Reinforcing thermostability and pH robustness of exo-inulinase facilitated by ReverseTag/ReverseCatcher tagging system

Enhancing protein stability is pivotal in the field of protein engineering. Protein self-cyclization using peptide a tagging system has emerged as an effective strategy for augmenting the thermostability of target proteins. In this study, we utilized a novel peptide tagging system, ReverseTag/ReverseCatcher, which leverages intramolecular ester bond formation. Initially, we employed GFP as a model to validate the feasibility of cyclization mediated by ReverseTag/ReverseCatcher in improving the protein thermostability. Cyclized GFP (cGFP) retained 30 % of its relative fluorescence after a 30-min incubation at 100 °C, while both GFP and linear GFP (lGFP) completely lost their fluorescence within 5 min. Additionally, we applied this method to exo-inulinase (EXINU), resulting in a variant named cyclized EXINU (cEXINU). The T₅₀ and t_1/2 values of cEXINU exhibited significant enhancements of 10 °C and 10 min, respectively, compared to EXINU. Furthermore, post-cyclization, EXINU demonstrated a broad operational pH range from 5 to 10 with sustained catalytic activity, and cEXINU maintained a half-life of 960 min at pH 5 and 9. Molecular dynamics simulations were conducted to elucidate the mechanisms underlying the enhanced thermostability and pH robustness of EXINU following cyclization. This study highlights that cyclization substanitially enhances the stability of both highly stable protein GFP and low-stable protein EXINU, mediated by the ReverseTag/ReverseCatcher tagging system. The ReverseTag/ReverseCatcher tagging system proves to be a potent conjugation method, with potential applications in improving thermostability, pH robustness, and other areas of protein engineering.