Composite carrier for preparing immobilized protein, polypeptide or oligopeptide and preparation method and application thereof

A composite carrier and protein technology, applied in the direction of chemicals used for biological control, immobilized on/in organic carriers, immobilized enzymes, etc., can solve the problems of high cost and industrial application limitations, and achieve simple production, Wide applicability, low cost effect

Active Publication Date: 2015-01-14
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used chromatography column carrier in affinity chromatography technology is agarose gel, but the cost of agarose gel is expensive and it needs to use (for example) highly toxic cyanogen bromide in the process of coupling with the ligand protein (CNBr) for activation and other shortcomings, so it is greatly limited in industrial applications

Method used

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  • Composite carrier for preparing immobilized protein, polypeptide or oligopeptide and preparation method and application thereof
  • Composite carrier for preparing immobilized protein, polypeptide or oligopeptide and preparation method and application thereof
  • Composite carrier for preparing immobilized protein, polypeptide or oligopeptide and preparation method and application thereof

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Experimental program
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Effect test

preparation example Construction

[0079] (4) Preparation method of the complex

[0080] The present invention also provides a method for preparing the complex as described in (3), which includes: reacting the complex carrier as described in (1) with a solution of protein, polypeptide or oligopeptide, thereby obtaining the Complex.

[0081] The principle of the above reaction is as follows: the amino group on the protein, polypeptide or oligopeptide reacts with the aldehyde group on the cross-linked product of chitosan and polyaldehyde compound to form a Schiff base, and covalently bonds the protein , polypeptide or oligopeptide immobilized on the complex as described in (1).

[0082] In the immobilization process of proteins, polypeptides or oligopeptides, the extrusion operation of the carrier can be done manually, or with equipment or machines; generally speaking, the concentration of the protein, polypeptide or oligopeptides used is 0.3-30% (W / V), the concentration of cross-linking agent is 0.01%-30% (V / ...

Embodiment 1

[0116] Example 1 Immobilized α-glucosidase

[0117]Take 0.02g of melamine flake sponge (Zhuhai Tianhong Special Sponge Factory), add 0.8g of 2% (w / v) chitosan (Shandong Laizhou Haili Biological Products Co., Ltd.) solution to the sponge, and repeatedly squeeze the chitosan The solution is evenly distributed on the sponge; soak in 5ml 1M sodium hydroxide solution for 30 minutes; wash the sponge with deionized water until the pH is neutral; soak in 5% (v / v) glutaraldehyde (Tianjin Damao Chemical Reagent Factory) Solution (containing 1% (v / v) acetic acid) 6ml for 1 hour; wash the sponge with 20mM pH7.4 phosphate buffer solution (i.e. potassium phosphate buffer solution, the same below) to remove the remaining unreacted glutaraldehyde solution; remove with filter paper Excess moisture, resulting in a carrier with a wet weight of 0.5 g.

[0118] Soak the carrier in 5ml α-glucosidase (Amano Enzyme Co., Ltd.) solution (20mM pH7.4 phosphate buffer solution to a concentration of 2mg / m...

Embodiment 2

[0122] Example 2 Immobilized polymyxin B

[0123] Take 0.04g of melamine flake sponge, add 1.6g of 2% (w / v) chitosan solution to the sponge, squeeze repeatedly to make the chitosan solution evenly distributed on the sponge; soak in 10ml of 1M sodium hydroxide solution for 30 minutes; use Wash the sponge with deionized water until the pH is neutral; soak in 12ml of 5% (v / v) glutaraldehyde solution for 1 hour; wash the sponge with 20mM pH7.4 phosphate buffer solution to remove the remaining unreacted glutaraldehyde solution; Filter paper was used to remove excess water and the resulting carrier had a wet weight of 1 g.

[0124] Soak the carrier in 20ml polymyxin B (Jiangsu World Industrial Co., Ltd.) solution (20mM pH7.4 phosphate buffer solution to a concentration of 1.8mg / ml) for 24 hours; use 10ml20mM pH7.4 phosphate The remaining unreacted polymyxin B solution was washed with buffer solution to remove the remaining unreacted polymyxin B solution, and the washing was repeate...

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Abstract

Provided are a composite carrier for immobilization of a protein, polypeptide or oligopeptide, a preparation method and an application thereof. The composite carrier is a porous material which comprises: (1) a porous organic foam material containing open pores; and (2) a crosslinked product having aldehyde groups and immobilized on the surface of the walls of one or more pores of the porous organic foam material, wherein the aldehyde groups are able to react with the protein, polypeptide or oligopeptide, and the crosslinked product are formed by chitosan via a crosslinking reaction with a polyaldehyde compound.

Description

technical field [0001] The invention belongs to the technical field of immobilized proteins, polypeptides or oligopeptides, and specifically relates to a composite carrier for preparing immobilized proteins, polypeptides or oligopeptides and a preparation method thereof; the composite and its preparation method and application. Background technique [0002] With the development of biotechnology, the application of enzymes is becoming more and more extensive. Due to the high cost of directly using liquid enzymes, enzymes are often used in industrial and other fields in immobilized form. In addition, immobilized enzymes are easy to separate from the product and can be reused, and are often more stable than free enzymes. There are many ways to immobilize enzymes, such as physical adsorption, affinity binding, covalent cross-linking, flocculation embedding, etc. [0003] The immobilized enzyme / immobilized cell is usually evaluated by the specific activity (the activity of immo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/10C07K17/08C07K17/06C12N11/10C12N11/08C12N11/06C08J9/36A01N37/46A01P1/00B01J20/24B01J20/26
CPCB01D15/3809B01J20/24B01J20/267B01J20/286B01J20/3212B01J20/3219B01J20/3274C07K1/22C07K17/04C07K17/06C07K17/08C12N9/1051C12N11/02C12N11/06C12N11/089A01N43/68B01J31/0234B01J31/06C07K5/0806C07K7/62C07K14/31C08J9/0061C08J2201/026C08J2361/28C08J2403/10C08J2405/08C12N11/10
Inventor 张仲康王骏
Owner BIORIGHT WORLDWIDE
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