Abstract: 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 (rSEM) were identified by circular dichroism (CD), fluorescence spectroscopy and sodium dodecyl sulfate/native-polyacrylamide gel electrophoresis (SDS/native-PAGE). Below 40 ℃, rSEM had a well-folded structure with high contents of α-helix (17%), β-sheet (32%) and β-turn (21%) and the single tryptophan residue at position 121 (Trp121) on its molecular surface was found be located in the hydrophobic environment of the β-grasp domain. As the heating temperature increased from 42 to 55 ℃, α-helix content decreased, and β-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 flexible β-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 the β-sheet/turn structure of rSEM and the formation of IS and the aggregation state were predominantly responsible for the structural stability at high temperature. Understanding the heat-induced unfolding process of rSEM will help in clarifying its heat resistance mechanism. In the future, using this method to study the heat inactivation mechanism of other types of staphylococcal enterotoxin will help in improving the food production process.

Key words: staphylococcal enterotoxin M; circular dichroism; fluorescence; sodium dodecyl sulfate/native polyacrylamide gel electrophoresis ; heat inactivation