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Preparation method of cross-linking enzyme

A technology of cross-linking enzymes and polyphenol oxidases, applied in biochemical equipment and methods, enzymes, transferases, etc.

Active Publication Date: 2020-10-30
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 public protocol, there is no report on the immobilization of enzymatic transglutaminase

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  • Preparation method of cross-linking enzyme
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  • Preparation method of cross-linking enzyme

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preparation example Construction

[0033] The purpose of the present invention is to provide a method for preparing a cross-linked enzyme, which uses polyphenol oxidase as a catalyst to cross-link the substrate (enzyme) to prepare cross-linked enzyme particles that are insoluble in aqueous solution. The size is measured by the diameter meter, and the vitality is measured by the color-visible light spectrophotometry. It includes the following steps:

[0034] (1) Select an enzyme containing tyrosine residues in the polypeptide chain such as transglutaminase as a substrate to prepare a first solution; prepare a polyphenol oxidase into a second solution to obtain a catalyst;

[0035] In this step, the polypeptide chain of the substrate (enzyme) contains tyrosine residues that can be specifically catalyzed by polyphenol oxidase, such as transglutaminase. These enzymes only need to be obtained through coarse separation, and no fine separation is required. The enzyme protein can contain ingredients such as dextrin added t...

Embodiment 1

[0043] The catalysis of laccase cross-links transglutaminase.

[0044] (1) Prepare a transglutaminase solution with a protein concentration of 10 mg / mL, and prepare a laccase solution of 1 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, and 10 mg / mL respectively. Among them, transglutaminase was donated by Jiangsu Yiming Biological Co., Ltd., containing dextrin and other ingredients, and the protein content in the enzyme preparation was analyzed by Coomassie brilliant blue method to about 10%. Laccase was provided by Sigma. Both enzyme solutions are filtered.

[0045] (2) Take 0.5 mL of transglutaminase solution and laccase solution, and mix them at 25°C. After reacting for 20min, 40min, 1h, 2h, 4h, 6h, 8h, 12h, 18h, 24h, at low temperature Centrifuge at 10,000 rpm for 20 minutes at 10°C. The precipitate was suspended in distilled water and then centrifuged, and repeated twice. Finally, the immobilized product (cross-linked transglutaminase) was dispersed in 1 mL of distilled water to de...

Embodiment 2

[0050] Catalysis of tyrosinase cross-linked transglutaminase

[0051] The present invention studies the feasibility of other polyphenol oxidase (tyrosinase) catalyzing cross-linking transglutaminase. Including the following steps:

[0052] (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 ingredients, and the protein content in the enzyme preparation was analyzed by Coomassie brilliant blue method about 10%. Tyrosinase is derived from mushrooms and provided by Mecoline. Both enzyme solutions are filtered.

[0053] (2) Take 0.2 mL of transglutaminase solution and tyrosinase solution and mix them at 25°C. After reacting for 1h, 2h, 4h, 8h, 18h, and 24h respectively, samples are taken for SDS-PAGE analysis. Furthermore, 2mM mediator dopa was added to study the effect of mediator on tyrosinase cross-li...

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Abstract

The invention discloses a preparation method of cross-linking enzyme. The preparation method comprises the following steps: mixing polyphenol oxidase with a glutamine transaminase solution for a reaction; the cross-linked enzyme microparticles with a particle size of several micrometers to tens of micrometers and different vigor sizes can be obtained,the cross-linked glutamine transaminase prepared by the method can be separated from a solution by means of filtration, centrifugation and the like, and the prepared cross-linked glutamine transaminase is used for polyethylene glycol (PEG) modification and the like of protein drug molecules. The cross-linked glutamine transaminase is easy to recover and can be repeatedly used in the use process, no burden is caused to product separation, and the production cost is greatly reduced. According to the method, the crosslinking process is environmentally friendly, mild and efficient, the reaction is controllable, a carrier is not needed, glutamine transaminase does not need to be purified, and multiple enzymes can be crosslinked and immobilized together.

Description

Technical field [0001] The invention belongs to the technical field of biochemical engineering and relates to a preparation method of a cross-linked enzyme. Background technique [0002] Natural enzymes (such as transglutaminase) are generally dissolved in an 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 (EC2.3.2.13) catalyzes the acyl transfer reaction between transglutaminase residues (acyl donor) on the polypeptide chain and various primary amino (acyl acceptor) compounds (including lysine residues) , The formation of isopeptide bonds, so as to achieve protein cross-linking or modification. Transglutaminase catalyzes protein cross-linking, which can improve the texture and function of food and increase the value of products. Therefore, it has been commercialized for food processing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/18C12N9/10C12N9/02
CPCC12N11/18C12N9/1044C12N9/0071C12N9/0061C12Y203/02013C12Y114/18001C12Y110/03002
Inventor 周建芹刘江云胡明刚
Owner SUZHOU UNIV
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