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Novel bionic affinity purification material and application thereof in purifying chitosanase

A chitosanase and affinity technology, applied in the direction of glycosylation enzymes, biochemical equipment and methods, enzymes, etc., can solve the problems of affecting the biological activity of chitosanase, increasing steps and costs, and achieving low cost, The effect is simple and easy to amplify

Active Publication Date: 2019-02-12
QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the particularity of metal chelating ligands, in the process of metal chelating chromatography, a large amount of toxic imidazole needs to be used in the elution process, which will affect the biological activity of the purified chitosan enzyme, and the imidazole is treated with a desalting column. Additional steps and costs will be added

Method used

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  • Novel bionic affinity purification material and application thereof in purifying chitosanase
  • Novel bionic affinity purification material and application thereof in purifying chitosanase
  • Novel bionic affinity purification material and application thereof in purifying chitosanase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The synthetic process of embodiment 1 biomimetic affinity material

[0023] The synthesis process of the biomimetic affinity material includes 4 steps: 1), epichlorohydrin activates the agarose gel, makes the agarose gel in an activated state to connect other groups; 2), amination of the agarose gel, Add amino groups to the activated agarose gel; 3), connect the cyanuric chloride connecting arm, and use the connected amino group to crosslink the cyanuric chloride connecting arm; 4), chelate chitobiose biomimetic affinity ligand, and connect Chitobiose biomimetic affinity ligand coupled to the arm. Specific steps are as follows:

[0024] 1.1 Epichlorohydrin activated agarose gel

[0025] The agarose gel (Sepharose 6B) was thoroughly washed with double distilled water at a ratio of 1:10 (v / v), so that the pH of the effluent was balanced to 7.0, and the washed agarose gel was dried at room temperature and dissolved In 100mL activation solution (1M sodium hydroxide, 2.5g...

Embodiment 2

[0032] Performance characterization of embodiment 2 biomimetic affinity material

[0033] The biomimetic affinity material synthesized in Example 1 is a macromolecular biomimetic affinity gel. In order to determine the ligand density of the synthesized biomimetic affinity material, the biomimetic affinity material synthesized in step 1.2 in Example 1 was used to The ketone method was used to detect the amino group density in the biomimetic affinity material, so as to characterize the ligand density of the synthesized biomimetic affinity material. The density of the synthesized biomimetic affinity ligand was found to be 20.9 μmol / ml by ninhydrin method.

[0034] In order to determine the structure of the bionic affinity ligand in the synthesized biomimetic affinity material, the synthesized biomimetic affinity gel was added into 6M concentrated hydrochloric acid in equal proportions (w / v), and after 6 hours of action, it was centrifuged at 4000rpm for 10min, and the supernatant...

Embodiment 3

[0035] Example 3 Dissociation constant and maximum binding capacity of biomimetic affinity material

[0036] Utilize Scatchard (Scatchard) equation method to carry out dissociation constant (K d ) and maximum binding capacity (Q max ) evaluation to determine the adsorption and dissociation abilities of the synthesized biomimetic affinity material to chitosanase CsnM. The chitosanase CsnM used was purchased from Qingdao Aifite Biotechnology Co., Ltd.

[0037] The specific assay method is: take 1 mL of chitosanase CsnM with different concentrations (0.1-0.9 mg / mL, 20 mM Gly-NaOH, pH 8.6) and mix it with 0.5 g of the biomimetic affinity material described in Example 1, and mix it at 4° C. Under the condition of 100rpm shaking culture for 2h, to reach the adsorption equilibrium. The mixture was centrifuged at 1500 g for 5 min, and the remaining protease activity and protein content in the supernatant were detected. The detected data is calculated according to the Scatchard equ...

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Abstract

The invention relates to a novel bionic affinity purification material and application thereof in purifying chitosanase, which belongs to the technical field of industry biology. An affinity ligand ofthe bionic affinity material is chitobiose, a connection arm is cyanuric chloride, and a basic medium is activated agarose 6B. A dissociation constant (Kd) and a maximum combination capacity (Qmax) of the bionic affinity material are respectively 24.2 microgram / mL and 24.1 mg / g. A chitosanase bionic affinity purification method is established by utilizing the bionic affinity material, so that thehigh-purity chitosanase can be produced in high efficiency and low cost, and the industrialized application potential is good.

Description

technical field [0001] The invention relates to a novel bionic affinity purification material and its application in chitosanase purification, belonging to the field of industrial biotechnology. Background technique [0002] Chitosan is a derivative of chitin after partial or complete deacetylation. It is mainly composed of D-glucosamine connected by β-1,4-glycosidic bonds. It is the only positively charged polysaccharide in nature and is known as the first Six life elements. Oligochitosan is a functional oligosaccharide, which has the functions of antibacterial, anti-tumor, lowering blood fat, lowering blood sugar, regulating immunity, and promoting crop production. It is one of the hot spots in the research and development of functional oligosaccharides in recent years. [0003] Chitosanase is a kind of glycoside hydrolase that catalyzes the cleavage of β-1,4 glycosidic bonds between glucosamine and specifically degrades chitosan. Traditional chitosan enzyme purification...

Claims

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Application Information

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IPC IPC(8): C08B37/12C12N9/24
CPCC12N9/2402C12Y302/01132C08B37/0039C08B37/003B01D15/3804B01J20/3212B01J20/289B01J20/3274B01J20/3255B01J20/267B01D15/22B01J20/3251
Inventor 李尚勇
Owner QINGDAO UNIV
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