CN110760497B - Co-crosslinking immobilization method of chloroperoxidase - Google Patents

Abstract

The invention relates to a co-crosslinking immobilization method of chloroperoxidase. Oil-soluble isocyanuric acid triacrylate is used as a cross-linking agent, reactants in a water phase are amino-containing chloroperoxidase and a supramolecular complex formed by aminated epoxy resin and beta-cyclodextrin, and the immobilized chloroperoxidase with different loading amounts is prepared by utilizing Michael addition reaction of double bonds and amino groups to carry out co-crosslinking polymerization reaction at a lower temperature. The cross-linking degree is controlled, the dispersibility is improved, the mass transfer microenvironment in the immobilized enzyme is improved, the immobilized enzyme has high catalytic activity, and the loading capacity has the highest specific activity when 66mg of enzyme/g of carrier, which reaches 91 percent of that of free enzyme.

Description

A co-crosslinking immobilization method of chloroperoxidase

technical field

The invention relates to the technical field of immobilized enzyme biocatalysis, in particular to a method for co-crosslinking and immobilizing chloroperoxidase. The novel immobilized chloroperoxidase can be specially used for efficient preparation of organic compounds.

Background technique

Chloroperoxidase (EC 1.11.1.10) is a heme glycoproteinase (isoelectric point 3.5) isolated from marine fungi by Hager et al. for the first time in the 1960s. In the peroxidase family, it is one of the most widely used enzymes and combines the catalytic activities of peroxidase, peroxidase and cytochrome. Pure chloroperoxidase is reddish brown, its molecular weight is about 42kDa, it is acidic, and it consists of 299 amino acids, including 9 α-helices and 2 β-sheets. , α-helices and β-sheets accounted for 34% and 1.34% of the total amino acids, respectively. Chloroperoxidase contains about 20% to 30% carbohydrates and about 30% non-protein components.

Chloroperoxidase uses H ₂ O ₂ and other peroxides as electron acceptors to catalyze the oxidation reactions of various substrates. Chloroperoxidase has a variety of catalytic properties due to its unique active site structure. It has high selectivity in biotransformation, high catalytic activity, and can be used at room temperature. At the same time, chloroperoxidase has a wide range of catalytic properties, which can catalyze heteroatom oxidation (S-oxidation and N-oxidation), epoxidation, carbon-hydrogen bond oxidation, alcohol oxidation and indole oxidation; Catalysis of peroxidase on reactions such as halogenation, epoxidation, hydroxylation of alkenes and sulfonation oxidation of organosulfur compounds. Due to its wide substrate adaptability and high stereoselectivity, it has gradually become a promising green catalyst in organic synthesis, pharmaceutical industry and industrial catalysis.

Immobilized enzyme is to change water-soluble free enzyme into insoluble solid enzyme by chemical means. Immobilization has many advantages: for example, immobilized chloroperoxidase can be reused, which improves the efficiency of enzyme use and reduces the cost of use; The immobilized chloroperoxidase is easily separated from the reaction system, which simplifies the operation process; the storage stability and thermal stability of the immobilized chloroperoxidase are improved; the catalytic reaction process of the immobilized enzyme is easier to control ; The immobilized enzyme has a certain mechanical strength and can act on the substrate solution by means of stirring or column packing, which is convenient for the continuous and automatic operation of the enzyme-catalyzed reaction. The cross-linking of enzymes is a very effective immobilization method, and the products formed are called cross-linked enzyme aggregates. The most commonly used cross-linking agent is water-soluble glutaraldehyde, which has high reactivity and is difficult to control the dosage. It is easy to cause excessive cross-linking of the enzyme and cause a great loss of enzyme activity. In addition, the traditional cross-linking method often Enzyme molecules must be precipitated and aggregated before cross-linking, which will not only cause waste of enzymes, but also block mass transfer channels, and cannot fully exert the catalytic efficiency of enzymes.

The patent of the present invention provides a method of co-crosslinking for the immobilization of chloroperoxidase, which uses the amino group on the chloroperoxidase molecule to undergo a Michael addition reaction with an acrylate crosslinking agent, and at the same time introduces a compound containing β- The structural unit of cyclodextrin can not only provide space for catalytic reactions, reduce mass transfer resistance, but also increase hydrophilicity and improve enzyme activity. Using this co-crosslinking method, the enzyme loading capacity and catalytic activity are high, the stability is good, the immobilized enzyme is granular, and the catalytic reaction is easy to operate.

Contents of the invention

The technical problem to be solved by this invention is to provide a method for the immobilization of chloroperoxidase, which is based on the co-crosslinking reaction of chloroperoxidase and another molecular complex containing organic amines. The basis of the reaction is the Michael addition of acrylate and amino groups, which can occur rapidly at room temperature, so it will not damage the overall structure of the enzyme. The co-crosslinking method has high loading efficiency, good stability, and can also regulate The microenvironment of the immobilized enzyme keeps its catalytic activity high.

1. The technical scheme adopted by the present invention to solve technical problems is: a kind of water/oil two-phase cross-linking reaction, the oil phase is a cross-linking agent isocyanurate triacrylate, its structure as shown in Figure 1, the water phase The reactant in the method is chloroperoxidase and the supramolecular complex of β-cyclodextrin and aminated epoxy resin, and the load of immobilized enzyme is regulated by the concentration of chloroperoxidase.

It is very beneficial that the degree of cross-linking can be controlled through heterogeneous reactions to avoid excessive cross-linking of enzymes. At the same time, the cross-linking agent contains multiple double bonds, so that the cross-linked products form a branched structure, which prevents the aggregation of enzymes to a greater extent. Enhance enzyme activity;

It is very beneficial that the molecular complex of β-cyclodextrin and aminated epoxy resin has a strong affinity with the enzyme molecule, resulting in a cross-linking reaction that enables the immobilization of chloroperoxidase with a utilization rate close to 100% , after the cross-linking reaction occurs, there is almost no residual chloroperoxidase in the liquid phase;

It is very beneficial that the molecular complex of β-cyclodextrin and aminated epoxy resin has a curved rigid structure, which brings sufficient free volume and provides mass transfer channels for the interaction between biomacromolecules and substrates, and at the same time Provides stability to the conformation of biomacromolecules, thereby improving the catalytic activity of the immobilized enzyme.

2, the technical scheme that the present invention adopts to solve another technical problem is: a kind of preparation method of above-mentioned immobilized enzyme, its characteristic step is: 1) bisphenol A epoxy resin (brand is E-51, epoxy value 0.51, the number average molecular weight is 392), methanol and diethylenetriamine are mixed according to the mass ratio of 2:2:1, stirred and reacted in the range of 25-35°C for 4-5 hours, and the mixture is poured into water , the precipitate was repeatedly washed with water to remove methanol and a small amount of amine, and then put into a vacuum oven and dried at room temperature to obtain an epoxy resin aminate; Add the molar ratio of 2.3 into water, heat and stir until the epoxy resin amides are all converted into molecular complexes and dissolved in water, and keep the total mass concentration of the aqueous solution in the range of 5 to 6wt.%; 3) chloroperoxidase Dissolved in sodium phosphate buffer solution with pH = 7.5, the concentration of the enzyme was kept in the range of 1.0-7.0 mg/mL; , 5.0mg/mL, 6.0mg/mL, 7.0mg/mL chloroperoxidase solution and the above-mentioned molecular complex aqueous solution were mixed according to the ratio of 60mL: 20mL, and the loading capacity of the immobilized enzyme was adjusted by changing the concentration of the enzyme solution ;5) Add 1.2g of isocyanuric acid triacrylate to the above mixed aqueous solution under stirring, keep the reaction temperature in the range of 25-30°C, and white gel particles will form after 10-15 minutes, stop stirring to make the reaction system After standing for 5-6 hours, the products of immobilized chloroperoxidase with different loads can be obtained after filtering.

It is very beneficial that a double bond in the cross-linking agent first reacts with the amino group on the molecular complex to form an emulsified product, and the oil phase will quickly disperse until it disappears after the reaction starts, and the chloroperoxidase first It enters the polymer by adsorption, and then the double bond on the crosslinking agent slowly reacts with the amino group on the enzyme, and finally becomes a co-crosslinked immobilized enzyme product;

It is very beneficial to use the interaction between β-cyclodextrin and hydrophobic benzene ring to introduce hydrophilic groups, avoid the use of chemical bonds, and make β-cyclodextrin unable to detach from the polymer through cross-linking reactions, so that the preparation of immobilized enzymes simplify;

It is very beneficial that no other organic solvents are added during the entire polymerization process, and higher temperatures are not required.

The present invention has the advantages of: 1) the use of water/oil biphasic reaction to realize the cross-linking of enzymes, which reduces the degree of cross-linking of enzymes; The microenvironment improves the catalytic activity of the enzyme; 3) The co-crosslinking immobilization method can immobilize chloroperoxidase with extremely high efficiency; 4) The use of multifunctional crosslinking agents can make the immobilized products form The branched structure prevents the aggregation of enzymes and improves the catalytic performance of enzymes.

Detailed ways

Enzyme immobilization

1) Bisphenol A epoxy resin (brand name is E-51, epoxy value is 0.51, number average molecular weight is 392), three kinds of components of methanol and diethylenetriamine are mixed according to the mass ratio of 2: 2: 1, Stir and react in the range of 25-35°C for 4-5 hours, pour the mixture into water, wash the precipitate repeatedly with water to remove methanol and a small amount of amine, then put it in a vacuum oven and dry it at room temperature to obtain an aminated epoxy resin;

2) Add epoxy resin amides and β-cyclodextrin into water at a molar ratio of 1:2.1 to 1:2.3, heat and stir until all epoxy resin amides are converted into molecular complexes and dissolved in water, and keep the The total mass concentration of the aqueous solution is in the range of 5 to 6wt.%.

3) dissolving chloroperoxidase in a sodium phosphate buffer solution with pH=7.5, and keeping the concentration of the enzyme in the range of 1.0-7.0 mg/mL;

4) Chloroperoxidase solutions with concentrations of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6.0mg/mL, and 7.0mg/mL were mixed with the above molecules The complex aqueous solution is mixed according to the ratio of 60mL: 20mL, and the loading capacity of the immobilized enzyme is adjusted by changing the concentration of the enzyme solution;

5) Add 1.2g of isocyanuric acid triacrylate into the above mixed aqueous solution under stirring, keep the reaction temperature in the range of 25-30°C for 10-15 minutes, white gel particles will form, and the oil phase will disappear at the same time, stop stirring The reaction system is allowed to stand for 5-6 hours, and the products of immobilized chloroperoxidase with different loads can be obtained after filtering.

Immobilized Enzyme Loading Determination:

After the chloroperoxidase was immobilized by the co-crosslinking method, the activity of chloroperoxidase could not be detected in the reaction residual liquid, indicating that all the chloroperoxidase had entered into the solid particles after crosslinking, so the calculation of the loading capacity Use the following formula:

Where: C is the concentration of the co-crosslinked enzyme solution (mg/mL); V is the volume of the co-crosslinked enzyme solution (mL); m is the dry mass of the immobilized enzyme (g).

Enzyme activity assay:

(1) Determination of free enzyme activity: use chloroperoxidase (CPO) to catalyze 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (MCD) to generate 2,2-dichloro-5 , 5-dimethyl-1,3-cyclohexanedione (DCD) chlorination reaction to detect the activity of CPO. Add 0.1M potassium phosphate buffer (pH=2.75) to a quartz cuvette with a total volume of 3mL, containing 20mM potassium chloride solution, 0.1mM MCD, 2mM H ₂ O ₂ and an appropriate amount of CPO to make the solution The final volume was brought to 3 mL. Add the reactants, shake well, and read the ΔA ₂₇₈ nm value at 278 nm, which is the reading at t=0. Immediately add H ₂ O ₂ to the cuvette, mix and time. Detect the decrease of the absorbance of MCD at ΔA ₂₇₈ nm at room temperature, and read every 20 seconds for a total of 3-5 minutes until the substrate basically reacts completely. The ΔA ₂₇₈ nm of the measured sample was plotted against the time-absorbance relationship curve. In the initial stage of the reaction, it is linear, and the ΔA ₂₇₈ nm within 1 min is calculated using the linear part.

(2) Determination of immobilized enzyme activity: put 10 mg of immobilized enzyme in a cuvette, add 3 mL of 0.1 M potassium phosphate buffer (pH=2.75) to react at 25 ° C, and measure it on a UV-visible spectrophotometer The change of absorbance at 278nm within 5 minutes of the reaction system was used to calculate the activity of the immobilized enzyme. Each value is the average of three numbers. The standard deviation of the results does not exceed 5%.

One activity unit U is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of MCD (e=12.2M ^-1 cm ^-1 ) into DCD within 1 min at 25°C (ie, the absorption at 278 nm disappears).

Relative activity:

The ratio of the activity of the immobilized enzyme to the activity of the free enzyme was defined as the relative activity.

Experimental results:

A total of 7 immobilized chloroperoxidase samples with different loads were obtained in the experiment, their activities were measured respectively, and their relative activities were calculated. Fig. 2 is the relation of relative activity and load, when load is 66mg enzyme/g carrier, its relative activity reaches maximum, and its specific activity is 91% of free enzyme, and this result shows that chloroperoxidase is in this range Very suitable for catalytic state. When the loading amount is less than 66 mg enzyme/g carrier, the activity of the immobilized enzyme gradually increases with the increase of the loading amount, which is mainly because the polymer structure is relatively compact when the enzyme content is low, and the catalytic activity of the enzyme is not easy. As the enzyme content increases, the structure of the polymer becomes looser, the chance of contact between the enzyme and the substrate increases, and its relative activity also increases. When the load was greater than 66mg enzyme/g carrier, the activity of the immobilized enzyme gradually decreased with the increase of the load. Generally speaking, the crosslinking reaction will make the conformation of the enzyme stiff, thereby reducing the activity. The co-crosslinking immobilization method of the patent of the present invention can improve the microenvironment of the enzyme, which is related to the introduction of the cyclodextrin supramolecular structural unit, which The structure of the immobilized enzyme becomes loose, and the internal hydrophilicity is also improved. In addition, the cross-linking agent with a high degree of branching can also improve the dispersibility of the enzyme, avoid the aggregation of the enzyme, and thus improve its catalytic activity. But when the load is too large, the aggregation of the enzyme becomes inevitable, so its activity will decrease again.

We took the sample with a load of 66mg enzyme/g carrier as the research object, and measured the storage stability of the immobilized enzyme and free enzyme solution. The results are shown in Figure 3, and the activity of the initial state with time as zero is 100%. , after 28 days of storage at 4°C and pH=7.0, only 47% of the activity of the free enzyme solution remains, and 78% of the activity of the immobilized enzyme remains, so in terms of storage stability, the immobilized enzyme is obviously better than free enzyme.

Description of drawings

Figure 1 Chemical structures of crosslinkers.

Figure 2 The dependence of the catalytic activity of immobilized chloroperoxidase on its load.

Fig. 3 comparison of immobilized and free chloroperoxidase storage stability.

Claims (1)

1. A chloroperoxidase co-crosslinking immobilization method is characterized in that a water/oil two-phase reaction system is used, and an oil phase is isocyanuric acid triacrylate serving as a crosslinking agent and has the following structure:

the reactant in the water phase is a molecular compound of chloroperoxidase and the following structure:

the chloroperoxidase co-crosslinking immobilization method comprises the following steps:

1) Mixing bisphenol A epoxy resin with the number average molecular weight of 392, methanol and diethylenetriamine according to the mass ratio of 2: 1, stirring and reacting for 4-5 hours at the temperature of 25-35 ℃, pouring the mixture into water, repeatedly washing precipitates with water to remove methanol and a small amount of amine, and then putting the precipitates into a vacuum oven to dry at normal temperature to obtain an epoxy resin amide;

2) Adding epoxy resin aminated substance and beta-cyclodextrin into water according to the mol ratio of 1: 2.1-1: 2.3, heating and stirring until the epoxy resin aminated substance is completely converted into molecular compound and dissolved in the water, and keeping the total mass concentration of the aqueous solution within the range of 5-6 wt%;

3) Dissolving chloroperoxidase in a sodium phosphate buffer solution with the pH =7.5, keeping the concentration of the enzyme within the range of 1.0-7.0 mg/mL, and mixing chloroperoxidase solutions with different concentrations with the molecular complex aqueous solution according to the ratio of 60mL to 20 mL;

4) Adding 1.2g of isocyanuric acid triacrylate into the mixed aqueous solution under stirring, keeping the reaction temperature within the range of 25-30 ℃, forming white gel particles after 10-15 minutes, stopping stirring to allow the reaction system to stand for 5-6 hours, and filtering to obtain the chloroperoxidase immobilized products with different loading amounts.

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