CN110760498B - Co-crosslinking immobilization method of glutamate decarboxylase - Google Patents

Abstract

The invention relates to a co-crosslinking immobilization method of glutamate decarboxylase. Oil-soluble 22112oxazine diacrylate is used as a cross-linking agent, reactants in a water phase are amino-containing glutamic acid decarboxylase and a supermolecular complex formed by aminated epoxy resin and beta-cyclodextrin, and the immobilized glutamic acid decarboxylase with different loading amounts is prepared by utilizing Michael addition reaction of double bonds and amino and carrying out co-crosslinking polymerization reaction at a lower temperature. By controlling the crosslinking degree, the dispersibility is improved, the mass transfer microenvironment in the immobilized enzyme is improved, the immobilized enzyme has higher catalytic activity, and the highest activity is achieved when the load is 75mg of enzyme/g of carrier, which is 94% of that of free enzyme.

Description

A co-crosslinking immobilization method for glutamic acid decarboxylase

technical field

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

Background technique

γ-aminobutyric acid is a four-carbon non-protein amino acid, which is recognized as an inhibitory transmitter in the mammalian nerve center and plays an important role in regulating the cardiovascular and cerebrovascular activities of mammals. It is widely used in the pharmaceutical industry and food industry. Drug development of γ-aminobutyric acid analogs and derivatives has become one of the current research hotspots. Drugs already on the market include baclofen, milnacipran, xipronin, piracetam, oxiracetam, etc. γ-aminobutyric acid also has the functions of treating epilepsy, assisting in the treatment of asthma, strengthening memory, anti-aging, activating liver and kidney functions, detoxifying, preventing obesity, promoting alcohol metabolism and deodorizing.

Glutamate decarboxylase (EC 4.1.1.15) is a kind of pyridoxal phosphate-dependent enzyme (isoelectric point is 5.61), and it is the only key enzyme for biocatalytic preparation 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. The method has high yield per unit area and high purity, and is suitable for large-scale industrial production. The catalysis of glutamic acid desmutase to glutamic acid is specific, and it has no effect on catalyzing the decarboxylation of other amino acids, and when the pH of the action is 4.0-5.0, the enzyme activity reaches the highest, but glutamic acid decarboxylase Dissociation readily occurs at pH above 7.0, protein concentration, and temperature.

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 efficiency of enzyme use and reduces the cost of use; The immobilized glutamic acid decarboxylase is easily separated from the reaction system, which simplifies the operation process; the storage stability and thermal stability of the immobilized glutamic acid 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 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 co-crosslinking method for the immobilization of glutamic acid decarboxylase, using the amino group on the glutamic acid decarboxylase molecule to undergo Michael addition reaction with an acrylate crosslinking agent, and at the same time introducing 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 immobilizing 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 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 solution adopted by the present invention to solve technical problems is: a kind of water/oil two-phase cross-linking reaction, the oil phase is cross-linking agent pyridazine diacrylate, its structure as shown in Figure 1, the water phase The reactant is glutamic acid decarboxylase and supramolecular complex of β-cyclodextrin and aminated epoxy resin, and the load of immobilized enzyme is regulated by the concentration of glutamic acid decarboxylase.

It is very beneficial that the degree of cross-linking can be controlled through heterogeneous reactions to avoid 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 glutamic acid decarboxylase to be immobilized at a utilization rate close to 100% , after the cross-linking reaction occurs, there is almost no residual glutamic acid 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 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 triethylenetetramine are mixed according to the mass ratio of 2:2:1.5, 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) Glutamic acid decarboxylase Dissolved in a phosphate buffer solution with pH = 6.5, the concentration of the enzyme was kept in the range of 1.0 to 7.0 mg/mL; 5.0mg/mL, 6.0mg/mL, 7.0mg/mL glutamic acid decarboxylase solution is mixed with the above-mentioned molecular complex aqueous solution according to the ratio of 55mL: 20mL, and the loading capacity of the immobilized enzyme is adjusted by changing the concentration of the enzyme solution; 5) Add 1.2g of pyridazine diacrylate 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 and let the reaction system stand for 4- After 5 hours, the products of immobilized glutamic acid decarboxylase with different loads 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 an emulsified product, and the oil phase will quickly disperse until it disappears after the reaction is initiated. Glutamic acid decarboxylase 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 the enzyme, which controls the cross-linking degree; 3) The co-crosslinking immobilization method can immobilize glutamic acid decarboxylase with extremely high efficiency.

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 triethylenetetramine are mixed according to the mass ratio of 2: 2: 1.5, 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 glutamic acid decarboxylase in a phosphate buffer solution with pH=6.5, and keeping the concentration of the enzyme in the range of 1.0-7.0 mg/mL;

4) Glutamic acid decarboxylase 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 was mixed according to the ratio of 55mL: 20mL, and the loading capacity of the immobilized enzyme was adjusted by changing the concentration of the enzyme solution;

5) Add 1.2g of pyridazine diacrylate to the above mixed aqueous solution under stirring, and keep the reaction temperature at 25-30°C for 10-15 minutes. After 10-15 minutes, white gel particles are formed, and the oil phase disappears at the same time. Stop stirring to allow the reaction The system is left for 4-5 hours, and the products of immobilized glutamic acid decarboxylase with different loads can be obtained after filtering.

Immobilized Enzyme Loading Determination:

Since glutamic acid decarboxylase was immobilized by the co-crosslinking method, the activity of glutamic acid decarboxylase could not be detected in the reaction residual liquid, indicating that glutamic acid decarboxylase all entered into the solid particles after crosslinking, so the calculation of 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: Take 400 μL of substrate solution (pH 4.8, 0.2mol/L citric acid-disodium hydrogen phosphate buffer, containing 0.01mmol/L PLP, 100mmol/L substrate L-MSG) and add it to Place in a 1.5mL centrifuge tube, preheat in a 37°C water bath, then add 20μL of pure glutamic acid 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 terminate the reaction, and after centrifugation, 0.5mL of the supernatant was taken, then an equivalent amount of DNS-C1 acetone (8g/L) solution was added, and it was derivatized at 30°C for 1h in the dark. After the sample was filtered through a 0.22 μm microporous membrane, the content of GABA generated by the reaction was measured by high performance liquid chromatography (HPLC method) to measure the enzyme activity.

(2) Determination of immobilized enzyme activity: use 0.05mol/L phosphate (pH 5.6) buffer as the reaction medium, add 20mg of immobilized glutamic acid decarboxylase, and then add 0.05mol/L sodium glutamate as the substrate , oscillating in a constant temperature water bath at the optimum temperature at a speed of 100r/min for 2h, inactivated at 90°C for 5min, centrifuged to take the supernatant, and after constant volume, use high performance liquid chromatography (HPLC) to measure γ-aminobutyric acid in the solution acid (GABA) content. 1 μmol GABA was generated every 30 min as a unit of enzyme activity.

Wherein, pre-column derivatization is carried out when the HPLC method measures the GABA content, and the specific method is:

(i) The method of pre-column derivatization of GABA:

1% 2,4-dinitrofluorobenzene in acetonitrile (FDNB) was used as the derivatizing agent. Take 0.2 mL of the sample supernatant after centrifugation at 10,000 r/min for 10 min in a 2 ml centrifuge tube, add 0.2 mL of 0.5 mol/L NaHCO ₃ (pH=9.0) solution, 0.2 mL of FDNB solution, and place in a 60°C water bath Heat in the dark for 1 hour, and cool with cold water quickly 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 membrane, centrifuge at 10,000 r/min for 10 min, and store in the dark at 4°C 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 acetonitrile aqueous solution with equal volume ratio; flow rate: 1mL/ min; injection volume: 10 μL.

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 samples of immobilized glutamic acid decarboxylase 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 75mg enzyme/g carrier, its relative activity reaches maximum value, and its specific activity is 94% of free enzyme, and this result shows that glutamic acid decarboxylase is in this range Very suitable for catalytic state. When the load is less than 75 mg enzyme/g carrier, the activity of the immobilized enzyme gradually increases with the increase of the load, which is mainly because the structure of the polymer becomes loose with the increase of the enzyme content, and the contact between the enzyme and the substrate As the opportunity increases, so does its relative activity. When the load was greater than 75mg enzyme/g carrier, the activity of the immobilized enzyme gradually decreased with the increase of the load. Generally speaking, 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 the cyclodextrin supramolecular structural unit. It loosens the structure of the immobilized enzyme and improves the internal hydrophilicity. In addition, the co-crosslinking can also improve the dispersion of the enzyme and avoid the aggregation of the enzyme, thereby improving its catalytic activity. But when the load is too large, the aggregation of the enzyme becomes inevitable, so its activity declines 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 free enzyme solution. The results are shown in Figure 3, and the activity of the initial state at time zero is 100%. , after 28 days of storage at 4°C and pH=6.0, only 57% of the activity of the free enzyme solution remains, and 84% 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 glutamic acid decarboxylase on its load.

Fig. 3 Comparison of storage stability of immobilized and free glutamic acid 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, and an oil phase is a crosslinking agent 22112oxazine diacrylate and has the following structure:

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 epoxy resin aminated substance and beta-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 phosphate buffer solution with pH =6.5, and keeping the concentration of the enzyme in 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 22112oxazine 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 immobilized products of the glutamic acid decarboxylase with different loading amounts.

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