BackgroundEnterococcus faecium(E. faecium) is a member of symbiotic lactic acid bacteria in gastrointestinal tract and it was successfully used to treat diarrhea cases in humans. For a lactobacteria to survive during the pasteurization process, resistance of proteins to denaturation at high temperatures is crucial. Pyruvate kinase (PYK) is one of the proteins possessing such property. It plays a major role during glycolysis by producing pyruvate and adenosine triphosphate (ATP).ObjectiveTo assess the acquired thermostability of PYK of ALE strain usingin silicomethods.MethodsFirst, we predicted and assessed tertiary structures of our proteins using SWISS-MODEL homology modelling server. Second, we then applied molecular dynamics (MD) simulation to simulate and assess multiple properties of molecules. Therefore, we implemented comparative MD to evaluate thermostability of PYK of recently developed high temperature resistant strain ofE.faeciumusing Adaptive Laboratory Evolution (ALE) method. After 20ns of simulation at different temperatures, we observed that ALE enhanced strain demonstrated slightly better stability at 300, 340 and 350 K compared to that of the wild type (WT) strain.ResultsWe collected the results of MD simulation at four temperature points: 300, 340, 350 and 400 K. Our results showed that the protein demonstrated increased stability at 340 and 350 K.ConclusionResults of these study suggest that PYK of ALE enhanced strain ofE. faeciumdemonstrates overall better stability at elevated temperatures compared to that of WT strain.