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Membrane-like immobilized cell, polypeptide, oligopeptide or protein and preparation method thereof

A technology for immobilizing cells and proteins, which is applied in the field of immobilized cells and proteins, can solve problems such as increasing production costs, and achieve the effects of easy operation, cost and time reduction, and safety and reliability

Pending Publication Date: 2022-07-19
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technology needs to design specific equipment for production in industrial production (Zeng Zhaojing, Ouyang Pingkai, Pan Guangliang, "Immobilized Cell Carrier Forming Machine", Chinese Patent Application Publication No.: CN2181511Y), which greatly increases production costs

Method used

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  • Membrane-like immobilized cell, polypeptide, oligopeptide or protein and preparation method thereof
  • Membrane-like immobilized cell, polypeptide, oligopeptide or protein and preparation method thereof
  • Membrane-like immobilized cell, polypeptide, oligopeptide or protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1: Preparation of immobilized E. coli cell membrane expressing glucose isomerase with chitosan

[0095] Get 1g of Escherichia coli cells expressing glucose isomerase (the preparation method of this Escherichia coli cell is as described in the Chinese patent application with publication number CN1982445A), resuspend in 9ml deionized water, add 0.1g shell after the cells are completely resuspended Polysaccharide powder (Weifang Kehai Chitin Co., Ltd., the degree of deacetylation is 95%); add 50 μl of acetic acid under stirring; add 0.25 ml of 1% (v / v) glutaraldehyde (Tianjin Da Mao Chemical Reagent) after thorough mixing After 10 minutes of reaction, the mixed solution was placed in a petri dish to dry at room temperature; after drying, soaked in 20ml of 5% (w / v) sodium trimetaphosphate (pH 6.0) (Shanghai Aladdin Biochemical Technology Co., Ltd.) solution 30 minutes; use deionized water to remove unreacted sodium trimetaphosphate, and dry at room temperature to ob...

Embodiment 2

[0097] Example 2: Preparation of immobilized glucose isomerase membrane with chitosan

[0098] Take 1 g of Escherichia coli cells expressing glucose isomerase and resuspend in 4 ml of deionized water. After the cells are completely resuspended, use an ultrasonic cell crusher to crush the cells in an ice bath (ultrasonic power 50W, ultrasonic time 5 seconds, gap time 5 seconds). s, cycle 30 times), centrifuge at 13,000 rpm for 20 min at 4 °C, and take the supernatant. Dilute the protein concentration of the supernatant to 10mg / ml with deionized water, add 0.1g chitosan powder to 10ml; add 50μl acetic acid under stirring; add 0.25ml 1% (v / v) glutaraldehyde solution after thorough mixing After 10 minutes of reaction, put the mixed solution in a petri dish to dry at room temperature; soak in 20ml of 5% (w / v) sodium trimetaphosphate (pH 6.0) solution for 30 minutes after drying; remove unreacted water with deionized water Sodium trimetaphosphate was dried at room temperature to ob...

Embodiment 3

[0100] Example 3: Preparation of immobilized glucose isomerase membrane with pre-crosslinked chitosan

[0101] Take 10g of chitosan powder, add 400ml of 1% sodium trimetaphosphate (pH 6.0); stir at room temperature for 30 minutes; filter and remove unreacted sodium trimetaphosphate with deionized water; dry at 60°C to obtain pre-crosslinked chitosan Sugar 9.7g.

[0102] Take 1 g of Escherichia coli cells expressing glucose isomerase and resuspend in 4 ml of deionized water. After the cells are completely resuspended, use an ultrasonic cell crusher to crush the cells in an ice bath (ultrasonic power 50W, ultrasonic time 5 seconds, gap time 5 seconds). s, cycle 30 times), centrifuge at 13,000 rpm for 20 min at 4 °C, and take the supernatant. Dilute the protein concentration of the supernatant with deionized water to 10mg / ml, take 10ml and add 0.1g of pre-crosslinked chitosan powder; add 50μl acetic acid under stirring; add 0.25ml 1% (v / v) after thorough mixing Glutaraldehyde s...

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Abstract

The invention provides a membrane-like immobilized cell, polypeptide, oligopeptide or protein and a preparation method thereof. The method comprises the following steps: 1) providing chitosan which is not subjected to film formation, wherein the chitosan is chitosan which is not subjected to pre-crosslinking or chitosan which is subjected to pre-crosslinking; (2) a mixed solution of the chitosan without film formation and cells, polypeptides, oligopeptides or proteins is provided, and in the mixed solution, the chitosan without film formation is in a dissolved state; (3) mixing a cross-linking agent with the mixed solution to obtain a co-cross-linking product of the chitosan and the cells, the polypeptides, the oligopeptides or the proteins; 4) drying the co-crosslinking product to obtain membrane-like immobilized cells, polypeptides, oligopeptides or proteins; wherein when chitosan which is not pre-crosslinked is used in the step (1), the method further comprises the step (5) of mixing the membrane-like immobilized cells, the polypeptide, the oligopeptide or the protein with phosphate, so that chitosan molecules in the membrane-like immobilized cells, the polypeptide, the oligopeptide or the protein are crosslinked. The method is a one-step method, the process is simple, and time and cost are saved.

Description

technical field [0001] The present invention belongs to the field of immobilized cells and immobilized proteins, and in particular, relates to membrane-like immobilized cells, polypeptides, oligopeptides or proteins and preparation methods thereof. Background technique [0002] The application of enzymes is becoming more and more extensive, and immobilized enzymes are often used in industrial applications to reduce production costs because they are easy to separate from products and can be reused. There are many immobilization methods for enzymes, such as physical adsorption, affinity binding, covalent cross-linking, flocculation, etc. (see the document "Roger A. Sheldon, 2007, Advanced Synthesis & Catalysis, 349: 1289-1307"). [0003] The manufacturing process of the existing carrier is complicated, for example: the preparation of silicon carrier requires the use of a variety of organic solutions (Yuan Zhongyi, Wu Xingjia, Li Shiyun, Ji Xinsong, "Preparation of a beaded chi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C07K17/10
CPCC12N11/10C07K17/10C12N1/20C12N9/92C12N2533/72C12N2537/10C12Y503/01018
Inventor 张仲康王骏
Owner BIORIGHT WORLDWIDE