Immobilized alkyl halide dehalogenase and preparation method thereof, battery system and dehalogenation method for catalyzing hydrolysis of halogenated organic matter

Abstract

The invention discloses an immobilized alkyl halide dehalogenase, a preparation method thereof, a battery system and a dehalogenation method for catalyzing hydrolysis of halogenated organic matters. The immobilized alkyl halide dehalogenase comprises a carrier and an alkyl halide dehalogenase mutant fixed on the carrier, the carrier is a conductive two-dimensional carbon material of which the surface contains carboxyl; the carbon terminal of the alkyl halide dehalogenase mutant has a cysteine residue, and the cysteine residue is the only cysteine residue on the protein surface; the carrier is connected with the alkyl halide dehalogenase mutant through a connecting agent, and end groups at two ends of the connecting agent are maleimide group and amino group respectively. According to the immobilized alkyl halide dehalogenase and the battery system, the problem of product inhibition caused by halide ion enrichment is solved, and the stability of the enzyme is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biocatalysis, and particularly relates to an immobilized alkyl halide dehalogenase, a preparation method thereof, a battery system, and a dehalogenation method for catalyzing the hydrolysis of halogenated organic compounds. Background Art

[0002] In manufacturing processes such as chemical synthesis, a large amount of wastewater and waste gas containing halogenated organic compounds are inevitably generated. Halogenated organic compounds are stable under natural conditions, difficult to degrade, highly lipophilic, can be enriched in the human body through the food chain, have chronic toxic side effects and genotoxicity, and pose a serious threat to environmental safety and human health. The treatment strategies for halogenated organic pollutants mainly include chemical oxidation methods (direct combustion method & catalytic oxidation method), separation and recovery methods (condensation method, absorption method, adsorption method, membrane separation method, etc.), and biological methods. At present, the chemical oxidation method is the most widely used, but it still has disadvantages such as the risk of secondary pollution, serious equipment corrosion, and expensive catalysts. The biological method has great prospects in the treatment of halogenated organic pollutants, especially low-concentration halogenated organic pollutants, due to its mild reaction conditions and broad substrate spectrum characteristics. Taking alkyl halide dehalogenase, which catalyzes the dehalogenation reaction through a hydrolysis reaction mechanism, as an example, it has a broad substrate spectrum containing more than 30 substrates, and no coenzyme / cofactor is required during the reaction process, showing relatively significant industrial application advantages.

[0003] However, there are some key problems in the practical application of the hydrolysis dehalogenation reaction catalyzed by alkyl halide dehalogenase. One is the stability problem of the enzyme: Since the catalytic object of alkyl halide dehalogenase is an organic compound insoluble in water, organic solvents are often introduced into the reaction system or organic co-solvents are added to water to improve the accessibility of substrate molecules, but this may have an adverse effect on the stability of the enzyme. The other is the inhibition of enzyme activity by the product halide ions during the hydrolysis reaction. The literature [Journal of Biological Chemistry, 2003, 278(46):45094-45100] explained the product inhibition phenomenon of halide ions from the perspective of the catalytic mechanism and proved that the intrinsic catalytic activity of alkyl halide dehalogenase can be restored after transferring the high-concentration halide ions in the environment.

[0004] Immobilized enzymes are one of the important methods to improve the stability of enzymes in non-aqueous phases. For example, the invention patent CN200880021830.5 discloses a technology for preparing biodiesel using lipase, and the stability of the immobilized enzyme is improved by regulating the hydrophilic-hydrophobic properties of the carrier; the literature [Journal of Molecular Catalysis B: Enzymatic 2001, 15(4-6), 147-153] reports the design of immobilizing horseradish peroxidase on silica microspheres, and all catalytic activities of the enzyme can be retained by immobilization in an aqueous reaction system containing 30% co-solvent DMSO. However, in the currently published reports and technical materials, there is still no good immobilization technical scheme for the catalytic stability of alkyl halide dehalogenase in non-aqueous phases. Moreover, there is also a lack of published technical solutions for the product inhibition phenomenon caused by halide ions. Summary of the Invention

[0005] The object of the present invention is to provide an immobilized alkyl halide dehalogenase, its preparation method, a battery system, and a dehalogenation method for catalyzing the hydrolysis of halogenated organic compounds, which alleviates the problem of product inhibition caused by halide ion enrichment and improves the stability of the enzyme during the hydrolysis of halogenated organic compounds.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides an immobilized alkyl halide dehalogenase, which includes a carrier and an alkyl halide dehalogenase mutant immobilized on the carrier;

[0008] The carrier is a conductive two-dimensional carbon material with carboxyl groups on its surface;

[0009] The C-terminus of the alkyl halide dehalogenase mutant has a cysteine residue, and it is the only cysteine residue on the protein surface;

[0010] The carrier and the alkyl halide dehalogenase mutant are connected by a linker, and the end groups at both ends of the linker are maleimide group and amino group respectively.

[0011] In the above immobilized alkyl halide dehalogenase, further, the conductive two-dimensional carbon material with carboxyl groups on its surface includes hydrophobic carbon paper and carboxylated carbon nanotubes loaded on one side of the hydrophobic carbon paper;

[0012] Further, the linker is N-(2-aminoethyl) maleimide.

[0013] In the above immobilized alkyl halide dehalogenase, further, the alkyl halide dehalogenase mutant is a mutant of the hydrolytic dehalogenase LinB derived from Sphingomonas paucimobilis; preferably, the alkyl halide dehalogenase mutant has the amino acid sequence shown in SEQ ID NO.1.

[0014] In the above-mentioned immobilized alkyl halide dehalogenase, further, the loading amount of the alkyl halide dehalogenase mutant on the surface of the carrier is 0.1-0.2 mg / cm 2 .

[0015] In a second aspect, the present invention provides a method for preparing the immobilized alkyl halide dehalogenase described in any one of the above, comprising the following steps:

[0016] S1. Activate the carboxyl groups on the surface of the two-dimensional carbon material with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS);

[0017] S2. Replace the EDC and NHS on the surface of the two-dimensional carbon material activated in step S1 with the linker, so that the amino group in the linker is covalently bonded to the carboxyl group on the surface of the two-dimensional carbon material;

[0018] S3. Reduce with a reducing agent to make the sulfhydryl groups in cysteine in the alkyl halide dehalogenase mutant in a reduced state;

[0019] S4. React the sulfhydryl groups in the alkyl halide dehalogenase mutant treated in step S3 with the maleimide groups in the linker on the surface of the two-dimensional carbon material treated in step S2 to obtain the immobilized alkyl halide dehalogenase.

[0020] In the above-mentioned preparation method, further, the conductive two-dimensional carbon material containing carboxyl groups on the surface is obtained by the following steps: uniformly dropping a dispersion of carboxylated carbon nanotubes on a hydrophobic carbon paper and performing single-sided suction filtration to form a film.

[0021] In a third aspect, the present invention provides a battery system for catalyzing the hydrolysis of halogenated organic compounds, comprising a cathode, an anode and an electrolyte, wherein the cathode is the immobilized alkyl halide dehalogenase described in any one of the above or the immobilized alkyl halide dehalogenase prepared by the method described in any one of the above.

[0022] In the above-mentioned battery system for catalyzing the hydrolysis of halogenated organic compounds, further, the anode is a platinum electrode;

[0023] The electrolyte is a glycine buffer solution, the concentration of the glycine buffer solution is preferably 50 mmol / L, and the pH of the glycine buffer solution is preferably 8-10;

[0024] The battery system further includes a Hg / HgO reference electrode.

[0025] In a fourth aspect, the present invention provides a dehalogenation method for catalyzing the hydrolysis of halogenated organic compounds, comprising the following steps:

[0026] Add a halogenated organic compound to the electrolyte of the battery system for catalyzing the hydrolysis of halogenated organic compounds described above, and apply a constant voltage to the battery system with an external electric field to carry out the hydrolysis reaction.

[0027] In the above-mentioned dehalogenation method for catalyzing the hydrolysis of halogenated organic compounds, further, the halogenated organic compound is selected from one or more of chain halogenated hydrocarbons, cyclic halogenated hydrocarbons, halogenated esters, halogenated nitriles, and halogenated amides containing a benzene ring structure;

[0028] Use a cosolvent to dissolve the hydrophobic halogenated organic compound in the electrolyte. The cosolvent is preferably PEG1000, and the content of the cosolvent in the electrolyte is preferably 5-15 mg / mL;

[0029] The content of the halogenated organic compound in the electrolyte is 5-20 mg / mL;

[0030] Based on the mass of the alkyl halide dehalogenase, the concentration of the immobilized alkyl halide dehalogenase in the electrolyte is 30-80 mg / L;

[0031] The constant voltage < 1.1 V and the working current I ≤ 0.1 mA.

[0032] The method for immobilizing an enzyme on a conductive two-dimensional carbon material support provided by the present invention and the reaction system for catalyzing the hydrolysis of halogenated organic compounds in a microelectric field environment have the following advantages:

[0033] 1) Using a two-dimensional carbon material support modified with hydrophilic groups to immobilize the alkyl halide dehalogenase LinB can maintain the local hydrophilic microenvironment of the enzyme and maximize the protection of its catalytic activity and structural dynamics.

[0034] 2) By introducing cysteine at the C-terminus of the protein away from the cap domain and the active site to construct a mutant enzyme, it has been verified that this modification has no significant effect on the catalytic activity of the alkyl halide dehalogenase.

[0035] 3) Connecting the mutant enzyme to the support through a covalent bond, the structure is stable and not easy to fall off, which is beneficial to the recovery and reuse of the enzyme catalyst and has industrial application prospects.

[0036] 4) Providing suitable voltage and current working conditions (ΔE < 1.1 V and I ≤ 0.1 mA) will not only not cause electro-inactivation of the enzyme, but also eliminate the inhibition of enzyme activity by the product halide ions. Brief Description of the Drawings

[0037] Appendix Figure 1 It is a schematic molecular structure diagram of the directional immobilization of alkyl halide dehalogenase on a two-dimensional carbon-based support in Example 1 of the present invention.

[0038] Appendix Figure 2This is to explore the rate difference of bromide ion diffusion into the bulk water phase after applying a negative potential to the enzyme electrode (immobilized alkyl halide dehalogenase) in Example 1 of the present invention.

[0039] Appendix Figure 3 This is a schematic structural diagram of the battery system in the embodiment of the present invention.

[0040] Appendix Figure 4 This is the effect of the energized current intensity on the enzyme activity in Example 2 of the present invention.

[0041] Appendix Figure 5 This is to explore a) the catalytic activity and b) the stability of the immobilized enzyme under energized conditions in Example 3 of the present invention.

[0042] Appendix Figure 6 This is the degradation rate evaluation of the electroenzymatic system catalyzing the hydrolysis dehalogenation reaction of the model substrate 1-bromobutane in Example 4 of the present invention. Detailed implementation mode

[0043] As described in the background art, the catalytic stability of enzymes and the inhibition of enzyme activity by product halide ions during the hydrolysis reaction are the two key problems restricting the hydrolysis dehalogenation reaction catalyzed by alkyl halide dehalogenases. In order to improve the catalytic performance of alkyl halide dehalogenases and solve the problem of product halide ion inhibition of enzyme activity. Considering the charged nature of the product halide ions inhibited by alkyl halide dehalogenases, the present invention designs a reaction system that realizes the hydrolysis step of halogenated organic substrates and the removal process of inhibitory products in an electric field environment, while alleviating the product inhibition problem and enhancing the catalytic activity of enzymes.

[0044] The principle of the present invention is introduced below to better illustrate the intention and effect of the present invention.

[0045] The reaction equation of alkyl halide dehalogenase catalyzing halogenated hydrocarbon RX (R represents a hydrocarbon chain, X represents a halogen) is as follows:

[0046] RX + H2O = R-OH + HX

[0047] Enzyme catalysis occurs in an alkaline buffer environment, and the reaction products include alcohol R-OH and halide ion X - , and both are water-soluble substances. Among them, when the halide ion X - in the reaction system accumulates to a certain concentration, a relatively obvious product inhibition phenomenon will occur. The mechanism of product inhibition is mainly that: the product halide ions gradually enriched in the aqueous phase can return to the active pocket of the enzyme and occupy the substrate binding site, interfering with the targeting of subsequent substrate molecules to the substrate binding site and thus interfering with the enzyme catalytic activity. The present invention takes into account the charged nature of halide ions and uses the electromigration of charged ions under an electric field to resist the diffusion of halide ion X- to the enzyme located at the negative electrode, thereby realizing the high catalytic activity of alkyl halide dehalogenase.

[0048] In a first aspect, the present invention provides an immobilized alkyl halide dehalogenase, which comprises a carrier and an alkyl halide dehalogenase mutant immobilized on the carrier; the carrier is a conductive two-dimensional carbon material with carboxyl groups on its surface; the carbon terminus of the alkyl halide dehalogenase mutant has a cysteine residue, which is the only cysteine residue on the protein surface; the carrier and the alkyl halide dehalogenase mutant are connected by a linker, and the end groups at both ends of the linker are maleimide group and amino group respectively.

[0049] Based on the above technical solutions, the design concept of the immobilized enzyme of the present invention includes the following four points: 1) The carboxyl groups introduced in the conductive two-dimensional carbon material with carboxyl groups on its surface can not only serve as the connection sites for the immobilized enzyme, but also the hydrophilic environment constructed by it can ensure the structural flexibility and catalytic dynamics of the enzyme; 2) The selection of the grafting site on the protein surface should ensure that its negative impact on the enzyme catalytic activity is as small as possible. The C-terminal region of the alkyl halide dehalogenase far from the flexible region (Cap domain) and the catalytic core (Active site) meets the expectation; 3) A cysteine with a thiol structure is introduced at the carbon terminus of the protein to construct a mutant enzyme, and then the protein thiol group and the carboxyl group of the carrier are covalently connected by a linker molecule with maleimide group and amino group at both ends. At the same time, the linker molecule can keep a certain distance between the protein main body and the carrier plane, avoiding the destruction of the enzyme structure induced by the interfacial effect; 4) The introduction of the mutant site cysteine brings the only thiol group to the outer surface of the dehalogenase. While realizing the operation of the immobilized enzyme, it will also turn the catalytic core pocket of the dehalogenase away from the two-dimensional carrier surface. When a negative potential is applied to the enzyme electrode, it may accelerate the diffusion process of the hydrolyzed product halide ion from the active site to the bulk aqueous phase, thereby optimizing the enzyme catalytic activity. The combination of the above technical solutions significantly improves the activity of the enzyme-catalyzed hydrolysis dehalogenation reaction of halogenated organic compounds, and at the same time alleviates the problem of product inhibition caused by the enrichment of halide ions.

[0050] In an embodiment of the present invention, the conductive two-dimensional carbon material with carboxyl groups on its surface comprises a hydrophobic carbon paper and carboxylated carbon nanotubes loaded on one side of the hydrophobic carbon paper; wherein, the loading amount of the carboxylated carbon nanotubes on the surface of the hydrophobic carbon paper and the carboxyl modification rate of the carboxylated carbon nanotubes can be reasonably adjusted according to the loading amount of the target protein on the carrier; as an example, 5 mg of carboxylated carbon nanotubes are loaded on the surface of a hydrophobic carbon paper with a size of 1.5 cm in length × 1.0 cm in width, and the carboxyl modification rate of the carboxylated carbon nanotubes is 12%.

[0051] In an embodiment of the present invention, the linker is N-(2-aminoethyl) maleimide.

[0052] In one embodiment of the present invention, the alkyl halide dehalogenase mutant is a mutant of the hydrolytic dehalogenase LinB derived from Sphingomonas paucimobilis; as an example, the alkyl halide dehalogenase mutant has the amino acid sequence shown in SEQ ID NO.1. The alkyl halide dehalogenase mutant can be synthesized by a commercial institution or prepared by itself. In some embodiments, the coding gene of the alkyl halide dehalogenase mutant can be introduced into a recipient bacterium for expression. Preferably, the recipient bacterium is a stab bacterium, and the coding gene of the alkyl halide dehalogenase mutant can be determined according to the codon sequence shown in SEQ ID NO.2. In some embodiments, a tag can also be connected to the N-terminus and / or C-terminus of the alkyl halide dehalogenase mutant. The tag refers to a polypeptide or protein that is fused and expressed with the target protein by using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein.

[0053] In one embodiment of the present invention, the loading amount of the alkyl halide dehalogenase mutant on the surface of the carrier is 0.1 - 0.2 g / cm 2 , such as 0.13 mg / cm 2 . Among them, too low an enzyme loading amount leads to a slow catalytic reaction rate. However, when the enzyme loading amount is too high, due to the limitations of the accessibility of the substrate and the enzyme and the rate of removing bromide ions by electrification, the reaction rate cannot be correspondingly increased, thereby reducing the specific enzyme activity of the enzyme.

[0054] Second, as Figure 1 shown, the present invention provides a method for preparing the immobilized alkyl halide dehalogenase described in any one of the above, including the following steps: S1. Activate the carboxyl groups on the surface of the two-dimensional carbon material with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS); S2. Replace the EDC and NHS on the surface of the two-dimensional carbon material activated in step S1 with the linker so that the amino group in the linker covalently binds to the carboxyl group on the surface of the two-dimensional carbon material; S3. Reduce with a reducing agent to make the sulfhydryl group in cysteine in the alkyl halide dehalogenase mutant in a reduced state; S4. React the sulfhydryl group in the alkyl halide dehalogenase mutant treated in step S3 with the maleimide group in the linker on the surface of the two-dimensional carbon material treated in step S2 to obtain the immobilized alkyl halide dehalogenase.

[0055] In one embodiment of the present invention, the conductive two-dimensional carbon material with carboxyl groups on the surface is obtained by the following steps: uniformly drop the dispersion of carboxylated carbon nanotubes on a hydrophobic carbon paper and perform unilateral suction filtration to form a film. Among them, the concentration and solvent of the dispersion of carboxylated carbon nanotubes can be reasonably adjusted according to the dispersibility of carboxylated carbon nanotubes in the solvent. For example, the solvent is absolute ethanol and the concentration is 1 mg / mL.

[0056] In an embodiment of the present invention, the reactions in steps S1 - S4 are all carried out in HEPES buffer solution. As an example, the composition of the HEPES buffer solution is as follows: 10 mmol / L HEPES, 50 mmol / L NaCl, and 0.005% (volume fraction) of non - ionic surfactant Tween 20. The pH value of the buffer solution is adjusted with NaOH and controlled within the range of pH = 7.0 - 7.2.

[0057] In an embodiment of the present invention, 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide (EDC) and N - hydroxysuccinimide (NHS) are used to activate the carboxyl groups on the surface of the two - dimensional carbon material. The specific operation is as follows: Add the two - dimensional carbon material into the HEPES buffer solution, and make the side modified with carboxyl face upwards and float on the liquid surface. Then, add an excessive amount of EDC under stirring conditions for reaction (such as 5 min), and then add an excessive amount of NHS to continue the reaction (such as 25 min).

[0058] In an embodiment of the present invention, the EDC and NHS on the surface of the two - dimensional carbon material activated in step S1 are replaced by the linker, so that the amino group in the linker covalently binds to the carboxyl group on the surface of the two - dimensional carbon material. The specific operation is as follows: Add the linker into the system obtained in step S1 for reaction. Among them, the feeding amount of the linker can be reasonably adjusted according to the loading amount of carboxyl - functionalized carbon nanotubes and the carboxyl modification rate. Preferably, the amount of the linker (in terms of the amount of substance) is 2 times or more of the amount of carboxyl groups (in terms of the amount of substance) contained in the carboxyl - functionalized carbon nanotubes on the two - dimensional carbon material, to ensure that the carboxyl groups are fully linked. For example, when the linker is N - (2 - aminoethyl) maleimide, the mass ratio of the carboxyl - functionalized carbon nanotubes to the linker is 1:2, and the corresponding molar ratio of the linker to the amount of carboxyl groups contained in the carboxyl - functionalized carbon nanotubes on the two - dimensional carbon material is 2:1. Among them, the reaction time can be 4 - 10 h (such as 8 h) to enable the amino group in the linker to covalently bind fully with the carboxyl group on the surface of the two - dimensional carbon material.

[0059] In an embodiment of the present invention, a reducing agent is used to reduce the sulfhydryl group in cysteine of the alkyl halide dehalogenase mutant to a reduced state. The reducing agent can specifically be tris(2 - carboxyethyl)phosphine (TCEP), which can reduce the oxidized disulfide bond in sodium cysteine to a sulfhydryl group. Among them, the concentration of the reducing agent in the solution is 5 - 10 mmol / L, such as 5 mmol / L. The reduction reaction is stirred at a low temperature of 4 °C for 30 min - 60 min, such as 30 min.

[0060] In one embodiment of the present invention, the sulfhydryl group in the alkyl halide dehalogenase mutant treated in step S3 reacts with the maleimide group in the linker on the surface of the two-dimensional carbon material treated in step S2. The specific operation is as follows: Take out the two-dimensional carbon material treated in step S2 from the buffer system and place it in the solution of the alkyl halide dehalogenase mutant treated in step S3, and stir for reaction. Among them, the feeding amount of the mutant enzyme can be adjusted within a reasonable range according to the protein loading amount. For example, the mass ratio of the mutant enzyme to the carboxylated carbon nanotubes is 3:5. The mutant enzyme is a purified and freeze-dried mutant enzyme powder. It can be understood that before the reaction, there is also a step of washing off the EDC, NHS and linker adsorbed on the two-dimensional carbon material by non-covalent binding force using ultrapure water. The reaction time can be 4 to 10 hours of stirring (such as 8 hours), so that the maleimide group of the linker on the two-dimensional carbon material and the sulfhydryl group at the protein mutation site can fully undergo a specific affinity reaction.

[0061] In a third aspect, the present invention provides a battery system for catalyzing the hydrolysis of halogenated organic compounds, including a cathode, an anode and an electrolyte. The cathode is the immobilized alkyl halide dehalogenase described in any one of the above or the immobilized alkyl halide dehalogenase prepared by the method described in any one of the above.

[0062] In one embodiment of the present invention, the anode is a platinum electrode. It can be understood that the area of the platinum electrode is the same as that of the cathode electrode.

[0063] In one embodiment of the present invention, the electrolyte is a glycine buffer solution. The concentration of the glycine buffer solution is preferably 50 mmol / L, and the pH of the glycine buffer solution is preferably 8 to 10 (such as 8.8).

[0064] In one embodiment of the present invention, the battery system further includes a reference electrode, such as a Hg / HgO reference electrode.

[0065] In a fourth aspect, the present invention provides a dehalogenation method for catalyzing the hydrolysis of halogenated organic compounds, including the following steps: adding a halogenated organic compound to the electrolyte of the battery system for catalyzing the hydrolysis of halogenated organic compounds described above, and applying a constant voltage to the battery system with an external electric field for hydrolysis reaction.

[0066] According to the present invention, the halogenated organic compound is a halogenated alkane, such as a chain halogenated hydrocarbon (such as 1-bromobutane, 4-chloro-butene, etc.), a cyclic halogenated hydrocarbon (such as bromocyclopropane, chlorocyclopropane, 1-chlorocyclohexane, etc.), a halogenated ester (such as ethyl bromoacetate, ethyl chloroacetate, etc.), a halogenated nitrile (such as chloroacetonitrile, bromoacetonitrile, etc.), or one or more of them.

[0067] In one embodiment of the present invention, a solubilizer is used to dissolve the hydrophobic halogenated organic compound in the electrolyte. The solubilizer is preferably PEG 1000, and the content of the solubilizer in the electrolyte is preferably 5-15 mg / mL, such as 12.5 mg / mL.

[0068] In one embodiment of the present invention, the content of the halogenated organic compound in the electrolyte is 5-20 mg / mL, such as 9.5 mg / mL (1-bromobutane), 15 mg / mL (4-chloro-1-butene).

[0069] Based on the mass of the alkyl halide dehalogenase, the concentration of the immobilized alkyl halide dehalogenase in the electrolyte is 30-80 mg / L, such as 60 mg / L;

[0070] In one embodiment of the present invention, the constant voltage is <1.1 V and the working current I ≤ 0.1 mA. According to the Nernst equation, in the battery system of the present invention, the ΔE for the electrolysis reaction to occur is 1.1 V. To prevent the electrolysis reaction from occurring, the voltage applied to the electrode in the present invention is <1.1 V, such as 0.2 V, 0.8 V. At the same time, the introduction of the electric field will inevitably have a negative impact on the activity of the alkyl halide dehalogenase. In the practice of the present invention, the change in the activity of the immobilized enzyme after continuous power-on for 6 h at different currents was investigated, and it was found that when the current intensity I ≤ 0.1 mA, the reduction rate of enzyme activity did not exceed 10%. Therefore, the current working range of I ≤ 0.1 mA was determined, such as 0.1 mA, 0.01 mA, 0.001 mA.

[0071] Among them, the hydrolysis temperature can be reasonably adjusted according to the halogenated organic compound to be hydrolyzed. As an example, the hydrolysis of 1-bromobutane is at 36 °C; the hydrolysis time is determined according to the change of the hydrolysis rate with time.

[0072] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0073] The methods used in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0074] The sources of the various materials in the following examples are as follows:

[0075] Hydrophobic carbon paper (model HCP120) was purchased from Shanghai Hesen Electric Co., Ltd.;

[0076] Carboxylated carbon nanotubes (carboxyl mass fraction 12%, 9.5 nm × 1.5 μm) were purchased from Sigma Aldrich.

[0077] The alkyl halide dehalogenase contains the hydrolytic dehalogenase LinB derived from Sphingomonas paucimobilis and the mutant enzyme cLinB-cystein designed and constructed based on it. This mutant enzyme was expressed by the puncture bacterium E. coli BL21(DE3) containing the mutant enzyme cLinB-cystein gene (completed by entrusting Genewiz (Suzhou) Inc.) and separated by affinity chromatography, freeze-dried after purification to obtain the freeze-dried powder of the alkyl halide dehalogenase. The DNA sequence information of the mutant enzyme is shown in Table 1 below.

[0078] Table 1 Comparison of the gene sequences of the mutant enzyme and the wild-type enzyme

[0079]

[0080]

[0081] The composition of the HEPES buffer is as follows: 10 mmol / L HEPES, 50 mmol / L NaCl, and 0.005% (v / v) non-ionic surfactant Tween 20. The pH of the buffer was adjusted with NaOH and controlled within the range of pH = 7.0 - 7.2;

[0082] The composition of the glycine buffer is as follows: 50 mmol / L aqueous solution of glycine, adjusted to pH = 8.8 with sodium hydroxide;

[0083] All other chemicals are commercially available.

[0084] Example 1: Immobilization of alkyl halide dehalogenase on conductive two-dimensional carbon materials

[0085] I. Preparation of the conductive two-dimensional carbon-based membrane electrode

[0086] Take 5 mg of carboxylated carbon nanotubes in a sample tube, add 5 mL of absolute ethanol, and use an ultrasonic crusher to disperse CNTs-COOH into a suspension. The ethanol dispersion of carboxylated carbon nanotubes was evenly dropped on a hydrophobic carbon paper with a size of 1.5 cm × 1.0 cm, and a membrane was formed by unilateral suction filtration and air-dried for later use. Thus, a two-dimensional carbon-based membrane electrode with significantly different hydrophilic and hydrophobic properties on both sides was prepared. The carboxyl modification rate of CNTs-COOH was measured to be approximately 12% by energy dispersive spectroscopy (EDS) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR).

[0087] II. Immobilized enzyme construction process for covalently linking the mutant enzyme to the conductive two-dimensional carbon material carrier

[0088] As attached Figure 1As shown below. First, add 20 mL of HEPES buffer solution to a petri dish, and float 6 membrane electrodes prepared according to Example 1 with their hydrophilic sides (the sides modified with CNTs-COOH) facing the liquid surface; then, under magnetic stirring conditions, add 16 mg of excessive EDC to the HEPES buffer system and react for 5 min, then add 12 mg of excessive NHS and continue to react for 25 min; finally, add 10 mg of the linker molecule N-(2-aminoethyl) maleimide and react for 8 h to achieve the covalent connection process of the linker.

[0089] Dissolve 3 mg of the mutant enzyme in 15 mL of freshly taken HEPES buffer solution, add 20 mg of the reducing agent tris(2-carboxyethyl)phosphine (TCEP), and stir magnetically at a low temperature of 4 °C for 30 min to incubate the sulfhydryl group of the cysteine at the mutation site to the reduced state for subsequent reactions. At the same time, take out the membrane electrode modified with the linker from the original HEPES buffer system, wash off the EDC, NHS, and linker adsorbed by non-covalent binding force with ultrapure water, and place it in the above protein solution containing TCEP, and stir magnetically for 8 h to achieve the specific affinity reaction between the maleimide group of the two-dimensional carbon-based carrier linker and the sulfhydryl group of the protein mutation site, and achieve the covalent connection process of the mutant enzyme.

[0090] Measure the residual amount of protein in the HEPES buffer solution after the mutant enzyme grafting reaction, and calculate the protein loading amount on each 1.5 cm × 1.0 cm sized membrane electrode to be about 0.19 mg by the difference method, and convert it into the specific surface area data of 0.13 mg / cm 2 .

[0091] III. Influence of externally applied microelectric field on the distribution state of the charged inhibitory product bromide ion

[0092] First, soak the enzyme electrode (immobilized enzyme) prepared in II above with 1 mol / L sodium bromide aqueous solution for 1 h and take it out and place it in a glycine buffer system (50 mmol / L, pH = 8.75), equip a platinum electrode to form a closed circuit, and use a Hg / HgO electrode as a reference electrode. Apply a negative potential (ΔE = 0.2 V) to the enzyme electrode with an electrochemical workstation and measure the real-time change of the bromide ion concentration in the bulk solution within 30 s by the mercuric thiocyanate-ammonium ferric sulfate colorimetric method (measurement wavelength is 460 nm). Attached Figure 2 The increase in absorbance in it indicates the increase in the bromide ion concentration in the solution. Attached Figure 2 The results in it verify that the microelectric field can promote the faster diffusion of bromide ions into the bulk solution, and thus has the function of resisting the return of inhibitory products to the active pocket.

[0093] Example 2. Determination of the energized current

[0094] Fix the enzyme-immobilized conductive two-dimensional carbon-based film electrode prepared in Example 1 as the cathode electrode through an electrode clamp (with a size of 3 cm × 3 cm and an enzyme loading of 0.13 mg / cm 2 ), place the cathode electrode and a platinum electrode (anode) of the same area in 20 mL of 50 mmol / L glycine buffer solution (pH = 8.75) to form a single cell, and use a Hg / HgO electrode as the reference electrode (as shown in the appendix Figure 3 ). Use an electrochemical workstation to provide a constant negative voltage (negative potential of 0.8 V) as an external electric field. At I = 10 - 3 mA, 10 -2 mA, 10 -1 mA and 1 mA, measure the change in the activity of the immobilized enzyme after continuous power-on for 6 h. The results are as shown in the appendix Figure 4 . It can be seen that when the current intensity I ≤ 0.1 mA, the reduction rate of enzyme activity does not exceed 10%. Thus, the current working range of I ≤ 0.1 mA is determined.

[0095] Example 3, Construction of a Hydrolysis Reaction Single Cell System of Immobilized Enzyme and Determination of Catalytic Activity

[0096] Fix the enzyme-immobilized conductive two-dimensional carbon-based film electrode prepared in Example 1 as the cathode electrode through an electrode clamp. Place the cathode electrode and a platinum electrode (anode) of the same area in 20 mL of 50 mmol / L glycine buffer solution (pH = 8.75) to form a single cell, and use a Hg / HgO electrode as the reference electrode (as shown in the appendix Figure 3 ). Use an electrochemical workstation to provide a constant negative voltage (negative potential of 0.8 V) as an external electric field (I = 0.1 mA). Add 200 μL (256 mg) of 1-bromobutane as the substrate to the above solution, and add 200 μL (250 mg) of PEG1000 as a co-solvent. The size of the enzyme electrode is 3 cm × 3 cm, and the enzyme loading is 0.13 mg / cm 2 . The content of the immobilized enzyme in the solution is calculated to be 60 mg / L (based on the mass of alkyl halide dehalogenase). Use the mercury thiocyanate-ferric ammonium sulfate colorimetric method to measure the concentration of the product bromide ion in the reaction for 30 min, and evaluate the catalytic activity of the enzyme based on the change in the bromide ion concentration. With all other conditions being the same, only the group without power-on is used as a control. Taking the change amount Δc0 of the bromide ion concentration in the solution at 30 min in the control group as a benchmark, and taking the ratio of the change amount Δc E of the bromide ion concentration in the solution at 30 min during the power-on reaction to Δc0 as the relative enzyme activity. The results are as shown in the appendix Figure 5 a. The results in appendix Figure 5 a show that the reaction rate of the immobilized enzyme hydrolyzing the model substrate 1-bromobutane is significantly increased by applying an appropriate negative potential (ΔE = 0.8 V).

[0097] The free enzyme solution, the immobilized enzyme single cell (without power supply), and the immobilized enzyme single cell system (with power supply) were separately placed at room temperature for 48 h and 96 h, and the relative enzyme activity was measured at 48 h and 96 h according to the above method. The solution system used for the measurement of the free enzyme was the same as that used in the single cell system, both being glycine buffer solution, and the contents of the free enzyme and the immobilized enzyme on the electrode were the same; the power supply conditions for the immobilized enzyme solution were the same as above. The reference value of the enzyme activity was the change amount Δc0 of the bromide ion concentration in the free enzyme from the initial to 30 min of the reaction. The results are as Figure 5 shown in Fig. b. It can be seen that the immobilized enzyme constructed remained stable catalytic activity within 96 h, but the catalytic activity of the free enzyme was only 42% of the initial value after 96 h.

[0098] The changes in the reaction rate and substrate concentration within 30 min were measured using the free enzyme and the single cell system respectively (the solution system used for the measurement of the free enzyme was the same as that used in the single cell system, both being glycine buffer solution, and the amounts of the free enzyme and the immobilized enzyme on the electrode were the same; the substrate concentration was calculated by the increment of the bromide ion concentration in the solution; the power supply conditions were the same as above). The catalytic reaction kinetic constants K m and k cat， of the free enzyme and the immobilized enzyme were regressed with the Michaelis equation. The data results are shown in Table 2. The k cat of the immobilized enzyme in glycine buffer solution decreased significantly compared with the free enzyme, but increased by about 2 times under the power supply condition, indicating that the cumulative enrichment of the product bromide ion was the primary reason for the loss of the apparent activity of the immobilized enzyme. After applying a micro-electric field (the power supply condition was the same as above), the catalytic activity of the immobilized enzyme recovered to about 50% of that of the free enzyme in the aqueous phase system.

[0099] Table 2 Kinetic constants of enzyme-catalyzed reactions

[0100]

[0101] Example 4. Reaction effect of immobilized enzyme on two-dimensional carbon-based carrier in micro-electric field environment for catalyzing hydrolysis of 1-bromobutane

[0102] The single cell system and the catalytic system were the same as in Example 2, with 20 mL of glycine buffer solution. A constant negative voltage of ΔE = 0.8 V provided by an electrochemical workstation was used as the external electric field (I = 0.1 mA). 150 μL of the substrate 1-bromobutane (190 mg) was added to the reaction system, and 200 μL (250 mg) of the osmotic agent PEG1000 was added. The hydrolysis rate changing with time was measured under magnetic stirring and temperature control at 36 °C.

[0103] Taking the catalytic data of the immobilized enzyme under non-electrified conditions as a control (the measurement system and conditions are the same as in Example 2), the hydrolysis rate of the model substrate 1-bromobutane within 3 hours was measured. The specific measurement method is as follows: The concentration of the product bromide ion in the solution was determined by the mercury thiocyanate-ferric ammonium sulfate colorimetric method, and the change in the concentration of 1-bromobutane was calculated based on the change in the bromide ion concentration. The concentration of 1-bromobutane at the reaction time t is c st , and the initial concentration of 1-bromobutane is c s0 , then the hydrolysis rate x is: x = 1 - c st / c s0 . The experimental results are shown in the appendix Figure 6 . The hydrolysis rates of the electrified group at 60 min and 150 min were increased by 3.7% and 6.6% respectively compared with the non-electrified group

[0104] Example 5. Reaction effect of immobilized enzyme on two-dimensional carbon-based carrier catalyzing the hydrolysis of 4-chloro-1-butene in a microelectric field environment

[0105] The reaction system and its construction method are the same as in Example 2. The addition amount of the substrate 4-chloro-1-butene in the glycine buffer solution is 15 mg / mL, and 12.5 mg / mL of the osmotic agent PEG1000 is added. The content of the immobilized enzyme (calculated by the mass of LinB) is 0.03 mg / mL. An experiment on the hydrolysis of 4-chloro-1-butene catalyzed by the immobilized enzyme under non-electrified conditions was set as the control group, with the same other conditions, and the measurement method of the hydrolysis rate was the same as in Example 4. The hydrolysis rates at 90 min of the reaction were 29.6% (electrified group) and 21.3% (control group) respectively

[0106] The present invention has been described in detail above. For those skilled in the art, within the scope of not departing from the purpose and scope of the present invention, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although the present invention gives specific examples, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including those that deviate from the scope disclosed in this application and are made by using conventional techniques known in the art

Claims

1. An immobilized alkyl halide dehalogenase, characterized in that, It includes a carrier and an alkyl halide dehalogenase mutant immobilized on the carrier; The carrier is a conductive two-dimensional carbon material with carboxyl groups on its surface; The C-terminus of the alkyl halide dehalogenase mutant has a cysteine residue, which is the only cysteine residue on the protein surface; The carrier and the alkyl halide dehalogenase mutant are connected by a linker, and the end groups at both ends of the linker are maleimide group and amino group respectively.

2. The immobilized alkyl halide dehalogenase according to claim 1, characterized in that: The conductive two-dimensional carbon material with carboxyl groups on its surface includes a hydrophobic carbon paper and carboxylated carbon nanotubes loaded on one side of the hydrophobic carbon paper; and / or, The linker is N-(2-aminoethyl) maleimide.

3. The immobilized alkyl halide dehalogenase according to any one of claims 1-2, characterized in that: The alkyl halide dehalogenase mutant is a mutant of the hydrolytic dehalogenase LinB derived from Sphingomonas paucimobilis; preferably, the alkyl halide dehalogenase mutant has the amino acid sequence shown in SEQ ID NO.

1.

4. The immobilized alkyl halide dehalogenase according to any one of claims 1-3, characterized in that: The loading amount of the alkyl halide dehalogenase mutant on the surface of the carrier is 0.1 to 0.2 mg / cm 2 .

5. The preparation method of the immobilized alkyl halide dehalogenase according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Activate the carboxyl groups on the surface of the two-dimensional carbon material with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS); S2. Replace the EDC and NHS on the surface of the two-dimensional carbon material activated in step S1 with the linker, so that the amino group in the linker covalently binds to the carboxyl group on the surface of the two-dimensional carbon material; S3. Reduce with a reducing agent to make the sulfhydryl group in the cysteine of the alkyl halide dehalogenase mutant present in a reduced state; S4. React the sulfhydryl group in the alkyl halide dehalogenase mutant treated in step S3 with the maleimide group in the linker on the surface of the two-dimensional carbon material treated in step S2 to obtain the immobilized alkyl halide dehalogenase.

6. The preparation method of the immobilized alkyl halide dehalogenase according to claim 5, wherein: The conductive two-dimensional carbon material with carboxyl groups on its surface is obtained by the following steps: uniformly dropping a dispersion of carboxylated carbon nanotubes on a hydrophobic carbon paper and performing single-side suction filtration to form a film.

7. A battery system for catalyzing the hydrolysis of halogenated organic compounds, comprising a cathode, an anode, and an electrolyte, characterized in that, The cathode is the immobilized alkyl halide dehalogenase described in any one of claims 1-4 or the immobilized alkyl halide dehalogenase prepared by the method described in any one of claims 5-6.

8. The battery system for catalyzing the hydrolysis of halogenated organic compounds according to claim 7, characterized in that: The anode is a platinum electrode; The electrolyte is a glycine buffer solution, the concentration of the glycine buffer solution is preferably 50 mmol / L, and the pH of the glycine buffer solution is preferably 8-10; The battery system further includes a Hg / HgO reference electrode.

9. A dehalogenation method for catalyzing the hydrolysis of halogenated organic compounds, characterized in that, It includes the following steps: Add a halogenated organic compound to the electrolyte of the battery system for catalyzing the hydrolysis of halogenated organic compounds described in claim 7 or 8, apply a constant voltage to the battery system with an external electric field, and perform a hydrolysis reaction.

10. The dehalogenation method for catalytic hydrolysis of halogenated organic compounds according to claim 9, wherein: The halogenated organic compound is selected from one or more of chain halogenated hydrocarbons, cyclic halogenated hydrocarbons, halogenated esters, halogenated nitriles, and halogenated amides containing a benzene ring structure; Use a cosolvent to dissolve the hydrophobic halogenated organic compound in the electrolyte, the cosolvent is preferably PEG 1000, and the content of the cosolvent in the electrolyte is preferably 5-15 mg / mL; The content of the halogenated organic compound in the electrolyte is 5-20 mg / mL; Based on the mass of the alkyl halide dehalogenase, the concentration of the immobilized alkyl halide dehalogenase in the electrolyte is 30-80 mg / L; The constant voltage < 1.1 V and the working current I ≤ 0.1 mA.

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