蛋清溶菌酶的改造及其抑菌活性

doi:10.11843/j.issn.0366-6964.2021.04.025

Abstract

Abstract: The aim of this study is to modify the hen egg-white lysozyme (HEWL) and explore its effects on two gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and two gram-negative bacteria (Escherichia coli, Vibrio anguillarum). The egg-white lysozyme was modified under the condition of (57.0±0.1) ℃ at pH 2.0, the ultrastructure of modified hen egg-white lysozyme was observed by transmission electron microscope, and the hydrophobicity of the egg-white lysozyme before and after the modification was measured by 8-anilino-1-naphthalenesulfonic acid (ANS). Circular dichroism and BeStSel software analysis were used to monitor the secondary structure changes of modified HEWL, Oxford cup method was used to measure the bacteriostatic diameter of modified egg-white lysozyme against experimental bacteria, and growth curve determination method was used to determine the minimum inhibitory concentration of modified egg-white lysozyme and Western blot was used to detect the changes of lysozyme content in the four experimental bacteria supernatant and cells. Transmission electron microscopy results showed that the modified egg-white lysozyme generated short rod-shaped fibril structures. ANS and circular dichroism detection results showed that the modified egg-white lysozyme was more hydrophobic and the β-sheet content was increased by 31.7%. Oxford cup method showed that the inhibitory extent of the enzyme on the experimental bacteria was: Vibrio anguillarum>Bacillus subtilis>Staphylococcus aureus>Escherichia coli. The growth curve measurement results showed that the minimum inhibitory concentration was 8 μmol·L^-1 for Vibrio anguillarum, 6 μmol·L^-1 for Escherichia coli, Staphylococcus aureus and Bacillus subtilis. Western blot found that the molecular mass of the modified egg white lysozyme in the bacterial solution was not changed, but the content and molecular mass of the lysozyme in the bacteria were changed. Gram-positive bacteria and negative bacteriaboth showed bands at 10 ku, and gram-positive bacteria showed increased lysozyme content at 22 ku, however the supernatant of the bacteria only had a band at 14.3 ku. These results indicated modified egg-white lysozyme had a strong antibacterial effect on both gram-positive bacteria and negative bacteria，which provided basic data for the application of modified hen egg-white lysozyme in the field of animal husbandry.

Key words: modified hen egg-white lysozyme, hydrophobicity, secondary structure, antibacterial activity, lysozyme content

CLC Number:

S859.797

LI Ying, FENG Zili, BAI Yu, ZHANG Yuhang, REN Xueyi. The Modification of Hen Egg-White Lysozyme and Its Antibacterial Activity[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 1094-1102.

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The Modification of Hen Egg-White Lysozyme and Its Antibacterial Activity

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