单核细胞增生李斯特菌噬菌体裂解酶Lys039的活性分析及初步应用

Activity analysis and preliminary application of Listeria monocytogenes phage endolysin Lys039

为探究单核细胞增生李斯特菌噬菌体裂解酶的特性及在食品基质中的初步应用,从NCBI获取李斯特菌噬菌体裂解酶lys039基因序列,分析其生物信息学,并在大肠杆菌中表达纯化,研究其裂解活性、对生物被膜的影响以及在牛奶基质中的应用。结果表明:1)裂解酶Lys039由341个氨基酸组成,分子质量为36.4 ku,等电点为9.81,其结构具有酰胺酶活性,对本实验室22株单核细胞增生李斯特菌、2株无害李斯特菌、1株伊氏李斯特菌以及1株威氏李斯特菌具有裂解活性,但是对葡萄球菌、枯草芽孢杆菌以及沙门菌没有显示出裂解活性。2)随着Lys039浓度的升高,裂解活性逐渐增强,最适温度为30℃,最适pH值为6.0。3)Lys039能显著清除单核细胞增生李斯特菌形成的生物被膜,并且可以降低牛奶中单核细胞增生李斯特菌的细菌数量。综上,Lys039温度耐受范围广、耐酸耐碱且裂解谱较宽,可显著清除生物被膜以及初步应用效果良好。本研究为进一步防治及快速检测单核细胞增生李斯特菌提供理论基础。

To investigate the properties of Listeria monocytogenes phage endolysin and initial application in food matrix,the gene sequence of Listeria bacteriophage endolysin Lys039 was obtained from NCBI and subjected to bioinformatics analysis.The gene was then expressed and purified in E.coli,and its lytic activity,influence on biofilm,and application in milk matrix were investigated.The results showed that:1)Lys039 was composed of 341 amino acids,with a molecular weight of 36.4 ku and isoelectric point of 9.81.Its structure exhibited amidase activity and lyses the cell walls of 22 strains of Listeria monocytogenes,2 strains of Listeria innocua,1 strain of Listeria ivanovii,and 1 strain of Listeria welshimeri in our laboratory.However,it exhibit no lytic activity on Staphylococci spp,Bacillus subtilis,and Salmonella spp.2)The lytic activity of Lys039 increased gradually with concentration enrichment,the optimum temperature was 30℃and the optimal pH value was 6.0.3)Lys039 can effectively clear the biofilm formed by Listeria monocytogenes and reduce the number of Listeria monocytogenes in milk.In conclution,Lys039 has a wide range of temperature tolerance,acid and alkali resistance and wide lytic spectrum,which can significantly reduce biofilm and has good preliminary application.This study provides a theoretical basis for further prevention and rapid detection of Listeria monocytogenes.