CN110760503B - Co-crosslinking immobilization method of phospholipase D - Google Patents

Abstract

The invention relates to a method for co-crosslinking and immobilizing phospholipase D. Oil-soluble isocyanuric acid triacrylate is used as a cross-linking agent, reactants in an aqueous phase are amino-containing phospholipase D and a supramolecular complex formed by aminated epoxy resin and beta-cyclodextrin, and the immobilized phospholipase D with different loading amounts is prepared by utilizing Michael addition reaction of double bonds and amino groups to perform 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 93mg of enzyme/g of carrier, which reaches 92% of free enzyme.

Description

A co-crosslinking immobilization method of phospholipase D

technical field

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

Background technique

Phospholipase D (EC3.1.4.4) is phosphatidylcholine phospholipid hydrolase (isoelectric point 5.4), which is a general term for a class of enzymes that catalyze the hydrolysis of phosphodiester ester bonds and base exchange reactions. It was first isolated from carrots by Hananhan in 1947. Phospholipase D is distributed in many organisms from bacteria to higher animals and plants, has broad substrate specificity, and can widely act on many phospholipids and phospholipid derivatives. The role of phospholipase D is not only to affect the structure, function and stability of the membrane by hydrolyzing phospholipids in the cell membrane, but also in signal transduction, hormone action, cytoskeleton assembly, intracellular protein kinase and actin order Cell differentiation, cell division, transmembrane transport, secretion, defense response, seed germination and cell senescence all play an important role.

Phospholipase D has two characteristics of phospholipid transfer and high specificity, so some rare phospholipids can be obtained by converting crude phospholipids from a wide range of sources, which not only develops new varieties, but also improves the nutritional value of phospholipids. For example: Phospholipase D catalyzes soybean phospholipids to synthesize phosphatidylserine in the presence of serine, and can also prepare phosphatidylglycerol, phosphatidylinositol, etc.; in addition, lecithin with low purity can also be used to prepare high-purity lecithin. Phospholipase D is also very useful in agriculture. It exists in plants. Its reduction marks the decline of fruit, thus affecting the yield and harvest. It can be seen that if the content of phospholipase D is increased, not only the life of the fruit can be prolonged, but also in the fruit. There are also great benefits in terms of economic benefits. Under the action of phospholipase D, phospholipids can be modified to synthesize other rare phospholipids, thereby greatly improving and improving their nutritional value and various performances. This makes phospholipids widely used in food, medicine, cosmetics, textiles, etc. , The range of application in the leather technology industry is wider and the use value is higher.

Immobilized enzyme is to change water-soluble free enzyme into insoluble solid enzyme by chemical means. Immobilization has many advantages: for example, immobilized phospholipase D can be reused, which improves the efficiency of enzyme use and reduces the cost of use; immobilization The phospholipase D is easily separated from the reaction system, which simplifies the operation process; the storage stability and thermal stability of the immobilized phospholipase D have been improved; the catalytic reaction process of the immobilized enzyme is easier to control; the immobilized enzyme has a certain It 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 phospholipase D, using the amino group on the phospholipase D molecule to undergo Michael addition reaction with an acrylate crosslinking agent, and at the same time introducing β-cyclodextrin Structural units, which 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 phospholipase D, which is based on the co-crosslinking reaction of phospholipase D and another molecular complex containing organic amines, the basis of the crosslinking reaction is The Michael addition of acrylate and amino groups 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 and good stability, and can also regulate the activity of the immobilized enzyme. Microenvironment to maintain high catalytic activity.

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 reactants in the method are supramolecular complexes of phospholipase D and β-cyclodextrin and aminated epoxy resin, and the load of immobilized enzyme is regulated by the concentration of phospholipase D.

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 phospholipase D to be immobilized with a utilization rate close to 100%, cross-linking After the cascade reaction occurs, there is almost no residual phospholipase D 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 ethylenediamine are mixed according to the mass ratio of 2:2:0.8, 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 dried in a vacuum oven at room temperature to obtain an epoxy resin aminate; The mol ratio is added into water, heated and stirred until the epoxy resin aminate is completely converted into a molecular complex and dissolved in water, keeping the total mass concentration of the aqueous solution in the range of 5 to 6wt.%; 3) dissolving phospholipase D at pH =7.5 in the sodium phosphate buffer solution, the concentration of the enzyme is kept in the range of 1.0～7.0mg/mL; The phospholipase D solution of /mL, 6.0mg/mL, 7.0mg/mL is mixed with the above-mentioned molecular complex aqueous solution according to the ratio of 55mL: 20mL, and the load capacity of the immobilized enzyme is adjusted by changing the concentration of the enzyme solution; 5) stirring Add 1.2g of isocyanuric acid triacrylate to the above mixed aqueous solution. The reaction temperature is kept in the range of 25-30°C. After 10-15 minutes, white gel particles are formed. Stop stirring and let the reaction system stand for 4-5 hours. , Immobilized phospholipase D products 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. way into the polymer, and then the double bond on the cross-linking agent slowly reacts with the amino group on the enzyme, and finally becomes a co-cross-linked 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 reduces the degree of cross-linking of the enzyme; 2) the introduction of the β-cyclodextrin molecular complex improves the microenvironment of the immobilized phospholipase D , which improves the catalytic activity of the enzyme; 3) the co-crosslinking immobilization method can immobilize phospholipase D with extremely high efficiency; 4) the use of a multifunctional crosslinking agent can make the immobilized product form a branched structure, Prevent the aggregation of enzymes and improve 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 ethylenediamine are mixed according to the mass ratio of 2: 2: 0.8, in Stir and react at 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, and then put it in a vacuum oven to dry 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 phospholipase D in a sodium phosphate buffer solution with pH=7.5, keeping the enzyme concentration in the range of 1.0-7.0 mg/mL;

4) Phospholipase D 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 and the above molecular complex The aqueous solution is mixed 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 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 4-5 hours, and the products of immobilized phospholipase D with different loads can be obtained after filtering.

Immobilized Enzyme Loading Determination:

Since phospholipase D was immobilized by the co-crosslinking method, the activity of phospholipase D could not be detected in the reaction residual liquid, indicating that phospholipase D had all entered the solid particles after crosslinking, so the calculation of the loading capacity used 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: Add an appropriate amount of enzyme solution, mix and dissolve the Tris-HCl (pH8) solution of 10% Triton with 2.5mL of Tris-HCl (pH8) buffer and 2.5mL of mass content, put Preheat the constant temperature water bath at 37°C for 5 minutes, then absorb 100 μL of phosphatidyl p-nitrophenol, and measure the reading of the spectrophotometer at a wavelength of 405 nm as time changes. Calculate the absorbance per minute value. The enzyme activity calculation formula is:

Wherein: ΔA is the absorbance change value of enzymatic reaction; V is the measurement sample total volume (mL); _VS is the added enzyme solution volume (mL); K is the slope of the p-nitrophenol standard curve; t is the reaction time ( min); 277 is the conversion coefficient.

(2) Determination of immobilized enzyme activity: Accurately weigh 0.1 g of immobilized phospholipase D, suspend it in 5 mL of 0.05 mol/L Tris-HCl buffer solution containing Triton 5% at pH 8.0 in a constant temperature bath at 37°C for 5 min, Add 0.1mL of phosphatidyl p-nitrophenol, incubate accurately for 5min, centrifuge, take the supernatant, measure the absorbance at 405nm and calculate.

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 phospholipase D with different loads were obtained in the experiment, their activities were measured respectively, and their relative activities were calculated. Figure 2 is the relationship between relative activity and load. When the load is 93mg enzyme/g carrier, its relative activity reaches the maximum value, and its specific activity is 92% of free enzyme. This result shows that phospholipase D is very suitable in this range. catalytic state. When the load is less than 93 mg enzyme/g carrier, the activity of the immobilized enzyme gradually increases with the increase of the load, 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 93mg 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 93mg 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=7.0, only 37% 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 phospholipase D on its load.

Figure 3 Comparison of storage stability of immobilized and free phospholipase D.

Claims (1)

1. a phospholipase D co-crosslinking immobilization method is characterized in that using water/oil two-phase reaction system, oil phase is as the isocyanurate triacrylate of crosslinking agent, and its structure is as follows:

The reactants in the aqueous phase are phospholipase D and a molecular complex with the following structure:

The co-crosslinking and immobilization method of phospholipase D is carried out in the following steps: 1) Mix the three components of bisphenol A epoxy resin, methanol and ethylenediamine with a number average molecular weight of 392 according to the mass ratio of 2:2:0.8, and stir and react within the range of 25-35°C for 4-5 hours, The mixture is poured into water, and the precipitate is repeatedly washed with water to remove methanol and a small amount of amine, and then placed in a vacuum oven to dry 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) Dissolve phospholipase D in a sodium phosphate buffer solution with pH = 7.5, keep the enzyme concentration in the range of 1.0-7.0 mg/mL, mix different concentrations of phospholipase D solutions with the above-mentioned molecular complex aqueous solution according to the ratio of 55mL: 20mL Proportional mixing; 4) Add 1.2 g 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 and let the reaction system stand After 4-5 hours, the immobilized products of phospholipase D with different loads were obtained after filtration.

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