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Method for synthesizing protein skeleton nano-material and application of protein skeleton nano-material in immobilized enzyme

A technology of nanomaterials and immobilized enzymes, applied in the direction of immobilization on/in organic carriers, nanotechnology, nanotechnology, etc., can solve problems such as inhibition, and achieve low cost, convenient product separation and detection, and low price effects

Pending Publication Date: 2022-03-22
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the structure of traditional nanomaterials is rigid and inhibits enzyme activity to a certain extent.

Method used

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  • Method for synthesizing protein skeleton nano-material and application of protein skeleton nano-material in immobilized enzyme
  • Method for synthesizing protein skeleton nano-material and application of protein skeleton nano-material in immobilized enzyme
  • Method for synthesizing protein skeleton nano-material and application of protein skeleton nano-material in immobilized enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A method for synthesizing protein skeleton nanomaterials, comprising the following steps:

[0037] Step 1. At room temperature, take 1 mL of 60 mg / mL egg white solution, add 6 mL of DMSO at 200 rpm, and finally add 4 mL of 50 mmol / L, pH=7, Tris-HCl buffer solution to form a white precipitate Finally, increase the rotation speed to 400 rpm and react for 30 minutes. After the reaction is completed, the reaction system is placed at 4°C for 12 hours to obtain a white suspension, and the Tris-HCl buffer solution acts to initiate the reaction;

[0038] Step 2, centrifuge the white suspension at a speed gradient of 6000 rpm for 10 min, discard the supernatant, wash with an equal volume of water for 3 times, and centrifuge at 4000-8000 rpm for 5 min each time to obtain the protein Skeleton nanomaterials are difficult to wash if the speed is too high.

[0039] During the preparation process, by adding the tris buffer for 0-30 min, the protein skeleton nanomaterials were charact...

Embodiment 2

[0048] In order to compare the influence of different concentrations of Tris-HCl buffer solution on the material formation, the concentration of Tris-HCl buffer solution in step 1 is set to 25 mmol / L, 50 mmol / L, 75 mmol / L, 100 mmol / L, 125 mmol / L, other conditions are with embodiment 1.

[0049] From the experimental results, it can be seen that when the concentration of Tris-HCl buffer solution is too low, it cannot be formed smoothly. Although the amount of prepared material increases at high concentration, a large part of the material is no-load material, and the concentration is too high. Intrinsically active, when the concentration is 50 mmol / L-75 mmol / L, the synthesis of protein-skeleton nanomaterials is more stable, and when the Tris-HCl buffer solution is 50 mmol / L, the performance of protein-skeleton nanomaterials is more excellent, The result is as figure 1 (d) shown.

Embodiment 3

[0051] In Example 1, "after the reaction was completed, the reaction system was left to stand at 4°C for 12 h", the rest were the same as in Example 1.

[0052] We will find that the protein skeleton nanomaterial prepared in Example 3 is unstable in the aqueous phase and will decompose by itself. Methanol is often used for preservation, but after methanol preservation, the enzyme activity of the immobilized enzyme after application will be reduced.

[0053] Example 1 of the present invention used in the synthesis process to use the original system after standing for 12 h. The Coomassie Brilliant Blue method was used to measure the protein content of the solution after washing. The protein content in the material loss rate supernatant was 0.05 mg / ml. At the same time, the protein concentration of the total system before synthesis is 5.5 mg / ml, and the loss rate is less than 1%. Therefore, the synthesis method of the present invention can ensure that the material is stable, not ...

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Abstract

The invention discloses a method for synthesizing a protein skeleton nano-material and application of the protein skeleton nano-material to immobilized enzyme. 1 mL of a 10-100 mg / mL protein solution is taken, 6 mL of DMSO is added at the speed of 50-300 rpm, finally, a Tris-HCl buffer solution is added, after white precipitates are formed, the rotating speed is increased, the reaction is conducted for 30 min, after the reaction is completed, standing is conducted for 12 h at the temperature of 4 DEG C, white turbid liquid is obtained, and the volume of the Tris-HCl buffer solution needs to be larger than 3% of the total volume of a reaction system; and carrying out gradient centrifugation on the white suspension at a speed of 4000-8000 rpm for 10 min, discarding the supernatant, and washing with an isometric organic reagent or water for 3 times to obtain the protein skeleton nano-material. The preparation process is simple, the cost is low, only simple stirring is needed in the reaction process basically, reactants can be bought on the market and are low in price, toxic and harmful by-products cannot be generated, and the method is environmentally friendly.

Description

technical field [0001] The invention belongs to the technical field of nanomaterial preparation, and in particular relates to a method for synthesizing a protein skeleton nanomaterial and its application on immobilized enzymes. Background technique [0002] Lipase is one of the important industrial enzyme preparations, which can catalyze lipolysis, transesterification, ester synthesis and other reactions, and is widely used in oil processing, food, medicine, daily chemical and other industries. Lipases from different sources have different catalytic characteristics and catalytic activities. Among them, the large-scale production of lipase with transesterification or esterification function for organic phase synthesis is of great significance for the enzyme-catalyzed synthesis of fine chemical chiral compounds. At present, most esterification reactions are catalyzed by high-temperature acid and alkali, and there are problems such as toxic by-products, harsh reaction conditio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/02C12N9/20C12N9/04C12N9/88B82Y40/00B82Y5/00
CPCC12N11/02C12N9/20C12N9/0006C12N9/88B82Y40/00B82Y5/00C12Y301/01003C12Y101/03004C12Y401/02005
Inventor 李辉杨继宇曾金磊侯晨馨陆秋豪魏衍鹏陈可泉欧阳平凯
Owner NANJING UNIV OF TECH
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