热诱导潜在食物中毒威胁源重组M型金黄色葡萄球菌肠毒素去折叠及聚合过程  

Thermal Unfolding and Aggregation Process of Recombinant Staphylococcal Enterotoxin M(r SEM)Associated with Potential Toxin Responsible for Staphylococcal Food Poisoning

作　　者：刘骥[1] 田万帆 唐俊妮[1] 陈娟[1] 赵燕英[2] 于基成 LIU Ji;TIAN Wanfan;TANG Junni;CHEN Juan;ZHAO Yanying;YU Jicheng(College of Food Sciences and Technology,Southwest Minzu University,Chengdu 610041,China;College of Animal and Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;Key Laboratory of Biotechnology and Bioresources Utilization,Dalian Minzu University,Dalian 116600,China)

机构地区：[1]西南民族大学食品科学与技术学院,四川成都610041 [2]西南民族大学畜牧兽医学院,四川成都610041 [3]大连民族大学生物技术与资源利用教育部重点实验室,辽宁大连116600

出　　处：《食品科学》2021年第21期32-45,共14页Food Science

基　　金：“十三五”国家重点研发计划重点专项(2018YFD0500500);四川省科技计划项目(2019YJ0261,2019JDJQ0017);生物技术与资源利用教育部重点实验室开放课题(KF2020008);中央高校基本科研业务费专项资金项目(2016NZYQN37)。

摘　　要：热失活工艺是降低加工食品中潜在的细菌和毒素含量的有效手段。M型金黄色葡萄球菌肠毒素(staphylococcal enterotoxins M,SEM)归属于V型超抗原,具有较弱的催吐活性,是一种潜在的金黄色葡萄球菌所致食物中毒威胁源。本研究利用圆二色光谱、荧光光谱、变性/活性聚丙烯酰胺凝胶电泳监测重组M型金黄色葡萄球菌肠毒素(recombinant staphylococcal enterotoxin M,rSEM)热诱导变性过程。结果表明,在低于40℃时,rSEM具有富含α-螺旋(17%)、β-折叠(32%)、转角(21%)的天然折叠态结构,分子表面唯一的121号色氨酸残基位于β-掌型结构域中相对疏水的环境中。随着温度从42℃升高到55℃,rSEM二级结构中减少的α-螺旋含量被增加的β-折叠/转角含量所弥补,蛋白质分子之间聚集状态未发生明显改变,最大荧光发射波长明显蓝移,同时350 nm和340 nm波长处的荧光强度比表现出反S型曲线,说明42~55℃温和加热可导致另一种形式的rSEM折叠中间态(intermediate state,IS)形成。在55~90℃温度范围内,rSEM二级结构基本维持稳定。与此同时,当加热温度从65℃升高至80℃时,350 nm与340 nm波长处的荧光发射强度比并未显示出蛋白处于完全变性状态,说明在加热过程中rSEM二级结构基本维持稳定,而三级结构具有较大动态变化的可能性,这可能与β-掌型结构域可以通过诱导契合结合具有不同构象的主要组织性相容复合体等位基因产物有关系。此外,在70℃乃至更高的温度,rSEM的聚集程度明显增加并且在90℃时达到最大。综上,rSEM中间态形成、聚合体产生以及稳定的β-折叠/转角结构是该蛋白在高温下依然保持热稳定性的基础。通过对rSEM热诱导去折叠过程的研究有助于阐明rSEM耐热机理,未来利用该方法对其他各类金葡菌肠毒素热失活机制进行类似研究将利于食品安全性生产工艺过程的改善。Thermal processing is one of the most useful tools to reduce the amounts of bacteria and toxins that may potentially be present in processed foods.Staphylococcal enterotoxin M is a newly identified group V superantigen with mild emetic activity and has the potential risk of causing staphylococcal food poisoning.In this study,the heat-induced conformational changes of recombinant staphylococcal enterotoxin M(r SEM)were identified by circular dichroism(CD),fluorescence spectroscopy and sodium dodecyl sulfate/native-polyacrylamide gel electrophoresis(SDS/native-PAGE).Below 40℃,r SEM had a well-folded structure with high contents ofα-helix(17%),β-sheet(32%)andβ-turn(21%)and the single tryptophan residue at position 121(Trp^(121))on its molecular surface was found be located in the hydrophobic environment of theβ-grasp domain.As the heating temperature increased from 42 to 55℃,α-helix content decreased,andβ-sheet/turn contents increased to compensate for this.The aggregation state of the protein did not change markedly,while a distinct blue shift in the fluorescence emission maxima was observed accompanied by the reverse S-shaped curve for the ratio of fluorescence intensities at 350 and 340 nm,indicating the formation of an alternatively folded state,namely the intermediate state(IS).When the temperature was above 55℃,the secondary structure elements persisted even upon heating to 90℃.Meanwhile,upon heating from 65 to 80℃,the ratio of fluorescence intensities at 350 and 340 nm didn’t show that the protein was in the completely unfolded state.These results on well-folded secondary structure and tertiary structure variations imply stable integrated architecture of the protein and that the flexibleβ-grasp domain is responsible for binding to the diverse major histocompatibility complex(MHC)alleles.Besides,with increasing temperature from 70℃,the aggregation level increased visibly and reached its maximum at 90℃.Taken together,all data showed that theβ-sheet/turn structure of r SEM and the formati

关 键 词：M型金黄色葡萄球菌肠毒素 圆二色光谱 荧光光谱 变性/活性聚丙烯酰胺凝胶电泳 热失活 

分 类 号：TS201.6[轻工技术与工程—食品科学]