Insights into the Molecular-Level details of betaine interactions with Laccase under various thermal conditions

•The osmolyte showed better protecting effect at higher temperatures.•High temperatures decreased betaine’s number in the enzyme’s first hydration shell.•Betaine reduced the number of ion molecules in the enzyme’s hydration shell.•High temperatures positively affected hydrogen bonds and electrostatic interactions. Laccase is a blue enzyme with a remarkable ability to eliminate environmental pollutants. The use of the eco-friendly enzyme for removing ecological contaminants is extensive. In addition, there are some restrictions like its deactivation in various environmental conditions. Researchers have applied many strategies to improve the Laccase stability in different conditions, but those ways are expensive or demanding. Herein, we used betaine, a co-solvent, to increase the enzyme stability in various temperatures and pHs as an appropriate alternative strategy to improve the Laccase stability. The experimental results showed that the enzyme's stability would increase in the presence of betaine at high temperatures. Although no significant changes were detected for the enzyme secondary structure contents, betaine at high temperatures exhibited a protective effect on the enzyme's alpha-helical contents. The simulation results indicated that betaine and ion molecules could be excluded from the enzyme surface and help Laccase maintain its stability and activity. Increasing the betaine concentration up to 0.5 M increased the Tm value of laccase. The molecular dynamics (MD) simulation results also demonstrated that the enzyme's polar amino acids play essential roles in making hydrogen bonds with betaine molecules. Lys377 was also recognized as the most critical residue for electrostatic interactions with the osmolyte at low and high temperatures. At the same time, the higher temperatures could not considerably affect the electrostatic interactions between the carboxyl group of betaine and the basic residues. The results indicated that the negatively charged amino acids could strengthen electrostatic interactions with betaine at high temperatures. The computational analyses also demonstrated that the hydrated state of betaine would not change even at high temperatures. This study's outcomes could be valuable to researchers who seek to use a more affordable approach to increase Laccase activity in severe environmental conditions.