CN111849960B - A kind of preparation method of crosslinking enzyme - Google Patents

Abstract

The invention discloses a preparation method of cross-linking enzyme, which comprises the steps of mixing polyphenol oxidase with glutamine transaminase solution for reaction to obtain cross-linking enzyme particles with the particle size of several micrometers to tens of micrometers and different activity sizes, separating the prepared cross-linking glutamine transaminase from the solution by means of filtration, centrifugation and the like, wherein the prepared cross-linking enzyme is used for polyethylene glycol (PEG) modification of protein drug molecules. The crosslinked glutamine transaminase is easy to recycle and can be recycled in the use process, the burden on product separation is avoided, and the production cost is greatly reduced. The method has the advantages of green, environment-friendly, mild and efficient crosslinking process, controllable reaction, no need of a carrier, no need of purifying glutamine transaminase, and capability of crosslinking and immobilization of a plurality of enzymes together.

Description

A kind of preparation method of crosslinking enzyme

technical field

The invention belongs to the technical field of biochemical industry, and relates to a preparation method of a cross-linking enzyme.

Background technique

Natural enzymes (such as transglutaminase) are generally dissolved in aqueous solution to catalyze the reaction. After the reaction, the enzyme must be inactivated by heating, adding acid and alkali, etc. The expensive enzyme cannot be recovered and can only be used once, which is a huge waste. Transglutaminase (E.C.2.3.2.13) catalyzes the acyl transfer reaction between glutamine transamination residues (acyl donors) on polypeptide chains and various primary amino (acyl acceptor) compounds, including lysine residues , to form isopeptide bonds to achieve protein cross-linking or modification. Transglutaminase catalyzes protein cross-linking, which can improve food texture and function and increase product value, so it has been commercially used in food processing. Another important application of transglutaminase is to modify proteins, such as polyethylene glycol (PEG) modification and glycosylation modification of protein drugs, which are used to prolong the half-life of protein drugs and reduce the immunogenicity of protein drugs. Improving the clinical efficacy of protein drugs. The modification of protein drugs by transglutaminase method is characterized by highly specific modification sites, mild reaction, and no damage to protein drugs. Therefore, it is currently the most concerned and important method for site-specific modification of protein drugs. After the free transglutaminase modifies the protein drug reaction, the inactivation treatment is not only a waste, but also causes the denaturation of the protein drug. Immobilized enzymes can solve the above problems. The so-called immobilized enzyme is to turn the enzyme that can dissolve in the solution into an insoluble enzyme, but retain its catalytic activity. When the reaction is over, it can be easily separated from the solution by filtration, centrifugation, etc., which is convenient for controlling the reaction process and realizing Enzyme reuse. Cross-linking is one of the commonly used methods for immobilizing enzymes, usually using chemical cross-linking agents (such as glutaraldehyde) to covalently link enzyme proteins. The advantage of immobilizing enzymes by cross-linking is that no carrier is required and the enzyme activity will not be affected by the carrier. However, the chemical cross-linking method produces chemical damage to the enzyme molecule, which will reduce the catalytic ability of the enzyme, and there are chemical residues. Both tyrosinase (E.C.1.14.18.1) and laccase (E.C.1.10.3.2) belong to the family of polyphenol oxidases. Tyrosinase catalyzes the oxidation reaction of the phenolic hydroxyl group of the tyrosine residue on the polypeptide chain to generate catechol, and then catalyzes the catechol to generate active catechol, which undergoes self-polymerization or Reacts with lysine, histidine, cysteine residues, etc. to produce protein crosslinks. Laccase catalyzes the oxidation of tyrosine residues on the polypeptide chain to generate phenolic free radicals, and the spontaneous polymerization of the generated free radicals leads to protein crosslinking. Both enzymes act on tyrosine residues, but there are differences in the requirements for the tyrosine residue site and the reaction mechanism. At present, researchers have begun preliminary attempts to use tyrosinase and laccase for food protein crosslinking to improve food quality and food value. In conclusion, transglutaminase and polyphenol oxidase are enzymes that can catalyze protein crosslinking. In the application of transglutaminase-modified protein drugs, in order to facilitate the control of the reaction process and realize the recycling of enzymes, a mild and efficient enzymatic immobilization method is needed to cross-link and immobilize transglutaminase, but the existing In the disclosed scheme, there is no report on the immobilization of enzymatic transglutaminase.

Contents of the invention

The purpose of the present invention is to provide a preparation method of cross-linking enzyme, which utilizes the catalysis of polyphenol oxidase to carry out the enzymatic cross-linking of transglutaminase, which does not require a carrier and does not need to purify transglutaminase. The enzymes were cross-linked and immobilized together.

Technical scheme of the present invention is:

A method for preparing a cross-linked enzyme, the method comprising:

(1) preparing the substrate into a first solution with a protein concentration of 5-10 mg/mL, and preparing polyphenol oxidase into a second solution with a concentration range of 1-10 mg/mL;

(2) Mix the first solution and the second solution, react at a temperature of 20-50° C. for 20 minutes to 24 hours, centrifuge and collect the precipitate to obtain cross-linked enzyme particles.

Further, in step (1), the substrate is an enzyme containing a tyrosine residue in a polypeptide chain that can be specifically catalyzed by polyphenol oxidase.

Further, in step (1), the substrate is transglutaminase.

Further, in step (1), the polyphenol oxidase is any one of laccase and tyrosinase.

Further, in step (1), the concentration range of the second solution is 1-10 mg/mL.

Further, in step (2), the volume ratio of the first solution to the second solution is 1:1.

Further, in step (2), the reaction temperature is 20-30°C, and the reaction time is 12h-24h.

Further, in step (2), when the first solution is mixed with the second solution, 1-2 mmol/l mediator ferulic acid mediator or dopa is added.

Further, in step (2), the particle size of the cross-linked enzyme particles is 1-70 microns.

The invention provides a preparation method of a cross-linking enzyme, which selects transglutaminase containing tyrosine residues as a substrate, uses polyphenol oxidases such as laccase and tyrosinase as catalysts, and catalyzes transglutaminase Enzymatic cross-linking. For the first time, a technical solution based on cross-linked transglutaminase catalyzed by polyphenol oxidase is provided, and the cross-linked transglutaminase is used to catalyze protein drug modification. The method can reduce the chemical damage to the cross-linking enzyme, has mild reaction conditions, high efficiency and wide application range.

Description of drawings

Fig. 1 is the influence of laccase concentration and reaction time on transglutaminase cross-linking in the preparation method of a kind of cross-linking enzyme of the present invention in embodiment 1;

Fig. 2 is the SDS-PAGE result analysis after 1mg/mL laccase catalyzes different time in the preparation method of a kind of cross-linking enzyme of the present invention in embodiment 1,

Among them, the order of adding samples: Lane1: Marker26610; Lane2: laccase (1mg/ml); Lane 3: transglutaminase (10mg/ml); Lane 4-10: reaction time of laccase and transglutaminase 20min, 1h , 2h, 4h, 6h, 12h, 24h;

Fig. 3 is the SDS-PAGE result analysis after 7.5mg/mL laccase catalyzes different time in the preparation method of a kind of cross-linking enzyme of the present invention in embodiment 1,

Wherein, electrophoresis sample loading sequence: Lane1: Marker26610; Lane2: laccase (7.5mg/ml); Lane3: transglutaminase (10mg/ml); Lane 4-10: laccase and transglutaminase reaction time 20min, 1h, 2h, 4h, 6h, 12h, 24h;

Fig. 4 is that the preparation method of a kind of cross-linking enzyme of the present invention is in the influence of ferulic acid of intermediary in embodiment 1 on cross-linking enzyme activity;

Fig. 5 is the crosslinking SDS-PAGE result analysis of adding ferulic acid in the 7.5mg/ml laccase system in embodiment 1 of a kind of crosslinking enzyme preparation method of the present invention,

Wherein, sample addition order: Lane1:Marker26610; Lane2:laccase (7.5mg/ml); Lane 3:transglutaminase (10mg/ml); Lane 4-10: in the mediator of final concentration 1mmol/L, Laccase and transglutaminase reaction time 20min, 1h, 2h, 4h, 6h, 12h, 24h;

Fig. 6 is the particle size result of 1.0 mg/mL laccase-catalyzed transglutaminase cross-linked particles in Example 1 of a method for preparing a cross-linked enzyme according to the present invention;

Fig. 7 is the particle size result of the 2.5 mg/mL laccase-catalyzed transglutaminase cross-linked particles in Example 1 of a method for preparing a cross-linked enzyme according to the present invention;

Fig. 8 is the particle size result of 5.0mg/mL laccase-catalyzed transglutaminase cross-linked particles in the preparation method of a cross-linked enzyme according to the present invention in Example 1;

Fig. 9 is the particle size result of 7.5 mg/mL laccase-catalyzed transglutaminase cross-linked particles in the preparation method of a cross-linked enzyme according to the present invention in Example 1;

Fig. 10 is the SDS-PAGE result analysis chart of the crosslinking of tyrosinase catalyzed transglutaminase in the preparation method of a kind of crosslinking enzyme of the present invention in embodiment 2;

Among them, Figure A is the SDS-PAGE analysis chart without adding mediator dopa; Figure B is the SDS-PAGE analysis chart of adding mediator dopa. The order of adding samples is shown in the figure;

Fig. 11 is an SDS-PAGE analysis of the cross-linked transglutaminase catalyzed by the preparation method of a cross-linked enzyme according to the present invention in Example 3 to modify protein drugs by PEG derivatives.

Among them, the order of loading: Lane1: Marker26610; Lane2: transglutaminase; Lane 3: protein drug; Lane 7: cross-linked transglutaminase catalyzed PEGylation of protein drug.

Detailed ways

The purpose of the present invention is to provide a preparation method of cross-linked enzyme, which uses polyphenol oxidase as a catalyst to cross-link the substrate (enzyme) to prepare cross-linked enzyme particles insoluble in aqueous solution. The size was determined by a diameter meter, and the activity was determined by chromogenic-visible light spectrophotometry. Specifically include the following steps:

(1) Select enzymes containing tyrosine residues in polypeptide chains such as transglutaminase as substrates to prepare the first solution; polyphenol oxidase is prepared into the second solution to obtain the catalyst;

In this step, the polypeptide chains of the substrates (enzymes) all contain tyrosine residues that can be specifically catalyzed by polyphenol oxidase, such as transglutaminase, and these enzymes only need to be obtained by rough separation without fine separation , the enzyme protein can contain ingredients such as dextrin added to protect the enzyme protein during the freeze-drying process, and is prepared into a solution of 5-10 mg (protein)/mL. The tyrosine residues that can be specifically catalyzed by polyphenol oxidase should generally meet the following conditions: the tyrosine residues are located in a flexible polypeptide chain and exposed to solvents, and the three-dimensional structure of the enzyme generally cannot be too tight (that is, it should not rich in disulfide bonds). Enzyme proteins that can be catalyzed by polyphenol oxidase can be determined through preliminary experiments. The polyphenol oxidase can be selected from laccase or tyrosinase, and prepared in a concentration range of 1-10 mg/mL, preferably in a concentration range of 5-10 mg/mL.

(2) Mix the above two solutions at a volume ratio of 1:1, react at 20-50°C, add or not add 1mmol/l ferulic acid mediator, react for 20min-24h, and obtain the particle size by centrifuging and collecting the precipitate Cross-linked enzyme particles of 1-70 microns, and the particle size can be controlled by changing the concentration of polyphenol oxidase (the activity of cross-linked glutamine transaminase is in the range of 8-100U);

In this step, the volume ratio of the two solutions can be adjusted, preferably 1:1, and the reaction temperature is preferably 20-30°C. In high-concentration enzyme solution (≥5mg/mL), the mediator can be omitted, and it is recommended to add the mediator. The type of mediator can be changed, ferulic acid is preferred as the mediator of laccase, and dopa is preferred as the mediator of tyrosinase. The reaction time is preferably 12h. The method for separating cross-linked enzyme particles can be centrifugation, microporous filtration, etc., and the centrifugation method is preferred, and the centrifugation condition is below 10°C, 10000rpm, 20min. The cross-linked transglutaminase microparticles are measured by a laser particle sizer, and the measurement conditions are as follows: a Baxter laser particle sizer is used; water is used as the background. The cross-linking enzyme activity was determined by chromogenic-visible light spectrophotometry with the inactivated enzyme as the reference substance.

(3) The prepared cross-linked glutamine transaminase enzyme is used for polyethylene glycol (PEG) modification of protein drugs and the like.

In this step, the use is selected according to the catalytic properties of the cross-linking enzyme, such as cross-linking transglutaminase catalyzing the modification of protein drugs.

In order to make the above objects, features and advantages of the present invention more obvious and comprehensible, the technical solution of the present invention will be further described below in conjunction with specific examples. But the present invention is not limited to the listed embodiments, but also includes any other known changes within the claimed scope of the present invention.

Reference herein to "one embodiment" or "an embodiment" refers to a particular feature, structure or characteristic that can be included in at least one implementation of the present invention. "In one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

Example 1

Catalytic action of laccase to cross-link transglutaminase.

(1) Prepare transglutaminase solutions with a protein concentration of 10 mg/mL, and prepare 1 mg/mL, 2.5 mg/mL, 5 mg/mL, 7.5 mg/mL and 10 mg/mL laccase solutions respectively. Among them, transglutaminase was donated by Jiangsu Yiming Biological Co., Ltd., containing dextrin and other components, and the protein content in the enzyme preparation was analyzed by the Coomassie brilliant blue method to be about 10%. Laccase was provided by Sigma. Both enzyme solutions were filtered.

(2) Mix 0.5mL transglutaminase solution and laccase solution respectively, react at 25°C, react for 20min, 40min, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h respectively Centrifuge at 10000rpm for 20min at 10°C. The precipitate was suspended in distilled water and then centrifuged, repeating 2 times. Finally, the immobilized product (cross-linked transglutaminase) was dispersed in 1 mL of distilled water to measure the enzyme activity. The cross-linked transglutaminase microparticles are measured by a laser particle size analyzer, and the activity of the cross-linked enzyme is measured by a chromogenic-visible light spectrophotometric method.

In step (1), if the concentration of laccase is lower than 5mg/mL, the activity of cross-linked transglutaminase increases with the increase of laccase concentration; when the concentration of laccase rises to 5mg/mL, 7.5mg/mL mL, the activity of cross-linked transglutaminase reached the maximum, and the activity of cross-linked transglutaminase produced when the concentration of laccase was 10 mg/mL decreased instead. Therefore, it is determined that the concentration of laccase is 5-7.5mg/mL. In step (2), if the laccase catalysis time of 20min, 40min, 1h, 2h, 4h, 6h, 8h, 12h is selected, it is found that as the reaction time prolongs, the activity of cross-linked glutamine transaminase continues to rise until 12h After that, the vitality will no longer increase and reach a stable value. Therefore, it is more sufficient to choose a reaction time of 12h-24h for cross-linking. Please refer to Fig. 1 to Fig. 3, Fig. 1 is the preparation method of a kind of cross-linking enzyme described in the present invention in embodiment 1 in the impact of laccase concentration and reaction time on transglutaminase cross-linking; Fig. 2 is the cross-linking effect of the present invention The preparation method of a kind of cross-linking enzyme described in Example 1 is analyzed by SDS-PAGE results after 1mg/mL laccase catalyzes different time; Fig. 3 is the preparation method of a kind of cross-linking enzyme described in the present invention in embodiment Analysis of SDS-PAGE results after catalysis of 7.5mg/mL laccase in 1 for different time. As shown in Figure 1-3, according to the SDS-PAGE results in Figure 2, it can be seen that when the concentration of laccase is 1 mg/mL, the electrophoretic band of transglutaminase slightly weakens with the prolongation of the cross-linking reaction time, but after 24 hours of reaction The solution transglutaminase electrophoresis band is still obvious, indicating that there is still more transglutaminase remaining in the solution, and this electrophoresis result is consistent with the effect on cross-linking when the laccase concentration is 1 mg/mL in Figure 1; the laccase concentration At 7.5 mg/mL, the electrophoresis band of transglutaminase in the reaction for 20 minutes was obviously weakened, and no new band was produced in the electrophoresis, indicating that under the catalysis of this concentration of laccase, the cross-linking reaction of transglutaminase in the solution was rapid. The concentration decreases, and the molecular weight of the produced product is too large to enter the electrophoresis gel; after 12 hours of reaction, the electrophoretic band of transglutaminase basically disappears, indicating that after 12 hours of reaction, there is basically no remaining transglutaminase in the solution, and cross-linking occurs completely , this electrophoresis result is consistent with the effect of the laccase concentration of 7.5 mg/mL on cross-linking in Figure 1.

On the basis of clarifying the concentration and time of laccase in the catalytic reaction, the mediator was further added to investigate the effect of the mediator on the crosslinking. See Figures 4 through 5. Fig. 4 is that the preparation method of a kind of cross-linking enzyme of the present invention is in the influence of intermediary ferulic acid on cross-linking enzyme activity in embodiment 1; Fig. 5 is the preparation method of a kind of cross-linking enzyme of the present invention in Analysis of cross-linked SDS-PAGE results of ferulic acid added to the 7.5 mg/ml laccase system in Example 1. As shown in Figure 4, adding a mediator to the reaction solution of low concentration laccase (1 mg/mL) can promote the cross-linking of transglutaminase, and the activity of cross-linked transglutaminase is improved, while at high concentration laccase (7.5 mg/mL) in the reaction solution, within 2 hours of reaction, the presence of the mediator increases the activity of cross-linked transglutaminase, that is, promotes cross-linking, but as time prolongs, the presence of the mediator makes the cross-linked transglutaminase The activity is reduced, even lower than the result of no mediator reaction. As shown in Figure 5, when there is no mediator in the high-concentration laccase (7.5 mg/mL) reaction system, it takes 12 hours for the complete cross-linking of transglutaminase; The cross-linking reaction efficiency of amide transaminase is obviously improved, and the electrophoresis band of glutamine transaminase completely disappears when reacting for 20 minutes, that is, complete cross-linking has occurred. This is the reason why the activity of cross-linked glutamine transaminase in a short time is improved by adding mediator in the high concentration laccase (7.5mg/mL) reaction solution.

In step (2), the effect of various reaction conditions on particle size is measured by a laser particle size analyzer. See Figures 6 through 9. Fig. 6 is the particle size result of the 1.0 mg/mL laccase-catalyzed transglutaminase cross-linked microparticles in the preparation method of a cross-linking enzyme according to the present invention; Fig. 7 is a cross-linking particle size result of the present invention The preparation method of a kind of cross-linking enzyme in embodiment 1 in the 2.5mg/mL laccase catalyzed transglutaminase cross-linked particle size result; Fig. 8 is the preparation method of a kind of cross-linking enzyme of the present invention in implementation 5.0mg/mL laccase-catalyzed transglutaminase cross-linked particle size results in example 1; Figure 9 is the 7.5mg/mL lacquer in embodiment 1 of a preparation method of a cross-linked enzyme according to the present invention Enzyme-catalyzed transglutaminase cross-linked particle size results. As shown in Figures 6 to 9, the greater the concentration of laccase, the smaller the particle size, which shows that the concentration of laccase is a key factor in determining the particle size. Therefore, the particle size of cross-linked enzyme particles can be controlled by changing the concentration of laccase.

Example 2

Catalytic action of tyrosinase to cross-link transglutaminase

The present invention studies the feasibility of other polyphenol oxidase (tyrosinase) catalyzing cross-linked glutamine transaminase. Including the following steps:

(1) Prepare a transglutaminase solution with a protein concentration of 10 mg/mL, and prepare a 3 mg/mL tyrosinase solution. Among them, transglutaminase was donated by Jiangsu Yiming Biological Co., Ltd., containing dextrin and other components, and the protein content in the enzyme preparation was analyzed by the Coomassie brilliant blue method to be about 10%. Tyrosinase was derived from mushrooms and provided by Mecox Lane. Both enzyme solutions were filtered.

(2) Mix 0.2 mL of transglutaminase solution and tyrosinase solution respectively, react at 25°C, react for 1h, 2h, 4h, 8h, 18h, and 24h respectively, then take samples for SDS-PAGE analysis. Further, 2 mM mediator dopa was added to study the effect of mediator on tyrosinase cross-linked transglutaminase. Please refer to FIG. 10 . FIG. 10 is an analysis diagram of SDS-PAGE results of cross-linking of transglutaminase catalyzed by tyrosinase in the preparation method of a cross-linking enzyme according to the present invention in Example 2. As shown in Figure 10, in the presence of tyrosinase, the electrophoretic band of transglutaminase disappeared, which indicated that tyrosinase could also catalyze the cross-linking of transglutaminase.

Example 3

Uses of cross-linked transglutaminase

The cross-linked transglutaminase catalyzes the modification of the protein drug by the PEG derivative. The specific method is: the protein drug solution and the PEG derivative solution (0.05M, pH 8.0 Tris-HCl buffer) are evenly mixed, and the transglutaminase is added to start the reaction. After reacting for 2 hours in a water bath at 37°C, centrifuge to take the supernatant sample and add 2×SDS loading buffer to a boiling water bath for treatment, then perform SDS-PAGE. The gel imaging system takes pictures and analyzes the gel. See Figure 11 for the results. Fig. 11 is an SDS-PAGE analysis of the cross-linked transglutaminase catalyzed by the preparation method of a cross-linked enzyme according to the present invention in Example 3 to modify protein drugs by PEG derivatives. As shown in Figure 11, the cross-linked transglutaminase successfully catalyzed the modification of protein drugs by PEG derivatives, and there was no electrophoresis band of transglutaminase, indicating that the cross-linked transglutaminase and the product were successfully separated by centrifugation.

Polyphenol oxidase (including laccase, tyrosinase) and transglutaminase can both catalyze protein crosslinking, and both have a wide range of protein substrates. Laccase, tyrosinase, and transglutaminase have different amino acid sites and reaction mechanisms (see Table 1 for comparison), so there are differences in the range of protein substrates they act on. Both polyphenol oxidase and transglutaminase are potential substrates for each other, but the result of the mixed reaction between the two is that polyphenol oxidase successfully cross-links transglutaminase, while transglutaminase cannot catalyze the cross-linking of polyphenols oxidase. Therefore, this technology realizes the cross-linking of transglutaminase based on the catalysis of polyphenol oxidase, and solves the problem of restricting transglutaminase in protein drug modification.

The comparison of table 1 laccase, tyrosinase, transglutaminase

Table 1

In summary, the preparation method of a cross-linking enzyme according to the present invention uses polyphenol oxidase as a catalyst to catalyze the cross-linking of transglutaminase, and the cross-linking enzyme is easy to recycle and can be used repeatedly during use. , Will not cause a burden on product separation, greatly reducing production costs. This technology is environmentally friendly, mild and efficient, and the reaction is controllable. It does not require a carrier or purification of transglutaminase, and it can also be cross-linked and immobilized with multiple enzymes.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims (1)

1. The preparation method of the cross-linking enzyme is characterized by comprising the following specific steps:

(1) Preparing glutamine transaminase into a first solution with a protein concentration of 10mg/mL, and preparing laccase into a second solution with a concentration of 5-7.5 mg/mL;

(2) Mixing the first solution with the second solution, adding 1-2mmol/l mediator ferulic acid when the first solution is mixed with the second solution, reacting at 20-30 ℃ for 12-h-24 h, centrifuging, and collecting precipitate to obtain the cross-linked enzyme particles with the particle size of 1-70 microns.

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