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Lysozyme with covalent modification through acyl-free sophorolipid derivatives and modifying method and application of lysozyme

A technology of acyl sophorolipids and modification methods, which is applied in the field of food preservation, can solve the problems of limiting the application range of lysozyme, difficulty in dissolving, and inability to effectively inhibit activity, and achieve the expansion of antibacterial spectrum and loss of enzyme activity Small, simple and flexible effect

Active Publication Date: 2016-10-12
ZHEJIANG GONGSHANG UNIVERSITY
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  • 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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  • Lysozyme with covalent modification through acyl-free sophorolipid derivatives and modifying method and application of lysozyme
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  • Lysozyme with covalent modification through acyl-free sophorolipid derivatives and modifying method and application of lysozyme

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Experimental program
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Effect test

Embodiment 1

[0038] The preparation method of acyl-free sophorolipid derivatives:

[0039] To a 100mL round-bottomed flask, 29.80g (43.27mmol, 1eq) of the fermentation product of Candida C.bombicola ATCC22214, 50mL of dry methanol and 1.62mL (6.49mmol, 0.15eq) of sodium methoxide, CaCl 2 Protect, heat to reflux for 3 hours, lower to room temperature, then add 25mL 20% KOH aqueous solution, continue to reflux for 2 hours, remove the solvent under reduced pressure, dissolve in deionized water, use HCl to adjust the pH until the phenol is red, and place in an ice bath until precipitation occurs , and the product was separated by silica gel column chromatography, and the mobile phase was chloroform / methanol (3:22to 4:22, v / v). 1 H NMR (CH 3 OH-d 4 ):5.35(2H,m,H-9and H-10),4.63(1H,d,J)8.1Hz,H-1"),4.44(1H,d,J)7.5Hz,H-1'), 3.82-3.87(3H,m,H-6'b,H-6"b,and H-17),3.63-3.68(3H,m,H-6'a,H-6"a,and H-5 "),3.53-3.58(2H,m,H-2'and H-3'),3.21-3.41(5H,m,H-3",H-2",H-4",H-4', and H-5'),2.27(2H,t,J)7.3Hz,H-2...

Embodiment 2

[0041] Covalent modification of native egg white lysozyme by carboxylic acid type acyl-free sophorolipid (DSL-C18):

[0042] 1) Dissolve the N-hydroxysuccinimide ester of the acyl sophorolipid derivative in 5mL DMSO to form a solution with a final concentration of 0.7mM, and add 25mL, 0.2mM, 1% NaHCO dropwise under stirring 3 Egg white lysozyme aqueous solution, continue to stir slowly and react 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.

[0043] 2) Determination of lysozyme activity:

[0044] Place the enzyme solution to be tested and the substrate suspension in a water bath at 25°C for 20 m...

Embodiment 3

[0046] 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 a lysozyme with covalent modification through acyl-free sophorolipid derivatives and a modifying method and application of the lysozyme. The modifying method comprises the following steps that firstly, active esters of acyl-free sophorolipid derivatives are dissolved in DMSO, then a NaHCO3 aqueous solution of lysozyme is added, a condensation reaction is carried out under the stirring condition, and a mixture with covalent modification is obtained after the reaction is finished, wherein the sophorolipid derivatives are carboxylic acid type acyl-free sophorolipid; secondly, deionized water and a phosphate buffer solution are adopted in sequence to carry out dialysis on the butter solution system obtained in the first step, obtained dialysate is subjected to centrifugation separation, and the lysozyme with covalent modification is obtained. The modified lysozyme can effectively suppress the activity of gram-positive bacteria, can also remarkably suppress the activity of gram-negative bacteria, and can serve as a multifunctional food preservative.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to covalently modifying egg white lysozyme with biosurfactant sophorolipids, thereby expanding the antibacterial spectrum of natural lysozyme and further broadening the application range of lysozyme, especially in the field of food preservation. 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% to 0.4%. Lysozyme extract...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12P19/12C07H15/10C07H1/00A23L3/3571C12R1/72
CPCA23L3/3571A23V2002/00C07H1/00C07H15/10C12N9/2462C12P7/6436C12P19/12C12Y302/01017A23V2200/10
Inventor 石玉刚任月萍邵诗音蔡文强袁燕微汪琦朱陈敏
Owner ZHEJIANG GONGSHANG UNIVERSITY
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