CN111849960B

CN111849960B - Preparation method of cross-linking enzyme

Info

Publication number: CN111849960B
Application number: CN202010696371.3A
Authority: CN
Inventors: 周建芹; 刘江云; 胡明刚
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2023-05-23
Anticipated expiration: 2040-07-20
Also published as: CN111849960A

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

Preparation method of cross-linking enzyme

Technical Field

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

Background

The natural enzyme (such as glutamine transaminase) is generally dissolved in aqueous solution to catalyze the reaction, and the enzyme must be inactivated by heating, acid and alkali adding and other methods after the reaction is finished, so that the expensive enzyme cannot be recovered and can be used once, and the method is extremely wasteful. Glutamine transaminase (e.c. 2.3.2.13) catalyzes the acyl transfer reaction between glutamine transamination residues (acyl donors) and various primary amino (acyl acceptor) compounds, including lysine residues, on polypeptide chains to form isopeptidic linkages, thereby effecting protein cross-linking or modification. Glutamine transaminase catalyzes the crosslinking of proteins, which improves the texture and function of food products, and increases the value of the products, and has therefore been commercialized for use in food processing. Another important application of glutamine transaminase is modification of proteins, such as polyethylene glycol (PEG) modification, glycosylation modification, etc., of protein drugs, which has the effects of prolonging half-life of protein drugs, reducing immunogenicity of protein drugs, and thus improving clinical efficacy of protein drugs. The glutamine transaminase method for modifying the protein medicine has the characteristics of high specificity of modification sites, mild reaction and no damage to the protein medicine, so that the method is the most interesting and important method for site-directed modification of the protein medicine at present. After the reaction of the free glutamine transaminase modified protein medicine is finished, the inactivation treatment is wasteful, and the denaturation of the protein medicine can be caused. The immobilized enzyme can solve the above problems. The immobilized enzyme is an enzyme which can be dissolved in a solution and is changed into an insoluble enzyme, but the catalytic activity of the enzyme is maintained, and when the reaction is finished, the enzyme is easily separated from the solution by means of filtration, centrifugation and the like, so that the reaction process is conveniently controlled, and the reutilization of the enzyme is realized. Crosslinking is one of the commonly used methods of immobilizing enzymes, typically by covalent attachment of enzyme proteins using chemical crosslinking agents (e.g., glutaraldehyde). The immobilized enzyme of the crosslinking method has the advantages that a carrier is not needed, and the enzyme activity cannot be influenced by the carrier. However, chemical crosslinking causes chemical damage to enzyme molecules, which reduces the catalytic ability of the enzyme and causes chemical residues. Tyrosinase (e.c. 1.14.18.1) and laccase (e.c. 1.10.3.2) both belong to the polyphenol oxidase family. Tyrosinase catalyzes the phenolic hydroxyl of tyrosine residue on polypeptide chain to produce catechol, and then catalyzes catechol to produce active o-benzoquinone, and the o-benzoquinone is self-polymerized or reacts with lysine, histidine, cysteine residue, etc. to produce protein cross-linking. Laccase catalyzes the oxidation of tyrosine residues on polypeptide chains to phenolic free radicals, which spontaneously polymerize to cause protein cross-linking. Both enzymes act on tyrosine residues, but the requirements for the tyrosine residue sites and the reaction mechanisms differ. At present, researchers begin to make preliminary attempts to use tyrosinase and laccase for food protein crosslinking, improve food quality and increase food value. In summary, glutamine transaminase and polyphenol oxidase are enzymes that catalyze protein cross-linking. In the application of the glutamine transaminase modified protein medicine, a mild and efficient enzymatic immobilization method is needed to crosslink and immobilize the glutamine transaminase in order to conveniently control the reaction process and realize the recycling of enzymes, but in the prior published scheme, no report on the enzymatic immobilization of the glutamine transaminase is found.

Disclosure of Invention

The invention aims to provide a preparation method of a cross-linking enzyme, which utilizes the catalysis of polyphenol oxidase to carry out enzymatic cross-linking of glutamine transaminase, does not need a carrier or purification of glutamine transaminase, and can carry out cross-linking immobilization of a plurality of enzymes together.

The technical scheme of the invention is as follows:

a method of preparing a cross-linking enzyme, the method comprising:

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

(2) Mixing the first solution and the second solution, reacting for 20min-24h at 20-50 ℃, centrifuging, and collecting precipitate to obtain the cross-linked enzyme particles.

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

Further, in step (1), the substrate is glutamine transaminase.

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

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

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

further, in the step (2), the reaction temperature is 20-30 ℃ and the reaction time is 12-24 hours.

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

Further, in the step (2), the cross-linked enzyme particles have a particle size of 1 to 70. Mu.m.

The invention provides a preparation method of cross-linking enzyme, which selects glutamine transaminase containing tyrosine residue as a substrate, and uses polyphenol oxidase such as laccase and tyrosinase as a catalyst to catalyze the cross-linking of enzymes such as glutamine transaminase. The technical scheme of cross-linking glutamine transaminase based on polyphenol oxidase catalysis is provided for the first time, and the cross-linking glutamine transaminase is used for catalyzing protein drug modification. The method can reduce chemical damage to the cross-linking enzyme, and has mild reaction condition, high efficiency and wide application range.

Drawings

FIG. 1 shows the effect of laccase concentration and reaction time on glutamine transaminase crosslinking in example 1 in a method of preparing a cross-linking enzyme according to the invention;

FIG. 2 shows SDS-PAGE results analysis of 1mg/mL laccase in example 1 after various times in a preparation method of cross-linking enzyme according to the invention,

wherein, the sample adding sequence is as follows: lane1 Marker26610; lane2 laccase (1 mg/ml); lane3 glutamine transaminase (10 mg/ml); lane 4-10, wherein the reaction time of laccase and glutamine transaminase is 20min, 1h, 2h, 4h, 6h, 12h and 24h;

FIG. 3 shows SDS-PAGE analysis of 7.5mg/mL laccase in example 1 after various times,

wherein, electrophoresis loading sequence: lane1 Marker26610; lane2 laccase (7.5 mg/ml); lane3 glutamine transaminase (10 mg/ml); lane 4-10, wherein the reaction time of laccase and glutamine transaminase is 20min, 1h, 2h, 4h, 6h, 12h and 24h;

FIG. 4 shows the effect of mediator ferulic acid on the activity of the cross-linking enzyme in example 1 according to a method of preparing the cross-linking enzyme of the present invention;

FIG. 5 shows the analysis of the result of SDS-PAGE of ferulic acid in the laccase system of 7.5mg/ml in example 1,

wherein, the sample adding sequence is as follows: lane1 Marker26610; lane2 laccase (7.5 mg/ml); lane3 glutamine transaminase (10 mg/ml); lane 4-10, in the mediator with the final concentration of 1mmol/L, laccase and glutamine transaminase react for 20min, 1h, 2h, 4h, 6h, 12h and 24h;

FIG. 6 shows the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 1.0mg/mL laccase;

FIG. 7 is a graph showing the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 2.5mg/mL laccase;

FIG. 8 is a graph showing the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 5.0mg/mL laccase;

FIG. 9 is a graph showing the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 7.5mg/mL laccase;

FIG. 10 is a diagram showing SDS-PAGE analysis of tyrosinase-catalyzed glutamine transaminase crosslinking in example 2 according to a method of preparing a crosslinking enzyme of the present invention;

wherein, the A diagram is SDS-PAGE analysis diagram of the mediator-free dopa; panel B is an SDS-PAGE analysis of the mediator dopa. The sample adding sequence is shown in the figure;

FIG. 11 shows SDS-PAGE analysis of a cross-linked glutamine transaminase-catalyzed PEG derivative modified protein drug in example 3 according to a method of preparing a cross-linked enzyme of the present invention.

Wherein, the sample adding sequence is as follows: lane1 Marker26610; lane2, glutamine transaminase; lane3, protein medicine; lane 7, the PEG modification result of the protein drug catalyzed by the crosslinked glutamine transaminase.

Detailed Description

The invention aims to provide a preparation method of cross-linking enzyme, which uses polyphenol oxidase as a catalyst to cross-link a substrate (enzyme) to prepare cross-linking enzyme particles insoluble in aqueous solution, wherein the cross-linking enzyme is measured by a laser particle diameter meter, and the activity is measured by a color development-visible light spectrophotometry. The method specifically comprises the following steps:

(1) Selecting enzymes containing tyrosine residues in polypeptide chains such as glutamine transaminase and the like as substrates to prepare a first solution; preparing polyphenol oxidase into a second solution to obtain a catalyst;

in this step, the polypeptide chains of the substrate (enzyme) each contain tyrosine residues which can be specifically catalyzed by polyphenol oxidase, such as glutamine transaminase, and these enzymes are obtained only by coarse separation, and fine separation is not required, and the enzyme protein can contain ingredients such as dextrin added for protecting the enzyme protein during freeze drying, so as to prepare a solution of 5-10mg (protein)/mL. Tyrosine residues capable of being specifically catalyzed by polyphenol oxidase should generally satisfy the following conditions: tyrosine residues are located in flexible polypeptide chains and are exposed to solvents, the three-dimensional structure of the enzyme is generally not too tight (i.e., should not be rich in disulfide bonds). The enzyme proteins that can be catalyzed by polyphenol oxidase can be determined by pre-experiments. The polyphenol oxidase can be laccase or tyrosinase, and is prepared to be in a concentration range of 1-10mg/mL, preferably in a concentration range of 5-10 mg/mL.

(2) Mixing the two solutions according to a volume ratio of 1:1, mixing, reacting at 20-50 ℃, adding or not adding 1mmol/l ferulic acid mediator, reacting for 20min-24h, centrifuging, collecting precipitate to obtain cross-linked enzyme particles with the particle size of 1-70 microns, wherein 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 ratio, the reaction temperature is preferably 20-30 ℃. In the high concentration enzyme solution (more than or equal to 5 mg/mL), the mediator can be not added, and the mediator is recommended to be added. The type of mediator may vary, the laccase mediator is preferably ferulic acid, and the tyrosinase mediator is preferably dopa. The reaction time is preferably 12 hours. As a method for separating the crosslinked enzyme fine particles, centrifugation, microfiltration or the like can be used, and centrifugation is preferable, and centrifugation conditions are 10℃or less, 10000rpm,20 minutes. The cross-linked glutamine transaminase particles are measured by a laser particle size meter under the following conditions: selecting a Baite laser particle diameter instrument; against water as background. The activity of the cross-linking enzyme is determined by a color development-visible spectrophotometry method by taking the inactivated enzyme as a reference substance.

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

In this step, the use is selected according to the catalytic properties of the cross-linking enzyme, such as the modification of the protein drug by the cross-linking glutamine transaminase.

In order to make the above objects, features and advantages of the present invention more comprehensible, the following description further describes the technical solution of the present invention in connection with the embodiments. The invention is not limited to the embodiments listed but includes any other known modification within the scope of the claims that follow.

Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic may be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1

Catalysis by laccase crosslinks the glutamine transaminase.

(1) A glutamine transaminase solution with a protein concentration of 10mg/mL was prepared, and laccase solutions of 1mg/mL, 2.5mg/mL, 5mg/mL, 7.5mg/mL, and 10mg/mL were prepared, respectively. Wherein the glutamine transaminase is given by Jiangsu Yiming biological Co., ltd, contains dextrin and other components, and the protein content of the enzyme preparation is about 10% by using a Coomassie Brilliant blue method. Laccase was supplied by Sigma Co. Both enzyme solutions were filtered.

(2) Respectively taking and mixing 0.5mL of glutamine transaminase solution and laccase solution, reacting at 25 ℃ for 20min, 40min, 1h, 2h, 4h, 6h, 8h, 12h, 18h and 24h, and centrifuging at 10000rpm for 20min at a temperature lower than 10 ℃. The precipitate was suspended in distilled water and centrifuged again and repeated 2 times. Finally, the immobilized product (crosslinked glutamine transaminase) was dispersed in 1mL of distilled water, and the enzyme activity was measured. The cross-linked glutamine transaminase particles are measured by a laser particle size meter, and the activity of the cross-linked enzyme is measured by a chromogenic-visible spectrophotometry.

In step (1), if the laccase concentration is selected to be lower than 5mg/mL, the activity of the crosslinked glutamine transaminase increases with the increase of the laccase concentration; when the laccase concentration is increased to 5mg/mL and 7.5mg/mL, the activity of the cross-linked glutamine transaminase reaches the maximum, and the activity of the cross-linked glutamine transaminase generated when the laccase concentration is 10mg/mL is reduced. Thus, the laccase concentration was determined to be 5-7.5mg/mL. In the step (2), if laccase catalysis time of 20min, 40min, 1h, 2h, 4h, 6h, 8h and 12h is selected, the activity of the crosslinked glutamine transaminase is found to be continuously increased along with the extension of the reaction time, and the activity is not increased after 12h, so that a stable value is reached. Therefore, the reaction time is selected to be 12-24 hours, and the crosslinking is sufficient. Referring to FIGS. 1 to 3, FIG. 1 shows the effect of laccase concentration and reaction time on glutamine transaminase crosslinking in example 1 in a method of preparing a crosslinking enzyme according to the present invention; FIG. 2 shows SDS-PAGE results analysis of 1mg/mL laccase catalyzed in example 1 for various times in a preparation method of cross-linking enzyme according to the invention; FIG. 3 shows SDS-PAGE analysis of 7.5mg/mL laccase catalyzed samples of example 1 for various times in accordance with a method of preparing a cross-linking enzyme according to the invention. As shown in FIGS. 1-3, according to the SDS-PAGE result of FIG. 2, when laccase concentration is 1mg/mL, the electrophoresis band of glutamine transaminase is slightly weakened along with the extension of the crosslinking reaction time, but the electrophoresis band of glutamine transaminase in the solution is still obvious after 24 hours of reaction, which indicates that more glutamine transaminase still remains in the solution, and the electrophoresis result is consistent with the effect result of laccase concentration of 1mg/mL in FIG. 1 on crosslinking; when the laccase concentration is 7.5mg/mL, the electrophoresis band of glutamine transaminase is obviously weakened after 20min of reaction, no new band is generated in electrophoresis, which indicates that under the catalysis of laccase with the concentration, the glutamine transaminase in the solution has rapid crosslinking reaction, the concentration is reduced, and the molecular weight of the produced product is very large and can not enter electrophoresis gel; after 12h of reaction, the electrophoresis band of the glutamine transaminase is basically disappeared, which shows that after 12h of reaction, the glutamine transaminase basically does not remain in the solution and is completely crosslinked, and the electrophoresis result is consistent with the effect result of laccase concentration of 7.5mg/mL on crosslinking in FIG. 1.

On the basis of defining laccase concentration and time in the catalytic reaction, further adding a mediator, and examining the influence of the mediator on crosslinking. Please refer to fig. 4 to fig. 5. FIG. 4 shows the effect of mediator ferulic acid on the activity of the cross-linking enzyme in example 1 according to a method of preparing the cross-linking enzyme of the present invention; FIG. 5 is a cross-linked SDS-PAGE analysis of 7.5mg/ml laccase system in example 1 of the preparation method of cross-linked enzyme according to the invention. As shown in FIG. 4, the addition of mediator to the low concentration laccase (1 mg/mL) reaction solution can promote the cross-linking of the glutamine transaminase, the activity of the cross-linked glutamine transaminase is improved, while in the high concentration laccase (7.5 mg/mL) reaction solution, the presence of mediator improves the activity of the cross-linked glutamine transaminase, i.e., promotes the cross-linking, within 2 hours of reaction, but the presence of mediator rather reduces the activity of the cross-linked glutamine transaminase over time, even lower than the result of the reaction without mediator. As shown in FIG. 5, in the absence of mediator in the high concentration laccase (7.5 mg/mL) reaction system, 12h is required for complete crosslinking of glutamine transaminase; after the mediator ferulic acid is added into the reaction system, the crosslinking reaction efficiency of the glutamine transaminase is obviously improved, and the electrophoresis band of the glutamine transaminase completely disappears when the reaction is carried out for 20min, namely the complete crosslinking is carried out. This is the reason why the activity of the crosslinked glutamine transaminase is improved in a short period of time by adding a mediator to the reaction solution of laccase (7.5 mg/mL) at a high concentration.

In step (2), the effect of various reaction conditions on the particle size was measured by a laser particle diameter meter. Please refer to fig. 6 to fig. 9. FIG. 6 shows the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 1.0mg/mL laccase; FIG. 7 is a graph showing the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 2.5mg/mL laccase; FIG. 8 is a graph showing the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 5.0mg/mL laccase; FIG. 9 shows the particle size of the crosslinked glutamine transaminase particles produced by the method of the present invention in example 1 at a concentration of 7.5mg/mL laccase. As shown in fig. 6 to 9, the larger the laccase concentration, the smaller the particle size of the particles, which suggests that laccase concentration is a critical factor in determining particle size. Therefore, the particle size of the cross-linked enzyme microparticles can be controlled by varying the concentration of laccase.

Example 2

Catalytic action of tyrosinase cross-linking glutamine transaminase

The invention investigates the feasibility of other polyphenol oxidases (tyrosinase) to catalyze the crosslinking of glutamine transaminase. The method comprises the following steps:

(1) A glutamine transaminase solution with a protein concentration of 10mg/mL was prepared, and a tyrosinase solution with a protein concentration of 3mg/mL was prepared. Wherein the glutamine transaminase is given by Jiangsu Yiming biological Co., ltd, contains dextrin and other components, and the protein content in the enzyme preparation is about 10% by using a Coomassie Brilliant blue method. Tyrosinase was derived from mushrooms and is supplied by the company macolin. Both enzyme solutions were filtered.

(2) Mixing glutamine transaminase solution and tyrosinase solution respectively in 0.2mL, reacting at 25deg.C for 1 hr, 2 hr, 4 hr, 8 hr, 18 hr, and 24 hr, and sampling for SDS-PAGE analysis. Further 2mM mediator DOPA was added and the effect of the mediator on tyrosinase cross-linking glutamine transaminase was investigated. Referring to FIG. 10, FIG. 10 is a diagram showing SDS-PAGE analysis of tyrosinase-catalyzed glutamine transaminase crosslinking in example 2 according to a method of preparing a crosslinking enzyme of the present invention. As shown in fig. 10, the electrophoretic band of glutamine transaminase disappeared in the presence of tyrosinase, indicating that tyrosinase can also catalyze glutamine transaminase crosslinking.

Example 3

Use of a cross-linked glutamine transaminase

The specific method for modifying protein drugs by using the cross-linked glutamine transaminase to catalyze PEG derivatives comprises the following steps: the protein drug solution was mixed with PEG derivative solution (0.05M, pH 8.0Tris-HCl buffer) and the reaction was initiated by adding glutamine transaminase. After the reaction is carried out for 2 hours in a water bath at 37 ℃, a supernatant sample is centrifugally taken, after the supernatant sample is treated in a water bath with 2X SDS loading buffer boiling, SDS-PAGE is carried out, after the electrophoresis is finished, coomassie blue is used for dyeing, and after the decolorization, a Bio-Rad Gel Doc XR+ Gel imaging system is used for photographing and analyzing the Gel. The result is shown in FIG. 11. FIG. 11 shows SDS-PAGE analysis of a cross-linked glutamine transaminase-catalyzed PEG derivative modified protein drug in example 3 according to a method of preparing a cross-linked enzyme of the present invention. As shown in fig. 11, the cross-linked glutamine transaminase successfully catalyzes PEG derivative modification of protein drugs and has no glutamine transaminase electrophoresis band, indicating that the cross-linked glutamine transaminase is successfully separated from the product by centrifugation.

Polyphenol oxidase (including laccase and tyrosinase) and glutamine transaminase can catalyze protein crosslinking, and have a wide range of protein substrates. Laccase, tyrosinase, glutamine transaminase, etc. have different amino acid sites, reaction mechanisms and the like (see Table 1 for comparison), so that the range of protein substrates acted by the laccase, tyrosinase, glutamine transaminase and the like are different. Both polyphenol oxidase and glutamine transaminase are potential substrates for the other side, but the result shows that the mixed reaction result of the polyphenol oxidase and the glutamine transaminase is that the polyphenol oxidase successfully crosslinks the glutamine transaminase, and the glutamine transaminase cannot catalyze to crosslink the polyphenol oxidase. Therefore, the technology realizes the crosslinking of the glutamine transaminase based on the catalysis of the polyphenol oxidase, and solves the problem of limiting the glutamine transaminase in the aspect of protein drug modification.

TABLE 1 comparison of laccase, tyrosinase and glutamine transaminase

TABLE 1

In summary, the preparation method of the cross-linking enzyme takes polyphenol oxidase as a catalyst to catalyze the cross-linking of glutamine transaminase, and the cross-linking enzyme is easy to recycle, can be repeatedly used, does not bear the separation of products, and greatly reduces the production cost. The technology is environment-friendly, mild and efficient, has controllable reaction, does not need a carrier or purifying glutamine transaminase, and can be used for crosslinking and immobilizing a plurality of enzymes together.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.

Claims

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.