A method for weakening the inhibition of cyclodextrin glucosyltransferase product

A glucose-based, product-inhibiting technology, applied in the direction of glycosyltransferase, transferase, biochemical equipment and methods, etc., can solve the problems of weakening product inhibition, cumbersome operation, non-reusable use, etc. The effect of mild conditions and simple preparation process

Active Publication Date: 2021-07-27
JIANGNAN UNIV
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Problems solved by technology

However, although the use of site-directed mutagenesis technology-biotechnological means to transform cyclodextrin glucosyltransferase is simple and easy to implement and has good reproducibility, it often causes changes in other properties of the enzyme (such as enzyme activity, product specificity, thermal stability, etc.); Although the use of membrane separation technology can effectively separate the cyclodextrin in the product according to the molecular weight to weaken the product inhibition, but the starch can cause the blockage of the membrane, the effect is not significant, the operation is cumbersome, and the cost is high; on the one hand, the addition of organic reagents will lead to cost reduction. increase, on the other hand, the residue of reagents limits the application of cyclodextrin in the food field
[0004] At the same time, the enzymes used in these three methods are free and cannot be reused

Method used

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  • A method for weakening the inhibition of cyclodextrin glucosyltransferase product
  • A method for weakening the inhibition of cyclodextrin glucosyltransferase product
  • A method for weakening the inhibition of cyclodextrin glucosyltransferase product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Effect of Immobilized Enzyme on Cyclodextrin Glucosyltransferase Product Inhibition

[0047] Prepare 8% (w / w) gelatin solution with water, cool to 45°C, add cyclodextrin glucosyltransferase solution 10% (w / w) to mix evenly, pour it into a plate to a thickness of 3mm, and place it at 4°C Perform cooling and adsorption for 2 hours, cut into small pieces after forming a gel film, soak and cross-link with 0.25% glutaraldehyde solution at 25°C for 1 hour. Wash with water and set aside. The free enzyme and the prepared immobilized enzyme were analyzed for product inhibition. ( figure 1 Production of cyclodextrins for free and immobilized enzymes at different concentrations of cyclodextrin inhibitors)

[0048] from figure 1It can be seen that in the absence of cyclodextrin inhibitors, the cyclodextrin yield of free enzyme acting on maltodextrin at different time points is greater than that of gelatin-immobilized enzyme; the final concentration of 1mg / mL is added...

Embodiment 2

[0049] Example 2: Effect of Gelatin Concentration on Cyclodextrin Glucosyltransferase Product Inhibition

[0050] Prepare a 5%-15% (w / w) gelatin solution with water, cool to 45°C, add cyclodextrin glucosyltransferase solution 10% (w / w) to mix evenly, and immediately pour it into a plate to a thickness of 3mm. Place at 4°C for cooling and adsorption for 2h, cut into small pieces after forming a gel film, soak and cross-link with 0.25% glutaraldehyde solution at 25°C for 1h. Wash with water and set aside. The free enzyme and the prepared immobilized enzyme were analyzed for product inhibition. ( figure 2 Product inhibition of immobilized enzymes prepared for different concentrations of gelatin during reaction for 12 hours)

[0051] from figure 2 It can be seen that, when the gelatin concentration is low (5%-12%), the inhibition rate caused by the presence of 1mg / mL or 2mg / mLβ-cyclodextrin is lower than that of the free enzyme, and when the gelatin concentration is 15%, the...

Embodiment 3

[0053] Example 3: Effect of Temperature on the Activity of Immobilized Cyclodextrin Glucosyltransferase

[0054] Prepare 8% (w / w) gelatin solution with water, cool to 45°C, add cyclodextrin glucosyltransferase solution 10% (w / w) to mix evenly, pour it into a plate to a thickness of 3mm, and place it at 4°C Perform cooling and adsorption for 2 hours, cut into small pieces after forming a gel film, soak and cross-link with 0.25% glutaraldehyde solution at 25°C for 1 hour. Wash with water and set aside.

[0055] The free enzyme and the prepared gelatin-immobilized enzyme were respectively reacted at different temperatures (30-80°C) to measure the cyclization activity, and the highest cyclization activity was set as 100%, and the relative activity was calculated, taking the temperature as the horizontal Coordinates, relative activity is plotted as the ordinate to determine the optimum temperature of free enzyme and immobilized enzyme. ( Figure 12 Influence of temperature on th...

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Abstract

The invention relates to a method for weakening the inhibition of cyclodextrin glucosyltransferase products, which belongs to the technical field of enzymes. The invention provides a method for weakening the inhibition of cyclodextrin glucosyltransferase products. The method is to first prepare a gelatin solution, add the obtained gelatin solution to cyclodextrin glucosyltransferase and mix evenly, and then prepare the obtained Pour the mixed solution into the flat plate to a certain thickness, cool and absorb the solution poured into the flat plate to obtain a gel film, then cut the obtained gel film into small pieces, soak and cross-link with glutaraldehyde solution, and finally The gel fragments are washed to obtain the immobilized cyclodextrin glucosyltransferase that can weaken the inhibition of cyclodextrin glucosyltransferase products. The method can not only effectively weaken the product inhibition phenomenon of cyclodextrin glucosyltransferase, but also has simple preparation process, low cost, and the prepared immobilized enzyme has good stability and reusability.

Description

technical field [0001] The invention relates to a method for weakening the inhibition of cyclodextrin glucosyltransferase products, which belongs to the technical field of enzymes. Background technique [0002] Cyclodextrin can contain many hydrophobic guest molecules and change their physical and chemical properties by virtue of the nature of internal and external affinity, and has many extensive applications in various fields. At present, industrial cyclodextrins are all synthesized by cyclodextrin glucosyltransferase cyclization reaction on starch substrates. As the reaction progresses, the content of cyclodextrins in the system gradually increases, and some cyclodextrins will interact with enzymes. Self-combination affects its activity, which in turn affects the reaction efficiency, resulting in product inhibition. [0003] At present, researchers mainly solve the problem of product inhibition in the production process of cyclodextrins by means of site-directed mutagene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/02C12N9/10C12P19/18
CPCC12N9/1074C12N11/02C12P19/18C12Y204/01019
Inventor 李才明李兆丰陈双娣顾正彪李泽西洪雁程力
Owner JIANGNAN UNIV
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