【作者】 王玉平；

【导师】 刘爱平； 王利娜；

【作者基本信息】 四川农业大学 ， 农业硕士（专业学位）， 2020， 硕士

【摘要】 沙门氏菌是主要的食源性致病菌之一,噬菌体裂解酶作为控制致病菌的一种新方法,具有高效、特异性强、不易产生抗性等特点,在食品安全领域具有良好的应用前景。本研究根据鼠伤寒沙门氏菌ATCC14028的噬菌体LPST10功能基因注释,通过生物信息学技术分析其编码的裂解酶蛋白基本特性并预测裂解机制;在大肠杆菌表达系统中对该噬菌体编码的裂解酶LysWL59和LysWL60进行表达条件优化;鉴定了裂解酶的活性并研究部分环境条件对相对裂解活性的影响;探究了噬菌体裂解酶与外膜渗透剂的叠加作用及其在不同食品中的应用。研究结果如下:1.噬菌体LPST10的ORF59和ORF60分别编码2种不同的裂解酶LysWL59和LysWL60,它们的理论等电点分别为9.30和8.58,均无信号肽和跨膜区域,无疏水性。2种蛋白二级结构中α-螺旋和β-折叠占比相似,但三级结构差异较大。保守结构域皆属于Lysozyme-like domains家族,可能是只含球状结构域的糖苷酶。裂解酶LysWL59和LysWL60“自内裂解”时需借助穴蛋白或spanin蛋白Rz。2.LysWL59的最佳表达条件是37°C诱导大肠杆菌C41 4 h,LysWL60的最佳表达条件是30°C下诱导大肠杆菌BL21 4 h。两者的分子量分别约为18和11.6 k Da。3.LysWL59和LysWL60裂解谱广,分别在浓度为0.25和30μmol/L时具有最佳裂解活性。Zn²⁺和高浓度的Mn²⁺（1～10 mmol/L）对两者的相对裂解活性抑制作用明显。LysWL59在p H 8.0～10.0、4～90°C、10～500 mmol/L Na Cl的条件下能保持较高的活性,其热稳定性、Na Cl耐受性、保存稳定性、裂解活性均优于LysWL60,更适合应用在食品中。4.LysWL59与EDTA的叠加作用优于苹果酸和柠檬酸,最佳组合为0.25μmol/L LysWL59和0.50 mmol/L EDTA。最佳组合使培养液中鼠伤寒沙门氏菌ATCC14028在1 h时减少97.50%。将2.5μmol/L LysWL59和0.50 mmol/L EDTA联合作用于不同食品1 h时,对莴苣的杀菌效果最好,鼠伤寒沙门氏菌ATCC14028减少93.03%;其次是鸭肠;对哈密瓜、猪肉和脱脂奶粉中沙门氏菌的杀菌效果均较差,这可能是食品基质的复杂性所致。本研究有助于防控鼠伤寒沙门氏菌ATCC14028的感染,为G^-菌噬菌体裂解酶产品的研发提供理论依据,为解决噬菌体裂解酶的实际生产应用问题提供参考。更多还原

【Abstract】 Salmonella is one of the main foodborne pathogens.Bacteriophage endolysins,as a new method to control pathogenic bacteria,have the characteristics of high efficiency,strong specificity and low resistance,showing a good application prospect in the field of food safety.In this study,based on the functional gene annotation of bacteriophage LPST10 infecting S.Typhimurium ATCC14028,the basic characteristics of endolysins were analyzed through bioinformatics,and the cleavage mechanism was predicted;endolysins LysWL59 and LysWL60 were expressed in the Escherichia coli expression system through optimization;the activity of the recombinant protein was identified and the effects of partial environmental conditions on the relative lytic activity was studied;the synergies between endolysins and outer membrane permeabilizers and their application in different foods were studied.The results were as follows:1.ORF59 and ORF60 of bacteriophage LPST10 encoded two different endolysins LysWL59 and LysWL60,respectively,and their theoretical p I were 9.30 and 8.58.There were no signal peptides,transmembrane regions,and no hydrophobicity.The secondary structure of these two proteins had a similar proportion ofα-helix andβ-sheet,but the tertiary structure was quite different.Their conservative structure belonged to lysozyme-like domains,which might be glycosidases containing only globular domain.LysWL59 and LysWL60 require the holin or spanin protein Rz for"lysis from within".2.The optimal expression condition for LysWL59 was inducing E.coli C41 for 4 h at37°C,while for LysWL60 was inducing E.coli BL21 for 4 h at 30°C.The molecular weight of LysWL59 and LysWL60 were approximately 18 and 11.6 k Da,respectively.3.LysWL59 and LysWL60 had a wide lytic spectrum,and showed the best lytic activity at the concentration of 0.25 and 30μmol/L,respectively.Zn²⁺and high concentration of Mn²⁺（1～10 mmol/L）significantly inhibited the relative lytic activity of LysWL59 and LysWL60.LysWL59 could maintain high activity under the conditions of p H 8.0～10.0,4～90°C,10～500 mmol/L Na Cl,and its thermal stability,Na Cl tolerance,preservation stability and lytic activity were all better than LysWL60.Therefore,LysWL59was more suitable for application in food.4.The duplicate effect of LysWL59 with EDTA was better than malic acid and citric acid,and the optimal combination was 0.25μmol/L LysWL59 with 0.50 mmol/L EDTA,in which Salmonella was reduced by 97.50%within 1 h in the culture medium.When 2.5μmol/L LysWL59 and 0.50 mmol/L EDTA was used in different foods for 1 h,the best bactericidal effect was observed in lettuce,and S.Typhimurium ATCC14028 was reduced by 93.03%,followed by duck intestines.The bactericidal effects on cantaloupe,pork and skimmed milk powder were poor,which may be caused by the complexity of the food matrix.The present study provides insights into the prevention of the infection of S.Typhimurium ATCC14028,establishing a theoretical basis for the development of G^-endolysin products and a reference for solving actual production and application issues.更多还原