Alkali protease co-crosslinking immobilization method

A protease and co-cross-linking technology, applied in the field of co-cross-linking and immobilization of alkaline protease, can solve the problems of high reactivity, excessive cross-linking of enzymes, difficult to control dosage, etc., achieve high efficiency, improve micro-environment, and improve catalytic effect of reactivity

Active Publication Date: 2020-02-18
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used cross-linking agent is water-soluble glutaraldehyde, which has high reactivity and is difficult to control the dosage. It is easy to cause excessive cross-linking of the enzyme and cause a great loss of enzyme activity. In addition, the traditional cross-linking method often It is necessary to precipitate and aggregate enzyme molecules before cross-linking, which will not only cause waste of enzymes, but also block mass transfer channels, and cannot fully exert the catalytic efficiency of enzymes

Method used

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  • Alkali protease co-crosslinking immobilization method
  • Alkali protease co-crosslinking immobilization method
  • Alkali protease co-crosslinking immobilization method

Examples

Experimental program
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Embodiment Construction

[0015] Enzyme immobilization

[0016] 1) Bisphenol A epoxy resin (brand name is E-44, epoxy value is 0.44, number average molecular weight is 454), methanol and triethylenetetramine are mixed according to the mass ratio of 2: 2: 1.2, Stir and react in the range of 25-35°C for 4-5 hours, pour the mixture into water, wash the precipitate repeatedly with water to remove methanol and a small amount of amine, then put it in a vacuum oven and dry it at room temperature to obtain an aminated epoxy resin;

[0017] 2) Add epoxy resin amides and β-cyclodextrin into water at a molar ratio of 1:2.1 to 1:2.3, heat and stir until all epoxy resin amides are converted into molecular complexes and dissolved in water, and keep the The total mass concentration of the aqueous solution is in the range of 5 to 6wt.%.

[0018] 3) Dissolving alkaline protease in sodium phosphate buffer solution with pH=9.5, keeping the enzyme concentration in the range of 1.0-7.0 mg / mL;

[0019] 4) Alkaline proteas...

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Abstract

The invention relates to an alkali protease co-crosslinking immobilization method. Oil-soluble diethylene glycol diacrylate is used as a cross-linking agent, a reactant in an aqueous phase is a supermolecule compound prepared from amino containing alkali protease, aminated epoxy resin and beta-cyclodextrin, through a Michael addition reaction of dual bonds and amino, the co-crosslinking is generated at low temperature, and the immobilization alkali protease of different loading quantities can be prepared. The cross-linking degree is controlled, so that the dispersibility is improved, and internal mass transfer microenvironment can be improved. An immobilized enzyme has high catalytic activity, and when the loading quantity is 58-79mg of enzymes / 1g of carriers, the relative activity achieves 90% or above of free enzymes.

Description

technical field [0001] The invention relates to the technical field of immobilized enzyme biocatalysis, in particular to a method for co-crosslinking and immobilizing alkaline protease. The novel immobilized alkaline protease can be specially used for hydrolyzing protein and converting it into polypeptide. Background technique [0002] Alkaline protease refers to enzymes that hydrolyze protein peptide bonds under the condition of alkaline pH value (isoelectric point is 8.0-9.0). It is a kind of proteolytic enzyme and belongs to endo-serine protease. The pH value of microbial alkaline protease is generally active in the range of 7-11. When casein is used as the substrate, the optimum pH is mostly in the range of 9.5-10.5. In addition to catalyzing the hydrolysis of peptide bonds, protease also has the ability to catalyze the hydrolysis of ester bonds, amide bonds, transesterification and peptide conversion. The molecular weight of alkaline protease is between 20000 and 34000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12N11/089
CPCC12N11/10C12N11/08C12N9/50C12Y304/21Y02P20/50
Inventor 吴嘉沁张瑞丰李艳肖通虎龙能兵
Owner NINGBO UNIV
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