Co-crosslinking immobilization method of phospholipase D
A phospholipase and co-crosslinking technology is applied in the field of co-crosslinking and immobilization of phospholipase D, which can solve the problems of high reaction activity, excessive crosslinking of enzymes, and difficulty in controlling the dosage, so as to improve catalytic reaction activity and reduce crosslinking. degree, the effect of improving the catalytic performance
Active Publication Date: 2020-02-07
NINGBO UNIV
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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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[0016] Enzyme immobilization
[0017] 1) Bisphenol A epoxy resin (brand name is E-51, epoxy value is 0.51, number average molecular weight is 392), three kinds of components of methanol and ethylenediamine are mixed according to the mass ratio of 2: 2: 0.8, in Stir and react at 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, and then put it in a vacuum oven to dry at room temperature to obtain an aminated epoxy resin;
[0018] 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.%.
[0019] 3) Dissolving phospholipase D in a sodium phosphate buffer solution with pH=7.5, keeping the enzyme concentration in the range of 1.0-7.0 mg / mL;
[0020] 4) Ph...
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Abstract
The invention relates to a co-crosslinking immobilization method of phospholipase D. Oil-soluble isocyanuric acid triacrylate is used as a cross-linking agent, reactants in an aqueous phase are amino-containing phospholipase D and a supermolecular complex containing aminated epoxy resin and beta-cyclodextrin, through a Michael addition reaction of dual bonds and amino, co-crosslinking polymerization reaction happens at low temperature, and immobilized phospholipase D of different loading amounts can be prepared. The cross-linking degree is controlled, so that the dispersibility is improved, and internal mass transfer microenvironment can be improved. The immobilized enzyme has high catalytic activity, and when the loading amount is 93mg of enzymes for 1g of carriers, the activity is the highest, and achieves 92% of that of a free enzyme.
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 phospholipase D. The novel immobilized phospholipase D can be specially used for efficient preparation of phosphatidylserine. Background technique [0002] Phospholipase D (EC3.1.4.4) is phosphatidylcholine phospholipid hydrolase (isoelectric point 5.4), which is a general term for a class of enzymes that catalyze the hydrolysis of phosphodiester ester bonds and base exchange reactions. It was first isolated from carrots by Hananhan in 1947. Phospholipase D is distributed in many organisms from bacteria to higher animals and plants, has broad substrate specificity, and can widely act on many phospholipids and phospholipid derivatives. The role of phospholipase D is not only to affect the structure, function and stability of the membrane by hydrolyzing phospholipids in the cell membrane, but also in signal tran...
Claims
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IPC IPC(8): C12N11/10C12N11/089
CPCC12N11/10C12N11/08C12N9/16C12Y301/04004Y02P20/50
Inventor 吴嘉沁张瑞丰李艳肖通虎龙能兵
Owner NINGBO UNIV