CN110760497B - Co-crosslinking immobilization method of chloroperoxidase - Google Patents
Co-crosslinking immobilization method of chloroperoxidase Download PDFInfo
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- CN110760497B CN110760497B CN201910417452.2A CN201910417452A CN110760497B CN 110760497 B CN110760497 B CN 110760497B CN 201910417452 A CN201910417452 A CN 201910417452A CN 110760497 B CN110760497 B CN 110760497B
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/0101—Chloride peroxidase (1.11.1.10)
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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
Technical Field
The invention relates to the technical field of immobilized enzyme biocatalysis, in particular to a co-crosslinking immobilization method of chloroperoxidase.
Background
Chloroperoxidase (EC 1.11.1.10), a heme carbohydrase (isoelectric point 3.5) isolated from marine fungi by Hager et al for the first 60 s in the last century. Among the peroxidase family, it is one of the most widely used enzymes and combines the catalytic activities of peroxidases, and cytochromes. Pure chloroperoxidase is reddish brown, has a molecular weight of approximately 42kDa, is acidic, and consists of 299 amino acids, which contain 9 alpha helices and 2 beta sheets. The alpha-helix and beta-sheet account for 34% and 1.34% of the total amino acids, respectively. The chloroperoxidase enzyme contains approximately 20% to 30% carbohydrate compounds and also approximately 30% non-protein components.
The chloroperoxidase is prepared with H 2 O 2 And the peroxides are used as electron acceptors to catalyze various substrates to generate oxidation reactions. The chloroperoxidase has various catalytic performances due to the unique active site structure, and has the characteristics of high selectivity, high catalytic activity, normal temperature use and the like in the aspect of biotransformation. Meanwhile, the chloroperoxidase has wide catalytic performance, and can catalyze heteroatom oxidation (S-oxidation and N-oxidation), epoxidation, carbon-hydrogen bond oxidation, alcohol oxidation and indole oxidation; of interest is chlorineThe peroxidase has catalytic action on the halogenation, epoxidation, hydroxylation of olefin, sulfonation and oxidation of organic sulfur compound. And because of wide substrate adaptability and higher stereoselectivity, it has gradually become a green catalyst with great prospect in organic synthesis, pharmaceutical industry and industrial catalysis.
The immobilized enzyme is solid enzyme which changes water-soluble free enzyme into insoluble enzyme by chemical means, and has a plurality of advantages: for example, the immobilized chloroperoxidase can be reused, so that the use efficiency of the enzyme is improved, and the use cost is reduced; the immobilized chloroperoxidase is easy to separate from a reaction system, so that the operation process is simplified; the storage stability and the thermal stability of the immobilized chloroperoxidase are improved; the catalytic reaction process of the immobilized enzyme is easier to control; the immobilized enzyme has certain mechanical strength, can act on a substrate solution in a stirring or column packing mode, and is convenient for continuous and automatic operation of enzyme catalytic reaction. Crosslinking of enzymes is a very efficient immobilization process and the resulting product is called a crosslinked enzyme aggregate. The most commonly used cross-linking agent is water-soluble glutaraldehyde which has high reaction activity and difficult control of dosage, and can easily cause excessive cross-linking of enzyme, so that the activity of the enzyme has great loss.
The invention provides a co-crosslinking method for fixing chloroperoxidase, which utilizes amino on the molecule of the chloroperoxidase to generate Michael addition reaction with an acrylate crosslinking agent and introduces a structural unit containing beta-cyclodextrin, thereby not only providing space for catalytic reaction, reducing mass transfer resistance, but also increasing hydrophilicity and improving the activity of the enzyme. By using the co-crosslinking method, the loading capacity and catalytic activity of the enzyme are high, the stability is good, the immobilized enzyme is granular, and the catalytic reaction is easy to operate.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for immobilizing chloroperoxidase, which is based on the co-crosslinking reaction of the chloroperoxidase and another molecular compound containing organic amine, wherein the crosslinking reaction is based on Michael addition of acrylic ester and amino, and the reaction can quickly occur at normal temperature, so that the integral structure of the enzyme can not be damaged, the co-crosslinking method has high loading efficiency and good stability, and simultaneously, the microenvironment of immobilized enzyme can be regulated to ensure that the immobilized enzyme keeps high catalytic activity.
1. The technical scheme adopted by the invention for solving the technical problem is as follows: the cross-linking reaction of two phases of water and oil, oil phase is cross-linking agent isocyanuric acid triacrylate, its structure is shown in figure 1, reactant in water phase is chlorine peroxidase and beta-cyclodextrin and aminated epoxy resin supermolecular complex, the load of immobilized enzyme is regulated by the concentration of chlorine peroxidase.
The cross-linking degree can be controlled through multiphase reaction, excessive cross-linking of the enzyme is avoided, and meanwhile, the cross-linking agent contains a plurality of double bonds, so that a cross-linking product forms a branched structure, aggregation of the enzyme is prevented to the greater extent, and the activity of the enzyme is enhanced;
it is very beneficial that the molecular complex of beta-cyclodextrin and aminated epoxy resin generates strong affinity with enzyme molecules, resulting in a cross-linking reaction that enables chloroperoxidase to be immobilized with nearly 100% utilization, with little residual chloroperoxidase in the liquid phase after the cross-linking reaction has occurred;
the molecular compound of beta-cyclodextrin and aminated epoxy resin has a bent rigid structure, which brings sufficient free volume, provides a mass transfer channel for the interaction of biomacromolecules and substrates, and provides stability for the conformation of the biomacromolecules, thereby improving the catalytic activity of the immobilized enzyme.
2. The technical scheme adopted by the invention for solving another technical problem is as follows: a preparation method of the immobilized enzyme is characterized by comprising the following steps: 1) Mixing bisphenol A epoxy resin (with the brand number of E-51, the epoxy value of 0.51 and 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 pH =7.5, wherein the concentration of the enzyme is kept in a range of 1.0-7.0 mg/mL; 4) Mixing chloroperoxidase solutions with the concentrations of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6.0mg/mL and 7.0mg/mL with the molecular complex aqueous solution according to the ratio of 60mL to 20mL, and adjusting the loading amount of the immobilized enzyme by changing the concentration of the enzyme solution; 5) 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 products of immobilized chloroperoxidase with different loading amounts.
The method has the advantages that one double bond in the cross-linking agent firstly reacts with amino on a molecular compound to form a product with an emulsifying effect, an oil phase can be quickly dispersed until the oil phase disappears after the reaction is started, chloroperoxidase firstly enters a polymer in an adsorption mode, then the double bond on the cross-linking agent slowly reacts with the amino on the enzyme, and finally the co-crosslinked immobilized enzyme product is formed;
the method has the advantages that the interaction of the beta-cyclodextrin and the hydrophobic benzene ring is utilized to introduce the hydrophilic group, so that the use of chemical bonds is avoided, the beta-cyclodextrin cannot be separated from the polymer through crosslinking reaction, and the preparation of the immobilized enzyme is simplified;
advantageously, no additional organic solvent is added throughout the polymerization process and no higher temperatures are required.
The invention has the advantages that: 1) The cross-linking of the enzyme is realized by using water/oil double-phase reaction, so that the cross-linking degree of the enzyme is reduced; 2) The beta-cyclodextrin molecular compound is introduced to improve the microenvironment of the immobilized chloroperoxidase and improve the catalytic reaction activity of the enzyme; 3) The co-crosslinking immobilization method can lead the chloroperoxidase to be immobilized with extremely high efficiency; 4) The immobilized product can form a branched structure by adopting a multifunctional cross-linking agent, so that the aggregation of the enzyme is prevented, and the catalytic performance of the enzyme is improved.
Detailed Description
Immobilization of enzymes
1) Mixing bisphenol A epoxy resin (with the brand number of E-51, the epoxy value of 0.51 and 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 pH =7.5, wherein the concentration of the enzyme is kept in a range of 1.0-7.0 mg/mL;
4) Mixing chloroperoxidase solutions with the concentrations of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6.0mg/mL and 7.0mg/mL with the molecular complex aqueous solution according to the ratio of 60mL to 20mL, and adjusting the loading amount of the immobilized enzyme by changing the concentration of the enzyme solution;
5) Adding 1.2g of isocyanuric acid triacrylate into the mixed aqueous solution under stirring, keeping the reaction temperature within the range of 25-30 ℃ for 10-15 minutes, forming white gel particles, simultaneously eliminating an oil phase, stopping stirring to allow the reaction system to stand for 5-6 hours, and filtering to obtain the products of the immobilized chloroperoxidase with different loads.
And (3) measuring the loading capacity of the immobilized enzyme:
after the chloroperoxidase is immobilized by the co-crosslinking method, the activity of the chloroperoxidase cannot be detected in the reaction residual liquid, which indicates that all the chloroperoxidase enters the solid particles after crosslinking, so the load amount is calculated by the following formula:
wherein: c is the concentration of the co-crosslinking enzyme solution (mg/mL); v is the volume (mL) of the co-crosslinking enzyme solution; m is the dry mass (g) of the immobilized enzyme.
And (3) enzyme activity determination:
(1) And (3) measuring the activity of the free enzyme: CPO activity was measured by the chlorination reaction of 2-chloro-5, 5-dimethyl-1, 3-cyclohexanedione (MCD) catalyzed by Chloroperoxidase (CPO) to form 2, 2-dichloro-5, 5-dimethyl-1, 3-cyclohexanedione (DCD). To a quartz cuvette with a total volume of 3mL, 0.1M potassium phosphate buffer (pH = 2.75) containing 20mM potassium chloride solution, 0.1mM MCD,2mM H was added 2 O 2 And an appropriate amount of CPO to bring the final volume of the solution to 3mL. The reaction was added, shaken up and the.DELTA.A read at 278nm 278 nm values, read at t = 0. Immediately adding H 2 O 2 And (4) uniformly mixing in a cuvette and timing. At room temperature at Δ A 278 Detecting the reduction value of MCD absorbance at nm, reading every 20 seconds for 3-5 min until the substrate is basically reacted completely. Measuring the Delta A of the sample 278 And (5) making a time-absorbance relation curve at nm. The reaction was linear at the start of the reaction, and the value of Δ A within 1min was determined from the linear portion 278 nm。
(2) And (3) determining the activity of the immobilized enzyme: 10mg of the immobilized enzyme was placed in a cuvette, 3mL of 0.1M potassium phosphate buffer (pH = 2.75) was added to the cuvette to react at 25 ℃, the change in absorbance at 278nm in the reaction system for 5min was measured on an ultraviolet-visible spectrophotometer, and the activity of the immobilized enzyme was calculated. Each value is the average of three numbers. The standard deviation of the results did not exceed 5%.
1 unit of activity U is defined as 1. Mu. Mol MCD catalytically produced in 1min at 25 ℃ (e = 12.2M) -1 cm -1 ) The amount of enzyme required for conversion to DCD (i.e.the absorption at 278nm disappeared).
Relative activity:
the ratio of the activity of the immobilized enzyme to the activity of the free enzyme is defined as the relative activity.
The experimental results are as follows:
a total of 7 samples of immobilized chloroperoxidase with different loading amounts are obtained in the experiment, the activity of the samples is respectively measured, and the relative activity of the samples is calculated. FIG. 2 is a graph of relative activity versus loading, with the relative activity reaching a maximum at 66mg enzyme/g support and the specific activity being 91% of the free enzyme, indicating that chloroperoxidase is in a very catalytic state in this range. When the loading is less than 66mg of enzyme/g of carrier, the activity of the immobilized enzyme is gradually increased along with the increase of the loading, mainly because the polymer structure is compact when the content of the enzyme is lower, the catalytic activity of the enzyme is not easy to exert, the structure of the polymer becomes loose along with the increase of the enzyme content, the contact chance of the enzyme and the substrate is increased, and the relative activity is also improved along with the increase of the enzyme content. When the loading amount is more than 66mg of enzyme per gram of carrier, the activity of the immobilized enzyme gradually becomes smaller as the loading amount increases. Generally, the cross-linking reaction can make the conformation of the enzyme become rigid, so that the activity is reduced, the co-cross-linking immobilization method disclosed by the invention can improve the microenvironment of the enzyme, which is related to the introduction of cyclodextrin supermolecular structural units, the structure of the immobilized enzyme is loosened, the internal hydrophilicity is improved, and in addition, the cross-linking agent with high branching degree can also improve the dispersibility of the enzyme, avoid the aggregation of the enzyme and further improve the catalytic activity of the enzyme. However, when the amount is too large, aggregation of the enzyme becomes inevitable, so that the activity thereof becomes small.
As shown in fig. 3, the storage stability of the immobilized enzyme and the free enzyme solution was measured using a sample having a loading of 66mg of enzyme/g of carrier, and as a result, when the initial state activity of time zero was 100%, and the free enzyme solution was stored at 4 ℃ and pH =7.0 for 28 days, 47% of the activity remained in the free enzyme solution, and 78% of the activity remained in the immobilized enzyme, the immobilized enzyme was significantly superior to the free enzyme in terms of storage stability.
Drawings
FIG. 1 chemical structure of the crosslinker.
FIG. 2 dependence of immobilized chloroperoxidase catalytic activity on its loading.
FIG. 3 comparison of storage stability of immobilized and free chloroperoxidase.
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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| JP2008104359A (en) * | 2006-10-23 | 2008-05-08 | Dainippon Ink & Chem Inc | Carrier for immobilizing enzyme, immobilized enzyme and method for producing carrier for immobilizing enzyme |
| CN101818139A (en) * | 2010-04-22 | 2010-09-01 | 无锡市金坤生物工程有限公司 | Preparation method of immobilization peroxidase |
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| JP2003144147A (en) * | 2001-11-15 | 2003-05-20 | Kansai Paint Co Ltd | Method for producing granular formed product for immobilizing enzyme or microbial cell |
| EP2158627A2 (en) * | 2007-05-04 | 2010-03-03 | Akermin, Inc. | Immobilized enzymes and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2008104359A (en) * | 2006-10-23 | 2008-05-08 | Dainippon Ink & Chem Inc | Carrier for immobilizing enzyme, immobilized enzyme and method for producing carrier for immobilizing enzyme |
| CN101818139A (en) * | 2010-04-22 | 2010-09-01 | 无锡市金坤生物工程有限公司 | Preparation method of immobilization peroxidase |
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