Catalytic system and method for enzymatic hydrolysis of halogenated organic matters on oil-water interface

Abstract

The invention discloses a catalytic system and method for catalyzing hydrolysis of halogenated organic matters on an oil-water interface by enzyme. The catalytic system comprises an organic solvent, an aqueous solution and an enzyme-polymer coupling enzyme; the enzyme-polymer coupling enzyme is a conjugate formed by covalently binding halogenated alkane dehalogenase and an amphiphilic block copolymer; in the enzyme-polymer coupled enzyme, each molecule of enzyme is connected with 2-3 molecules of the amphiphilic block copolymer. According to the method, an oil-water two-phase reaction system is constructed, and the enzyme is enriched at an oil-water interface through a conjugate synthesized by an amphiphilic block polymer and the enzyme; the accessibility of a substrate in the organic solvent is improved, enzyme inactivation caused by the organic solvent is avoided, and the hydrolytic activity of enzyme to halogenated organic matters is improved; by constructing an oil-water two-phase reaction system, an enzyme catalytic reaction product hydrogen halide can be dissolved in an aqueous solution, and reduction of enzyme catalytic activity caused by retention of halide ions in an enzyme product pocket is reduced.

Description

Technical Field

[0001] The invention relates to a catalytic system and method for enzymatically hydrolyzing halogenated organic matter at an oil-water interface, belonging to the field of biocatalysis. Background Art

[0002] Halogenated organic compounds are often used as raw materials, intermediates, solvents, etc. in the chemical industry, which inevitably leads to the discharge of many halogenated organic compounds into the environment, becoming a common type of industrial pollutants. Halogenated organic pollutants are highly 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 incineration is the most widely used. The incineration method decomposes halogenated organic pollutants at high temperatures and may produce dioxins / furans, which enhance the toxicity of organic matter. Although high-temperature and high-pressure incineration can inhibit the formation of dioxins / furans, the technology is costly and the equipment is severely corroded. Catalytic oxidation and chemical catalytic reduction usually require precious metal catalysts, as well as oxidizing or reducing substances such as oxygen or hydrogen.

[0004] Dehalogenation is the main way to reduce the toxicity of halogenated organic pollutants. Dehalogenation by hydrolysis catalyzed by haloalkane dehalogenase is a simple and effective way to reduce the toxicity of halogenated organic pollutants. The reaction is carried out at room temperature and pressure, does not require oxygen or hydrogen, and does not require additional coenzymes / cofactors.

[0005] The process of enzyme-catalyzed hydrolysis and dehalogenation needs to be carried out in an aqueous environment, but most halogenated organic substances are poorly soluble in water. If catalyzed in an aqueous phase, the accessibility of the substrate is poor and the reaction rate is very low; while catalyzed in an organic solvent, the enzyme's basic water removal results in very low enzyme activity. In addition, the hydrogen halide produced by the hydrolysis of halogenated organic matter is a polar molecule and is also difficult to dissolve in organic solvents, resulting in product inhibition. The ideal situation for enzyme-catalyzed hydrolysis and dehalogenation reactions is to be carried out at the interface of the oil-water phase.

[0006] Common oil-water two-phase enzyme catalysis technology includes the use of free enzymes and immobilized enzymes for catalysis. When using free enzymes, an oil-water dispersion system is usually prepared, and then the enzyme is dissolved in the water phase for catalysis. For example, invention patent CN201610529540.8 discloses a technology for catalyzing transesterification reactions with free lipase in an oil-water two-phase system; invention patent CN202311447751.3 discloses a technology for catalyzing debenzylation and synthesizing key intermediates of sitafloxacin in an oil-water two-phase system with free laccase. The use of immobilized enzymes can reduce the effect of organic solvents on enzymes to a certain extent, and also facilitates the recovery and reuse of enzymes. For example, invention patent CN202211616988.5 reports a two-phase catalysis method of immobilizing enzymes in a packed tower. This technology loads the enzyme on porous particles and fixes the porous particles on the packing, while the oil-water two-phase is operated in the manner of a packed extraction tower to achieve two-phase dispersion and contact it with the enzyme. The above technologies are difficult to avoid the reduction of enzyme activity caused by direct contact of organic solvent with enzyme. At the same time, the enzyme in the main aqueous phase is difficult to fully contact the substrate in the organic phase, resulting in low enzyme activity.

[0007] In order to achieve catalysis at the oil-water interface more efficiently, some new technologies for constructing oil-water two-phase systems and immobilized enzymes have also been developed. For example, the immobilized enzyme Pickering emulsion reaction system immobilizes the enzyme on solid particles that serve as Pickering emulsion stabilizers, so that the enzyme catalyzes at the oil-water interface. For example, the document [Acs SustainableChemistry&Engineering 2019, 7(8): 7619-7629] reported that lipase was adsorbed and fixed on mesoporous carbon spheres to prepare Pickering emulsions, which were applied to the esterification reaction of plant sterols and α-linolenic acid. The document and invention patent CN202210057297.X reported the use of amphiphilic polymer-enzyme-inorganic hybrid nanoflowers as immobilized enzyme preparations to catalyze reactions at the oil-water interface. Although these methods can obtain higher enzyme activity and catalytic efficiency, they require very complex preparation technology for immobilized enzymes, which is very difficult to prepare on a large scale, and the stability of these immobilized carriers is still facing challenges.

[0008] In summary, the technology of enriching enzymes at the oil-water interface, maintaining high enzyme activity and obtaining high enzyme catalytic efficiency without the use of additional emulsifiers or additives and adopting a relatively simple, reliable and scalable method of immobilized enzymes still needs to be further developed. Summary of the invention

[0009] The purpose of the present invention is to provide a catalytic system and method for enzymatically hydrolyzing halogenated organic matter at an oil-water interface, which can significantly improve the activity of enzymatically catalyzing the dehalogenation reaction of halogenated hydrocarbons.

[0010] In order to construct an oil-water interface enzyme catalysis system, the present invention first provides an enzyme-polymer coupled enzyme that can be enriched at the oil-water interface, which is a covalently bonded conjugate of an enzyme and an amphiphilic block copolymer.

[0011] Wherein, 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, which is used to catalyze the hydrolysis reaction of haloalkane at the oil-water interface;

[0014] Preferably, the haloalkane dehalogenase is the Sphingomonas paucimobilis hydrolysis dehalogenase LinB;

[0015] In the enzyme-polymer coupled enzyme, each molecule of the enzyme is connected 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 obtained amphiphilic block copolymer modified with terminal aldehyde groups is covalently linked to the free amino groups on the surface of the enzyme in a solution.

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

[0019] The covalent linking step is as follows: dissolving the enzyme in a phosphate buffer solution, then adding it to an organic solution containing the terminal aldehyde-modified amphiphilic block copolymer, stirring the reaction, and then adding a reducing agent (such as sodium cyanoborohydride) to continue the reaction.

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

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

[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 the present invention can catalyze the hydrolysis of halogenated organic matter, and can be carried out according to the following steps:

[0025] S1, dissolving the enzyme-polymer coupled enzyme in the aqueous solution of the catalytic system as the aqueous phase;

[0026] S2, dissolving the halogenated organic matter in the organic solvent of the catalytic system as the oil phase;

[0027] S3, mixing the oil phase and the water phase to form an oil-water dispersion system, and performing enzyme-catalyzed hydrolysis of the halogenated organic matter 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 (calculated by the mass of the enzyme contained therein) to the mass concentration ratio of the halogenated organic matter 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 the present invention has the following characteristics: the substrate (reactant) of the enzyme-catalyzed reaction is a substance that is insoluble in water and is dissolved in an organic solvent for reaction; the product of the enzyme-catalyzed reaction is a substance that is insoluble in organic solvents and needs to be dissolved in an aqueous solution; the catalytic reaction occurs at the oil-water interface.

[0031] There are many ways to prepare the oil-water two-phase of the catalytic system: an emulsion formed by suspending an organic solvent (aqueous solution) in an aqueous solution (organic solvent) in the form of droplets; an oil-water dispersion system formed by the aqueous solution (organic solvent) forming a flowing liquid film along the surface of the filler and the organic solvent adhering to the surface of the aqueous solution film.

[0032] The enzyme-catalyzed reaction at the oil-water interface of the present invention has the following advantages:

[0033] 1) By constructing an oil-water two-phase reaction system and synthesizing a conjugate of an amphiphilic block polymer and an enzyme, the enzyme is enriched at the oil-water interface, thereby improving the accessibility of the substrate in the organic solvent, while avoiding the enzyme inactivation caused by the organic solvent and improving the enzyme's hydrolysis activity for halogenated organic matter.

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

[0035] 3) The conjugate synthesized by the amphiphilic block polymer and the enzyme also acts as a surfactant to stabilize the oil-water interface, and there is no need to add additional surfactant to stabilize the oil-water dispersion system.

[0036] The hydrolysis reaction equation of the halogenated hydrocarbon RX (R represents a hydrocarbon chain, and X represents a halogen) catalyzed by the halogenated alkane dehalogenase of the present invention is: RX+H2O=R-OH+HX; wherein the reactant halogenated hydrocarbon RX is usually insoluble in water and needs to be dissolved in an organic solvent for reaction, and the reaction requires water, and the generated product HX is insoluble in an organic solvent and also needs to be dissolved in an aqueous solution. Therefore, the reaction needs to construct an oil-water two-phase dispersion reaction system. Usually in an oil-water two-phase dispersion system, the free enzyme is usually evenly dispersed in the aqueous phase, so that the accessibility to the organic reaction substrate RX is poor, and the apparent catalytic activity of the enzyme is very low. The present invention combines the enzyme with an amphiphilic block copolymer to construct an enzyme-copolymer conjugate, which can be distributed at the oil-water interface (but mainly located on the water phase side), greatly improving the accessibility of the enzyme to the substrate RX in the organic phase. At the same time, the amphiphilic enzyme-copolymer conjugate has the function of a surfactant and can also stabilize the oil-water interface.

[0037] In the present invention, by the specific selection of polymer and organic solvent, the protein main part of the coupled enzyme molecule is located in the water environment, rather than in the organic solvent, at the oil-water two-phase interface to ensure the structural flexibility and activity of the enzyme. For this reason, for the amphiphilic copolymer for constructing the enzyme-copolymer conjugate, the amphiphilic polymer Pluronic F127 (hydrophile-lipophile balance coefficient HLB is 22) with slightly strong hydrophilicity is selected in the present invention. At the same time, the organic solvent is preferably selected, and the preferred organic solvent has the following characteristics in addition to being able to dissolve the halogenated hydrocarbon substrate well: 1) less influence on the enzyme activity; 2) suitable viscosity and density, easy to construct a more stable oil-water dispersion system. 3) suitable solubility for the copolymer connected with the enzyme, so that the coupled enzyme is mainly enriched at the oil-water interface, and mainly on the water phase side. The invention simulates the distribution of various types of polyoxyethylene-polyoxypropylene block copolymers at the oil-water interface through molecules, and finds that the use of Pluronic F127 with poor solubility in organic phase n-dodecane can make the main part of the coupled enzyme molecule distributed in the water phase, thus ensuring the water environment necessary for the enzyme. Figure 1 The laser confocal microscopy photograph in Figure a shows that the fluorescently labeled coupled enzyme is distributed at the interface between the oil and water phases, and mainly on the water phase side.

[0038] In addition, the product HX generated by the dehalogenation reaction of halogenated hydrocarbons will be retained in the active pocket of the enzyme and show relatively obvious product inhibition; placing the main body of the coupled enzyme molecule in an aqueous solution is conducive to the detachment of HX and reduces product inhibition. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1Confocal microscopy photos of the distribution of enzymes in oil-water phases; Figure a shows the distribution of LinB-PluronicF127 labeled with rhodamine B in n-dodecane-glycine buffer, and Figure b shows the distribution of LinB-PluronicF127 labeled with FITC in toluene-glycine buffer.

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

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

[0042] Figure 4 is the hydrolysis rate of 1-bromobutane catalyzed by the coupled enzyme in the oil-water two-phase reaction system. DETAILED DESCRIPTION

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

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] The haloalkane dehalogenases in the following examples are all Sphingomonas paucimobilis hydrolysis dehalogenase B (LinB, obtained from commercial channels), and the other chemicals are all commercially available.

[0046] Example 1. Synthesis of LinB-Pluronic F127 Conjugate

[0047] First, the polymer Pluronic terminal aldehyde group was modified: 10g Pluronic F127 was dissolved in 500mL of dichloromethane, and 5 times the amount of the substance (1.7g) of Dess-Martin periodinane was added, and stirred at room temperature for 24 hours. Most of the organic solvent was removed by a rotary evaporator to obtain a white turbid liquid, and cold ether was added to form a precipitate. The precipitate was collected by filtration and washed with cold ether several times to remove excess Dess-Martin periodinane to obtain Pluronic F12 modified with terminal aldehyde groups. The aldehyde group modification rate was determined by the Purpald method, and the aldehyde group modification rate was about 30%.

[0048] Halogenated alkane dehalogenase LinB is dissolved in 10mmol / L phosphate buffer (pH=8.0) to obtain a solution with an enzyme content of 2mg / L. The terminal aldehyde-modified Pluronic F127 solid powder is dissolved in the above phosphate buffer to ensure that the molar ratio of the terminal aldehyde group of the Pluronic F127 added to the surface amino group of LinB is 1.5-2. Then stir at room temperature for 2h, add sodium cyanoborohydride, a reducing agent, which is 10% of the mass of Pluronic F127, and continue stirring at room temperature for 12h. The product is separated by a protein purification system to obtain LinB-Pluronic F127 conjugate.

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

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

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

[0052] LinB-Pluronic F127 was labeled with the fluorescent dye Rhodamine B, and 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 an enzyme mass content of 0.05 mg / mL. 200 μL of the above enzyme-containing solution was added dropwise to a 96-well plate with optical precision, and then 200 μL of n-dodecane (without reactants) was continued to be added dropwise to construct an oil-water two-phase system. The sample in the well plate was horizontally sliced ​​and scanned along the oil-water interface in the z-stack mode of a laser confocal microscope. The excitation wavelength was 595 nm and the detection excitation wavelength was 561 nm. The laser confocal photograph was obtained as shown below. Figure 1 As shown in Figure a, the red fluorescent spots in the figure are LinB-PluronicF127 labeled with rhodamine B, and it can be seen that these fluorescent spots are focused at the oil-water interface. For comparison, the same oil-water two-phase system was constructed with the same mass content of free LinB enzyme solution, and it can be seen that the free LinB enzyme is evenly distributed in the glycine buffer.

[0053] In a two-phase reaction system of n-dodecane-glycine buffer with different oil-water ratios, 1-bromobutane was used as the substrate to determine the relative activity of the coupled enzyme (relative to the activity of the free enzyme in the aqueous phase). The bromide ions produced by the reaction were determined by the mercuric thiocyanate-ammonium ferric sulfate colorimetric method to determine the reaction rate (the reaction rate in the initial 30 minutes of the reaction was determined). The results are shown in Figure 2As shown. It can be seen that by constructing the LinB-Pluronic F127 conjugate, the relative enzyme activity of LinB in the oil-water two-phase reaction system was significantly improved. Determination of the kinetic constant K of the enzyme catalytic reaction m and k cat The results are shown in Table 1 (oil-water ratio is 1:10). It can be seen that compared with the free enzyme, the K m significantly decreased, indicating that the accessibility to the substrate was significantly improved; at the same time, k cat k of free enzyme in water cat This indicates that the activity of the enzyme was significantly reduced.

[0054] Table 1 Kinetic constants of enzyme catalyzed reactions

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

[0056] Using the same method as above, paraffin oil was used as solvent to construct an oil-water two-phase system (oil-water ratio of 1:10), and the LinB-PluronicF127 coupled enzyme was also enriched in the water phase. The enzyme activity was determined by the same method as above, and the relative enzyme activities of the coupled enzyme and the free enzyme in the oil-water two-phase system (with the free enzyme activity in the pure water phase as 100%) were 114% and 72%, respectively. It can be seen that in the paraffin oil-water system, the catalytic activity is also significantly improved.

[0057] Comparative Example 1: Effect of organic solvents

[0058] Toluene, in which Pluronic F127 has a high solubility, was used as an organic solvent to construct an oil-water two-phase reaction system. The composition of the water phase and the ratio of the oil-water phase were the same as those in Example 2.

[0059] LinB-PluronicF127 was labeled with fluorescent dye FTIC, and the distribution of the coupled enzyme LinB-PluronicF127 in the oil-water phase was observed by laser confocal microscopy in the manner described in Example 2. Figure 1 As shown in Figure b, it can be seen that the coupled enzyme LinB-Pluronic F127 is distributed in large quantities in the organic solvent and is not enriched at the oil-water interface. The enzyme activity was determined using the method of Example 2. The results are ( Figure 3 ) 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, and was significantly lower than the activity 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 enzyme-constructing copolymer is an important factor affecting the distribution of the coupled enzyme in the reaction system. An organic solvent with a lower solubility for the selected copolymer must be selected according to the selected copolymer to ensure that in the constructed oil-water two-phase reaction system, the coupled enzyme is enriched at the oil-water interface and its main body exists in the water phase.

[0061] Comparative Example 2: Effect of polymer

[0062] The polymer-enzyme conjugate was prepared according to the method in Example 1 using a more lipophilic polyoxyethylene-polyoxypropylene block copolymer Pluronic P123 (the hydrophilic-lipophilic balance coefficient HLB of Pluronic P123 is 8, and the HLB of Pluronic F127 is 22) and a non-amphiphilic polyethylene glycol PEG 10K (relative molecular weight 10000, close to Pluronic F127). An oil-water two-phase reaction system was constructed according to the method in Example 2, wherein the ratio of the oil-water two phases was 1:10, and the enzyme activity and kinetic constant K were determined by the same method as in Example 2. m and k cat The results are shown in Table 2. It can be seen that the selection of a polymer with strong lipophilicity significantly reduces the relative enzyme activity in the oil-water two-phase catalytic system because the enzyme-polymer body is dissolved in the oil phase; similarly, the use of a non-amphiphilic polymer PEG10K to construct a polymer-enzyme conjugate cannot be enriched at the oil-water interface, has poor accessibility to the substrate, and significantly reduces the relative enzyme activity in the oil-water two-phase catalytic system (even lower than the free enzyme).

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

[0064] polymer HLB Relative enzyme activity (%) <![CDATA[K m (mmol / L)]]> <![CDATA[k cat (s -1 )]]> Pluronic P123 8 89 6.33 5.88 Pluronic F127 22 121 3.21 7.39 PEG10K — 47 18.32 3.47

[0065] Example 3: Construction of LinB-Pluronic F127 conjugate oil-water reaction system and catalysis of 1-bromobutane hydrolysis

[0066] 50mmol / 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-water phase was 1:10. The LinB-Pluronic F127 conjugate was dissolved in the glycine buffer, with a content (based on the mass of LinB) of 0.03mg / L, and 1-bromobutane was dissolved in n-dodecane with a content of 15mg / mL. n-Dodecane was dropped into the glycine buffer and slowly stirred to form an oil-water dispersion system in which oil droplets were dispersed in the glycine buffer. The hydrolysis rate of 1-bromobutane was measured, and the results are shown in the attached figure. Figure 4 shown.

[0067] As a comparison, the same amount of free LinB was used to replace the LinB-Pluronic F127 conjugate, and other conditions were the same. It can be seen that by constructing an oil-water reaction system and using the LinB-Pluronic F127 conjugate to achieve interfacial catalysis, the hydrolysis rates at 60 min and 90 min were increased by 21% and 17% respectively compared with the hydrolysis rates catalyzed by free LinB in the water phase.

[0068] Example 4: Hydrolysis of 4-chloro-1-butene catalyzed by LinB-Pluronic F127 coupled enzyme oil-water reaction system

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

[0070] The content of substrate 4-chloro-1-butene in the organic phase is 10mg / mL, 15mg / mL and 25mg / mL respectively, and the content of LinB-Pluronic F127 in the aqueous phase (based on the mass of LinB) is 0.03mg / L. As a comparison, the free LinB with the same content catalyzes the hydrolysis of 4-chloro-1-butene in a glycine buffer, and other conditions are the same. The hydrolysis rate is measured after 90min of the hydrolysis reaction, and the results are shown in Table 3. It can be seen that for different substrate concentrations, the conversion rate of the coupled enzyme oil-water two-phase reaction is higher than that of the free enzyme aqueous phase reaction, and when the substrate concentration is low, the difference is clearer.

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

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

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

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

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

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

[0077] Oil-water volume ratio Specific activity <![CDATA[K m (mmol / L)]]> <![CDATA[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 hydrolyzing halogenated organic matter by enzyme at an oil-water interface, comprising an organic solvent, an aqueous solution and an enzyme-polymer coupled enzyme; The enzyme-polymer coupled enzyme is a conjugate of a haloalkane dehalogenase and an amphiphilic block copolymer covalently bonded; The amphiphilic block copolymer is a polyoxyethylene-polyoxypropylene block copolymer; In the enzyme-polymer coupled enzyme, each molecule of the enzyme is connected to 2 to 3 molecules of the amphiphilic block copolymer.

2. The catalytic system according to claim 1, characterized in that: In the catalytic system: 1) the organic solvent and the aqueous solution constitute an oil-water dispersion system; 2) 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 molecules is dispersed in the water phase; 3) The reactant halogenated organic compound is dissolved in the organic solvent, and at least one product is dissolved in the aqueous phase.

3. The catalytic system according to claim 1 or 2, characterized in that: The organic solvent is a high boiling point organic solvent partially miscible with water, selected from n-dodecane or paraffin oil; The aqueous solution is pure water, glycine buffer solution or phosphate buffer solution; The volume ratio of the organic solvent to the aqueous solution is 1:20 to 1:

5.

4. The catalytic system according to any one of claims 1 to 3, characterized in that: The preparation method of the enzyme-polymer coupled enzyme comprises the following steps: The amphiphilic block copolymer is modified with terminal aldehyde groups; the obtained amphiphilic block copolymer modified with terminal aldehyde groups is covalently linked to the free amino groups on the surface of the enzyme in a solution.

5. The catalytic system according to claim 4, characterized in that: The terminal aldehyde group was modified using Dess-Martin periodinane; The covalent connection step is as follows: dissolving the enzyme in a phosphate buffer solution, then adding it into an organic solution containing the terminal aldehyde-modified amphiphilic block copolymer, stirring for reaction, and then adding a reducing agent to continue the reaction.

6. Use of the catalytic system according to any one of claims 1 to 5 in catalyzing the hydrolysis of halogenated organic compounds.

7. A method for enzymatically hydrolyzing halogenated organic matter at an oil-water interface, comprising the following steps: S1, dissolving the enzyme-polymer coupled enzyme in the catalytic system according to any one of claims 1 to 5 in the aqueous solution as the aqueous phase; S2, dissolving the halogenated organic compound in the organic solvent as the oil phase; S3, mixing the oil phase and the aqueous solution to form an oil-water dispersion system, and performing enzyme-catalyzed hydrolysis of the halogenated organic matter at the oil-water interface.

8. The method according to claim 7, characterized in that: The halogenated organic matter is a halogenated alkane, including chain halogenated hydrocarbons, cyclic halogenated hydrocarbons, halogenated esters, halogenated nitriles and halogenated amides containing a benzene ring structure.

9. The method according to claim 7 or 8, 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 molecules is located in the water phase.

10. The method according to any one of claims 7 to 9, characterized in that: At least one product of the hydrolysis is dissolved in the aqueous phase.

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