Improving the thermostability of trehalose synthase from Thermomonospora curvata by covalent cyclization using peptide tags and investigation of the underlying molecular mechanism

One of the most desirable properties for industrial enzymes is high thermotolerance, which can reduce the amount of biocatalyst used and lower the production cost. Aiming to improve the thermotolerance of trehalose synthase (TreS, EC 5.4.99.16) from Thermomonospora curvata, four mutants (G78D, V289L, G322A, I323L) and four cyclized TreS variants fused using different Tag/Catcher pairs (SpyTag-TreS-SpyCatcher, SpyTag-TreS-KTag, SnoopTag-TreS-SnoopCatcher, SnoopTagJR-TreS-DogTag) were constructed. The results showed that cyclization led to a much larger increase of thermostability than that achieved via site-directed mutagenesis. The t_1/2 of all four cyclized TreS variants at 55 °C increased 2- to 3- fold, while the analysis of kinetic and thermodynamic stability indicated that the T₅₀ of the different cyclized TreS variants increased by between 7.5 °C and 15.5 °C. Molecular dynamics simulations showed that the Rg values of cyclized TreS decreased significantly, indicating that the protein maintained a tight tertiary structure at high temperatures, avoiding exposure of the hydrophobic core to the solvent. Cyclization using a Tag/Catcher pair is a simple and effective method for improving the thermotolerance of enzymes.