A kind of lysozyme covalently modified by short aliphatic chain sophorolipid derivatives and its modification method and application

A chain sophorolipid and modification method technology, which is applied in the field of food preservation, can solve the problems of inability to effectively inhibit the activity, difficult to dissolve, limit the application range of lysozyme, etc., and the method is simple and flexible, with little loss of enzyme activity, The effect of expanding the antibacterial spectrum

Active Publication Date: 2019-09-20
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, since there is a layer of lipopolysaccharide LPS on the outer wall of the cell membrane of Gram-negative bacteria, it is difficult for natural lysozyme to dissolve the membrane layer, so it cannot effectively inhibit the activity of this type of bacteria, which limits the scope of application of lysozyme

Method used

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  • A kind of lysozyme covalently modified by short aliphatic chain sophorolipid derivatives and its modification method and application
  • A kind of lysozyme covalently modified by short aliphatic chain sophorolipid derivatives and its modification method and application
  • A kind of lysozyme covalently modified by short aliphatic chain sophorolipid derivatives and its modification method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The preparation method of carboxylic acid type short aliphatic chain sophorolipid derivatives (PSL-C9):

[0040] (1) Add 29.80g (43.27mmol, 1eq) sophorolipids, 50mL dry methanol and 1.62mL (6.49mmol, 0.15eq) sodium methoxide, CaCl to a 100mL round bottom flask sequentially 2 Protect, heat to reflux for 3 hours, lower to room temperature, adjust pH to neutral with acid, remove solvent under reduced pressure, dissolve in deionized water, and place in ice bath at 0°C until powder precipitates, filter, wash with water, and dry under reduced pressure to obtain the product 22.5 g (82%).

[0041] (2) 21.56g (33.86mmol, 1eq) of step (1) product, 50mL of absolute dry organic solvent THF, 25.4mL (270.84mmol, 8eq) of acetic anhydride and 1.654g (13.54mmol, 0.4eq) DMAP, stirred at room temperature for 1h, CaCl 2 Protected, dissolved in ethyl acetate after removing the solvent under reduced pressure, and using saturated NaHCO 3 Wash the mixture 3 times with the solution, recover ...

Embodiment 2

[0045] Covalent modification of natural egg white lysozyme by carboxylic acid type short aliphatic chain sophorolipid derivative (PSL-C9):

[0046] 1) Dissolve the N-hydroxysuccinimide ester of the carboxylic acid type short aliphatic chain sophorolipid derivative (PSL-C9) in 5mL DMSO to form a solution with a final concentration of 0.7mM, and add 25mL dropwise while stirring, 0.2mM, 1% NaHCO 3 The egg white lysozyme solution was stirred slowly and reacted at 30°C for 6h. After the reaction, add 25mL of 100mM glycine solution to the system, keep the temperature at 30°C for 10min, dialyze with deionized water at room temperature, then dialyze with 20mM phosphate buffer, pH 7.0, and maintain at 4°C for about 1 day. In order to remove unreacted acid-type peracetyl sophorolipids, the dialyzate was centrifuged at 12,000 rpm at 5°C for 10 min, and the suspension was collected as a seed.

[0047] 2) Determination of lysozyme activity:

[0048] Place the enzyme solution to be teste...

Embodiment 3

[0050] Determination of the inhibition zone: using the cup and saucer method. Take 20mL of melted solid culture-based culture dish, after solidification, take 0.2mL with a concentration of 10 6 -10 7 Each bacterial suspension of CFU / mL was evenly spread in the solid medium, and after 10 minutes, the Oxford cup was placed on the surface of the petri dish, the sample solution was added, the medium was covered, and cultured at a constant temperature of 37°C for 24 hours, and the inhibition zone was measured. diameter. Each sample solution experiment was repeated 3 times, and the average value was taken.

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Abstract

The invention discloses lysozyme covalently modified by carboxylic acid type short-aliphatic-chain sophorolipid derivatives as well as a modification method and an application of lysozyme. The modification method comprises the following steps: (1) dissolving active esters of sophorolipid derivatives in DMSO (dimethyl sulfoxide), then adding the dissolved active esters in a NaHCO3 water solution of the lysozyme, and conducting a condensation reaction under a stirring condition, so that a reaction mixture is obtained after the reaction, wherein the sophorolipid derivatives are carboxylic acid type short-aliphatic-chain sophorolipid derivatives; and (2) dialyzing a buffer solution system obtained in the step (1) sequentially by virtue of de-ionized water and a phosphate buffer solution, and conducting centrifugal separation on an obtained dialysate, so that the modified lysozyme is obtained. The modified lysozyme not only can effectively inhibit the activity of Gram-positive bacteria but also can significantly inhibit the activity of Gram-negative bacteria; therefore, the antibacterial spectrum of natural lysozyme is expanded; and the lysozyme can serve as a multifunctional food preservative.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to covalently modifying egg white lysozyme by using biosurfactant sophorolipid derivatives, thereby expanding the antibacterial spectrum of natural lysozyme and further broadening the application range of lysozyme, especially in the field of food preservation application. Background technique [0002] Lysozyme is an alkaline enzyme that can hydrolyze mucopolysaccharides. Lysozyme mainly breaks the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposes the insoluble mucopolysaccharide of the cell wall into soluble glycopeptide, and causes the contents of the cell wall to break and escape. lyse the bacteria. It exists in the exocrine fluid of animals, plants and humans. Currently, lysozyme can be isolated from the milk of cattle, sheep, horses, etc., among which the content of egg white lysozyme is the most abundant, about 0.3%...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/36C12P19/12C07H15/10C07H1/00A23L3/3571
Inventor 石玉刚陆海霞陆旭阳卞立晴卢腾蔡文强吴煜孙锦程
Owner ZHEJIANG GONGSHANG UNIVERSITY
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