CN110760498A - A kind of co-crosslinking immobilization method of glutamic acid decarboxylase - Google Patents

Abstract

The present invention relates to a co-crosslinking immobilization method of glutamic acid decarboxylase. Oil-soluble pyridazine diacrylate is used as a cross-linking agent, and the reactants in the aqueous phase are amino-containing glutamic acid decarboxylase and supramolecular complexes formed by aminated epoxy resin and β-cyclodextrin. The Michael addition reaction between the bond and the amino group, and the co-crosslinking polymerization reaction occurs at a lower temperature to prepare the immobilized glutamate decarboxylase with different loadings. By controlling the degree of cross-linking, improving dispersibility, and improving the internal mass transfer microenvironment, the immobilized enzyme has high catalytic activity, and the loading amount of 75mg enzyme/g carrier has the highest activity, reaching 94% of the free enzyme. .

Description

A kind of co-crosslinking immobilization method of glutamic acid decarboxylase

technical field

The invention relates to the technical field of immobilized enzyme biocatalysis, in particular to a co-crosslinking and immobilization method of glutamic acid decarboxylase. The novel immobilized glutamic acid decarboxylase can be specially used for the efficient preparation of γ-aminobutyric acid.

Background technique

Gamma-aminobutyric acid is a four-carbon non-protein amino acid, which is confirmed to be an inhibitory transmitter in mammalian nerve center and plays an important role in regulating the activity of mammalian cardiovascular and cerebrovascular. It has a wide range of uses in the pharmaceutical industry and the food industry. The drug development of γ-aminobutyric acid analogs and derivatives has become one of the current research hotspots. The drugs that have been listed are baclofen, milnacipran, xiborine, piracetam, oxiracetam and so on. Gamma-aminobutyric acid also has the functions of treating epilepsy, adjuvant treatment of asthma, strengthening memory, anti-aging, activating liver and kidney function, detoxification, preventing obesity, promoting alcohol metabolism and deodorizing.

Glutamate decarboxylase (EC 4.1.1.15) is a class of pyridoxal phosphate-dependent enzymes (with an isoelectric point of 5.61), and is the only key enzyme for biocatalytic production of γ-aminobutyric acid. γ-aminobutyric acid is formed by removing the α carboxyl group from glutamic acid under the action of glutamic acid decarboxylase. This method has high yield and high purity, and is suitable for large-scale industrial production. The catalytic effect of glutamate decarboxylase on glutamate is specific, and it has no effect on catalyzing the decarboxylation reaction of other amino acids, and the enzyme activity reaches the highest when the pH of the action is 4.0 to 5.0, but glutamate decarboxylase Dissociation readily occurs at pH above 7.0, protein concentration and temperature are low.

Immobilized enzyme is to change water-soluble free enzyme into insoluble solid enzyme by chemical means. Immobilization has many advantages: for example, immobilized glutamic acid decarboxylase can be reused, which improves the use efficiency of the enzyme and reduces the use cost; The immobilized glutamate decarboxylase is easily separated from the reaction system, which simplifies the operation process; the storage stability and thermal stability of the immobilized glutamate decarboxylase have been 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 stirring or packing a column, which is convenient for the continuous and automated operation of the enzyme-catalyzed reaction. Enzyme cross-linking is a very efficient immobilization method, and the resulting products 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 in dosage, which can easily cause excessive cross-linking of enzymes and cause great loss of enzyme activity. In addition, traditional cross-linking methods often It is necessary to precipitate and aggregate the enzyme molecules before cross-linking, which will not only cause the waste of the enzyme, but also block the mass transfer channel, so that the catalytic efficiency of the enzyme cannot be fully utilized.

The patent of the present invention provides a co-crosslinking method for the immobilization of glutamate decarboxylase, which utilizes the Michael addition reaction between the amino group on the glutamate decarboxylase molecule and the acrylate cross-linking agent, and also introduces β- The structural unit of cyclodextrin can not only provide space for the catalytic reaction, reduce the mass transfer resistance, but also increase the hydrophilicity and improve the activity of the enzyme. Using this co-crosslinking method, the loading capacity and catalytic activity of the enzyme are high, the stability is good, the immobilized enzyme is in the form of granules, and the catalytic reaction is easy to operate.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a method for immobilization of glutamic acid decarboxylase, which is based on the co-crosslinking reaction of glutamic acid decarboxylase and another molecular complex containing organic amines. The basis of the reaction is the Michael addition of acrylate to amino group. The reaction 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 adjust The microenvironment of the immobilized enzyme keeps it high catalytic activity.

1. The technical scheme adopted by the present invention to solve the technical problem is: a cross-linking reaction of water/oil two-phase, and the oil phase is a cross-linking agent pyridazine diacrylate, and its structure is shown in Figure 1, and the The reactants were glutamate decarboxylase and supramolecular complexes of β-cyclodextrin and aminated epoxy resin, and the loading of the immobilized enzyme was adjusted by the concentration of glutamate decarboxylase.

Very beneficially, the degree of cross-linking can be controlled through a heterogeneous reaction, avoiding excessive cross-linking of enzymes;

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 glutamate decarboxylase to be immobilized with nearly 100% utilization. , after the cross-linking reaction occurs, there is almost no residual glutamate decarboxylase 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 to provide a mass transfer channel for the interaction of biological macromolecules and substrates, and simultaneously Provides stability to the conformation of biological macromolecules, thereby enhancing the catalytic activity of the immobilized enzyme.

2. The technical solution adopted by the present invention to solve another technical problem is: a preparation method of the above-mentioned immobilized enzyme, wherein the characteristic steps are: 1) bisphenol A epoxy resin (brand name is E-51, epoxy value is 0.51, the number average molecular weight is 392), methanol and triethylenetetramine are mixed according to the mass ratio of 2:2:1.5, and the reaction is stirred in the range of 25 to 35 ° C for 4 to 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 dried at room temperature in a vacuum oven to obtain epoxy resin aminate; The molar ratio of 2.3 is added to water, heated and stirred until all epoxy resin aminates are converted into molecular complexes and dissolved in water, keeping the total mass concentration of the aqueous solution in the range of 5-6 wt.%; 3) Glutamate decarboxylase Dissolved in phosphate buffer solution with pH=6.5, the concentration of enzyme was kept in the range of 1.0-7.0mg/mL; The glutamate decarboxylase solution of 5.0mg/mL, 6.0mg/mL and 7.0mg/mL was mixed with the above-mentioned molecular complex aqueous solution according to the ratio of 55mL: 20mL, and the loading of the immobilized enzyme was adjusted by changing the concentration of the enzyme solution; 5) 1.2g of pyridazine diacrylate was added to the above mixed aqueous solution under stirring, and the reaction temperature was kept in the range of 25 to 30 ° C. After 10 to 15 minutes, white gel particles were formed. Stop stirring and allow the reaction system to stand for 4 to After 5 hours, different loadings of immobilized glutamate decarboxylase products were obtained after filtration.

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 a product with emulsification, the oil phase will disperse quickly after the reaction starts until it disappears, and glutamate decarboxylase first It enters the polymer by adsorption, and then the double bond on the crosslinker reacts slowly with the amino group on the enzyme, and finally becomes a co-crosslinked immobilized enzyme product;

It is very beneficial to use the interaction of β-cyclodextrin with the hydrophobic benzene ring to introduce a hydrophilic group, avoid the use of chemical bonds, and make the β-cyclodextrin unable to dissociate from the polymer through the cross-linking reaction, enabling the preparation of immobilized enzymes. simplify;

It is very beneficial that no other organic solvent is added during the whole polymerization process, and higher temperature is not required.

The advantages of the invention are: 1) the cross-linking of the enzyme is realized by using the water/oil biphasic reaction, and the degree of cross-linking is controlled; 2) the introduction of the β-cyclodextrin supramolecular complex improves the microscopic efficiency of the immobilized glutamate decarboxylase 3) The co-crosslinking immobilization method can immobilize glutamic acid decarboxylase with extremely high efficiency.

Detailed ways

Immobilization of enzymes

1) bisphenol A epoxy resin (brand name is E-51, epoxy value is 0.51, number-average molecular weight is 392), methanol and triethylene tetramine three components are mixed according to the mass ratio of 2: 2: 1.5, The reaction was stirred at 25 to 35°C for 4 to 5 hours, the mixture was poured into water, the precipitate was repeatedly washed with water to remove methanol and a small amount of amine, and then placed in a vacuum oven for drying at room temperature to obtain epoxy resin aminate;

2) Add epoxy resin aminate and β-cyclodextrin into water according to the molar ratio of 1:2.1～1:2.3, heat and stir until epoxy resin aminate is completely converted into molecular complex and dissolve in water, keep this The total mass concentration of the aqueous solution is in the range of 5 to 6 wt.%;

3) Dissolving glutamate decarboxylase in a phosphate buffer solution with pH=6.5, the concentration of the enzyme is kept in the range of 1.0-7.0 mg/mL;

4) Glutamate decarboxylase solutions with concentrations of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL, 5.0mg/mL, 6.0mg/mL, 7.0mg/mL were respectively mixed with the above molecules. The complex aqueous solution is mixed according to the ratio of 55mL: 20mL, and the loading amount of the immobilized enzyme is adjusted by changing the concentration of the enzyme solution;

5) 1.2 g of pyridazine diacrylate was added to the above mixed aqueous solution under stirring, and the reaction temperature was kept in the range of 25 to 30 ° C for 10 to 15 minutes. After 10 to 15 minutes, white gel particles were formed, and the oil phase disappeared at the same time. Stop stirring to make the reaction The system was placed for 4 to 5 hours, and the products of different loadings of immobilized glutamic acid decarboxylase were obtained after filtration.

Loading determination of immobilized enzyme:

Since glutamate decarboxylase was immobilized by the co-crosslinking method, the activity of glutamate decarboxylase could not be detected in the reaction residue, indicating that all glutamate decarboxylase entered the solid particles after cross-linking, so the calculation of the loading amount Use the following formula:

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

Enzyme activity assay:

(1) Determination of free enzyme activity: Take 400 μL of substrate solution (pH 4.8, 0.2 mol/L citric acid-disodium hydrogen phosphate buffer, containing 0.01 mmol/L PLP, 100 mmol/L substrate L-MSG) and add it to In a 1.5mL centrifuge tube, place it in a 37°C water bath to preheat, then add 20μL of pure glutamate decarboxylase, mix quickly, and react at 37°C for 40min. After the reaction, take 0.1mL of the sample and add 0.9mL of NaHCO ₃ solution (0.2mol/L, pH 9.8) was used to stop the reaction. After centrifugation, 0.5 mL of the supernatant was taken, and an equal amount of DNS-C1 acetone (8 g/L) solution was added. After the sample was filtered through a 0.22 μm microporous membrane, the content of GABA generated by the reaction was determined by high performance liquid chromatography (HPLC method) to determine the enzyme activity.

(2) Determination of immobilized enzyme activity: use 0.05mol/L phosphate (pH 5.6) buffer as the reaction medium, add 20 mg of immobilized glutamate decarboxylase, and then add 0.05mol/L sodium glutamate as the substrate , shake at a constant temperature of 100 r/min in a constant temperature water bath for 2 hours, inactivate at 90 °C for 5 minutes, take the supernatant by centrifugation, and use high performance liquid chromatography (HPLC) to determine the γ-aminobutane in the solution after constant volume. Acid (GABA) content. 1 μmol GABA was generated every 30 min as an enzyme activity unit.

Wherein, pre-column derivatization is performed when the GABA content is determined by HPLC, and the specific method is:

(i) Method for pre-column derivatization of GABA:

1% 2,4-dinitrofluorobenzene in acetonitrile (FDNB) was used as a derivatizing agent. Take 0.2mL of the sample supernatant after centrifugation at 10000r/min for 10min in a 2ml centrifuge tube, then add 0.2mL of 0.5mol/L NaHCO ₃ (pH=9.0) solution, 0.2mL of FDNB solution, and place it in a 60°C water bath Heated in the dark for 1h, and quickly cooled with cold water after taking it out. Add 1.4 mL of 0.05 mol/L phosphate buffer (pH=7.0), mix well, pass through a 0.45 μm filter, centrifuge at 10,000 r/min for 10 min, and store at 4°C in the dark for later use.

(ii) HPLC chromatographic conditions:

UV detection wavelength: 360nm; Chromatographic column: Lichrospher C18 (4.6×250mm, 5μm); Column temperature: 35°C; Mobile phase: A is 20mmol/L ammonium acetate aqueous solution, B is an equal volume ratio of acetonitrile aqueous solution; flow rate: 1mL/ min; injection volume: 10 μL.

Relative activity:

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

Experimental results:

A total of 7 samples with different loadings of immobilized glutamate decarboxylase were obtained in the experiment, their activities were measured respectively, and their relative activities were calculated. Figure 2 is the relationship between the relative activity and the loading amount. When the loading amount is 75 mg enzyme/g carrier, its relative activity reaches the maximum value, and its specific activity is 94% of the free enzyme. This result shows that glutamic acid decarboxylase is in this range. Very suitable for catalytic state. When the loading amount is less than 75mg enzyme/g carrier, the activity of the immobilized enzyme gradually increases with the increase of the loading amount, which is mainly because with the increase of the enzyme content, the structure of the polymer becomes loose and the contact between the enzyme and the substrate increases. As the opportunity increases, so does its relative activity. When the loading amount was greater than 75 mg enzyme/g carrier, the activity of the immobilized enzyme gradually decreased with the increase of the loading amount. In general, the cross-linking immobilization method will make the conformation of the enzyme stiff, thereby reducing the activity. The co-cross-linking immobilization method of the patent of the present invention can improve the microenvironment of the enzyme, which is related to the introduction of cyclodextrin supramolecular structural units. It loosens the structure of the immobilized enzyme and improves the internal hydrophilicity. In addition, the co-crosslinking can improve the dispersibility of the enzyme, avoid the aggregation of the enzyme, and improve its catalytic activity. But when the load is too large, the aggregation of the enzyme becomes inevitable, so its activity decreases rapidly.

We took the sample with a load of 61 mg enzyme/g carrier as the research object, and measured the storage stability of the immobilized enzyme and the free enzyme solution. The results are shown in Figure 3. The activity in the initial state with time zero is 100%. , after 28 days of storage at 4°C and pH=6.0, the free enzyme solution has only 57% of the activity remaining, and the immobilized enzyme can remain 84% of the activity, so in terms of storage stability, the immobilized enzyme is significantly better than free enzyme.

Description of drawings

Figure 1. Chemical structures of crosslinkers.

Fig. 2 The dependence of the catalytic activity of immobilized glutamate decarboxylase on its loading.

Figure 3 Comparison of storage stability of immobilized and free glutamate decarboxylase.

Claims (1)

1. A glutamic acid decarboxylase co-crosslinking immobilization method is characterized in that a water/oil two-phase reaction system is used, the oil phase is a crosslinking agent oxazine diacrylate, and the structure is as follows:

the reactant in the water phase is glutamate decarboxylase and a molecular compound with the following structure:

the glutamic acid decarboxylase co-crosslinking immobilization method comprises the following steps:

1) mixing bisphenol A epoxy resin with the number average molecular weight of 392, methanol and triethylene tetramine according to the mass ratio of 2: 1.5, 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 for drying at normal temperature to obtain an epoxy resin amide;

2) adding the epoxy resin aminated substance and β -cyclodextrin into water according to the molar ratio of 1: 2.1-1: 2.3, heating and stirring until the epoxy resin aminated substance is completely converted into a 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 glutamate decarboxylase in a phosphate buffer solution with the pH value of 6.5, and keeping the concentration of the glutamate decarboxylase within the range of 1.0-7.0 mg/mL;

4) mixing glutamate decarboxylase solutions with different concentrations with the molecular compound aqueous solution according to the ratio of 55mL to 20 mL;

5) adding 1.2g of oxazine diacrylate 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 4-5 hours, and filtering to obtain the glutamate decarboxylase immobilized products with different loading amounts.

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