CN110760503B - Co-crosslinking immobilization method of phospholipase D - Google Patents
Co-crosslinking immobilization method of phospholipase D Download PDFInfo
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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
Technical Field
The invention relates to the technical field of immobilized enzyme biocatalysis, in particular to a co-crosslinking immobilization method of phospholipase D, and the novel immobilized phospholipase D can be specially used for efficiently preparing phosphatidylserine.
Background
Phospholipase D (EC 3.1.4.4), a phosphatidylcholine phospholipid hydrolase (isoelectric point 5.4), is a generic term for enzymes that catalyze phosphodiester bond hydrolysis and base exchange reactions. 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 a plurality of phospholipids and phospholipid derivatives. Phospholipase D functions not only to affect the structure, function and stability of the membrane by hydrolyzing phospholipids in the cell membrane, but also plays an important role in signal transduction, exertion of hormonal action, cytoskeletal assembly, ordering of intracellular protein kinases and actin, occurrence of cell division, transmembrane transport, secretion, defense reaction, and seed germination and cell senescence.
Phospholipase D has two characteristics of phospholipid transferability and high specificity, so that some rare phospholipids can be obtained by converting crude phospholipids with wide sources, and not only new varieties are developed, but also the nutritional value of phospholipids is improved. Such as: phospholipase D catalyzes soybean phospholipid to synthesize phosphatidylserine in the presence of serine, and phosphatidylglycerol, phosphatidylinositol, etc. can also be prepared; in addition, high-purity lecithin can be prepared from low-purity lecithin. Phospholipase D is also known in agriculture, and its decrease in plant growth is indicative of fruit decay, which affects yield and harvest, and it is seen that if the content of phospholipase D is increased, not only the life of fruit can be prolonged, but also the fruit can be harvested economically. Under the action of phospholipase D, phospholipid is modified to synthesize other rare phospholipids, so that the nutritional value, various performances and other aspects of the phospholipids are greatly improved and enhanced, and the phospholipids have wider application range and higher use value in the technical industries of foods, medicines, cosmetics, textiles and leather.
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 phospholipase D can be reused, so that the use efficiency of the enzyme is improved, and the use cost is reduced; the immobilized phospholipase D 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 phospholipase D 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 immobilizing phospholipase D, which utilizes amino on the molecule of the phospholipase D to perform 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 enzyme. By using the co-crosslinking method, the loading capacity and the 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 phospholipase D, which is based on the co-crosslinking reaction of the phospholipase D and another molecular compound containing organic amine, wherein the crosslinking reaction is based on the Michael addition of acrylate and amino, and the reaction can rapidly 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, so that the immobilized enzyme can keep 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, the reactant in water phase is phospholipase D and supermolecular complex of beta-cyclodextrin and aminated epoxy resin, the load of immobilized enzyme is regulated by the concentration of phospholipase D.
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 phospholipase D to be immobilized with nearly 100% utilization, after the cross-linking reaction has occurred, there is little residual phospholipase D in the liquid phase;
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) and methanol with ethylenediamine according to the mass ratio of 2: 0.8, 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 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 phospholipase D 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) Mixing phospholipase D 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 with the molecular complex aqueous solution according to a ratio of 55mL 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 4-5 hours, and filtering to obtain products of immobilized phospholipase D with different loading amounts.
Very beneficially, a double bond in the cross-linking agent reacts with amino on the molecular compound first, form the product with emulsification, the oil phase will disperse very fast until disappearing after the reaction starts, phospholipase D enters the polymer through the absorption way at first, then the double bond on the cross-linking agent reacts with amino on the enzyme slowly, become the immobilized enzyme product of co-crosslinking finally;
the method has the advantages that the interaction of the beta-cyclodextrin and the hydrophobic benzene ring is utilized to introduce hydrophilic groups, so that the use of chemical bonds is avoided, and the beta-cyclodextrin can not be separated from the polymer through a crosslinking reaction, so that 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 enzyme crosslinking is realized by using water/oil two-phase reaction, so that the crosslinking degree of the enzyme is reduced; 2) The beta-cyclodextrin molecular compound is introduced to improve the microenvironment of the immobilized phospholipase D and improve the catalytic reaction activity of the enzyme; 3) The co-crosslinking immobilization method can immobilize the phospholipase D with extremely high efficiency; 4) The immobilized product can form a branched structure by adopting a polyfunctional cross-linking agent, so that the aggregation of 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) and methanol with ethylenediamine according to the mass ratio of 2: 0.8, 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 phospholipase D 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) Mixing phospholipase D 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 with the molecular complex aqueous solution according to a ratio of 55mL 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 the oil phase, stopping stirring to allow the reaction system to stand for 4-5 hours, and filtering to obtain the immobilized phospholipase D products with different loading amounts.
And (3) measuring the load of the immobilized enzyme:
after the phospholipase D is fixed by the co-crosslinking method, the activity of the phospholipase D cannot be detected in the reaction residual liquid, which shows that the phospholipase D completely enters 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: adding appropriate amount of enzyme solution, mixing and dissolving with 2.5mL Tris-HCl (pH 8) buffer solution and 2.5mL Tris-HCl (pH 8) solution with 10% of Triton by mass, placing in a 37 deg.C constant temperature water bath for preheating for 5 minutes, then sucking 100 μ L phosphatidyl p-nitrophenol, and measuring the reading of spectrophotometer with wavelength of 405nm along with the change of time. The value of absorbance per minute was calculated. The enzyme activity calculation formula is as follows:
wherein: delta A is the absorbance change value of the enzymatic reaction; v is the total volume (mL) of the sample; v S Volume of enzyme solution added (mL); k is the slope of the p-nitrophenol standard curve; t is reaction time (min); 277 is the conversion factor.
(2) And (3) determining the activity of the immobilized enzyme: accurately weighing 0.1g of immobilized phospholipase D, suspending the immobilized phospholipase D in 5mL of 0.05mol/L Tris-HCl buffer solution (pH8.0) containing Triton 5% and pre-keeping in a constant temperature bath at 37 ℃ for 5min, adding 0.1mL of phosphatidyl p-nitrophenol, accurately incubating for 5min, centrifuging, taking a supernatant, measuring the absorbance at 405nm and calculating.
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 phospholipase D 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 showing the relative activity as a function of loading, which is maximal at a loading of 93mg enzyme/g support, and a specific activity of 92% of that of the free enzyme, which indicates that phospholipase D is in a state very suitable for catalysis in this range. When the loading capacity is less than 93mg of enzyme/g of carrier, the activity of the immobilized enzyme is gradually increased along with the increase of the loading capacity, which is mainly because when the content of the enzyme is lower, the structure of the polymer is tighter, the catalytic activity of the enzyme is not easy to exert, and as the content of the enzyme is increased, the structure of the polymer is looser, the contact chance of the enzyme and a substrate is increased, and the relative activity of the enzyme and the substrate is also improved. When the supported amount is more than 93mg of the enzyme per g of the carrier, the activity of the immobilized enzyme gradually becomes smaller as the supported 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 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 supported amount of 93mg of enzyme/g of carrier, and as a result, the free enzyme solution retained only 37% of the activity and the immobilized enzyme retained 78% of the activity after 28 days of storage at 4 ℃ and pH =7.0, taking the initial state activity of time zero as 100%.
Drawings
FIG. 1 chemical structure of the crosslinker.
FIG. 2 dependence of the catalytic activity of immobilized phospholipase D on its loading.
FIG. 3 storage stability of immobilized versus free phospholipase D.
Claims (1)
1. A phospholipase D co-crosslinking immobilization method is characterized in that a water/oil two-phase reaction system is used, the oil phase is isocyanuric acid triacrylate which is used as a crosslinking agent, and the structure is as follows:
the reactant in the water phase is phospholipase D and a molecular compound with the following structure:
the phospholipase D co-crosslinking immobilization method comprises the following steps:
1) Mixing bisphenol A epoxy resin with the number average molecular weight of 392, methanol and ethylenediamine according to the mass ratio of 2: 0.8, 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 phospholipase D in sodium phosphate buffer solution with pH =7.5, keeping the concentration of enzyme in the range of 1.0-7.0 mg/mL, and mixing phospholipase D solutions with different concentrations with the molecular complex aqueous solution according to the ratio of 55 mL: 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 4-5 hours, and filtering to obtain phospholipase D immobilized products with different loading amounts.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053001A1 (en) * | 1998-04-08 | 1999-10-21 | Novo Nordisk A/S | An enzymatic oil-degumming process |
| WO2007036235A1 (en) * | 2005-09-30 | 2007-04-05 | Novozymes A/S | Immobilization of enzymes |
| CN103966277A (en) * | 2014-05-22 | 2014-08-06 | 南京工业大学 | Method for preparing phosphatidylserine under catalysis of immobilized phospholipase D |
| CN104155291A (en) * | 2014-05-19 | 2014-11-19 | 上海大学 | Determination method for activity of phospholipase D alpha |
| CN107099562A (en) * | 2017-06-27 | 2017-08-29 | 南京工业大学 | A kind of method that utilization immobilized phospholipase D produces phosphatidylserine |
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- 2019-05-07 CN CN201910417713.0A patent/CN110760503B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053001A1 (en) * | 1998-04-08 | 1999-10-21 | Novo Nordisk A/S | An enzymatic oil-degumming process |
| WO2007036235A1 (en) * | 2005-09-30 | 2007-04-05 | Novozymes A/S | Immobilization of enzymes |
| CN104155291A (en) * | 2014-05-19 | 2014-11-19 | 上海大学 | Determination method for activity of phospholipase D alpha |
| CN103966277A (en) * | 2014-05-22 | 2014-08-06 | 南京工业大学 | Method for preparing phosphatidylserine under catalysis of immobilized phospholipase D |
| CN107099562A (en) * | 2017-06-27 | 2017-08-29 | 南京工业大学 | A kind of method that utilization immobilized phospholipase D produces phosphatidylserine |
Non-Patent Citations (1)
| Title |
|---|
| 刘倩倩等.大孔树脂固定化磷脂酶D.2016,第46卷(第46期),全文. * |
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