is dissolved in the aqueous phase; The organic solvent is a high-boiling-point organic solvent that is partially miscible with water, and is selected from n-dodecane.

2. The catalytic system according to claim 1, characterized in that: The volume ratio of the organic solvent to the aqueous solution is 1:20 to 1:

5.

3. The catalytic system according to claim 1 or 2, characterized in that: The preparation method of the enzyme-polymer coupled enzyme includes the following steps: The amphiphilic block copolymer is modified with terminal aldehyde groups; the resulting terminally aldehyde-modified amphiphilic block copolymer is covalently linked to the free amino groups on the surface of the enzyme in solution.

4. The catalytic system according to claim 3, characterized in that: Terminal aldehyde modification was performed using Dys-Martin oxidant; The covalent linkage steps are as follows: the enzyme is dissolved in a phosphate buffer solution, then added to an organic solution containing the amphiphilic block copolymer with terminal aldehyde modification, the reaction is stirred, and then a reducing agent is added to continue the reaction to obtain the final product.

5. The application of the catalytic system according to any one of claims 1-4 in the catalytic hydrolysis of halogenated organic compounds.

6. A method for the enzymatic hydrolysis of halogenated organic compounds at the oil-water interface, comprising the following steps: S1. Dissolve the enzyme-polymer coupled enzyme in the catalytic system according to any one of claims 1-4 in the aqueous solution as the aqueous phase; S2. Dissolve the halogenated organic compound in the organic solvent to form the oil phase; S3. The oil phase and the aqueous solution are mixed to form an oil-water dispersion system, and the enzymatic hydrolysis of the halogenated organic compound is carried out at the oil-water interface.

7. The method according to claim 6, characterized in that: The halogenated organic compounds include chain halogenated hydrocarbons, cyclic halogenated hydrocarbons, halogenated esters, halogenated nitriles, and halogenated amides containing a benzene ring structure.

8. The method according to claim 6 or 7, characterized in that: The enzyme-polymer coupled enzyme is dispersed at the oil-water interface of the oil-water dispersion system, and the main body of the enzyme-polymer coupled enzyme molecule is located in the aqueous phase.

9. The method according to claim 6 or 7, characterized in that: At least one product of the hydrolysis dissolves in the aqueous phase.

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