Reversible peptide tagging system to regulate enzyme activity

Peptide tagging systems are widely used across scientific fields. Here, to obtain a peptide tagging system with high covalent linkage efficiency, we rationally designed the ReverseTag/ReverseCatcher system, building upon the ester-bond-based peptide tagging system Cpe0147 439 −563 /Cpe0147 565−587 . Our optimized system exhibits a second-order rate constant of 1.92 ± 0.002 × 10 4 M −1 s −1 , a 21.7-fold increase over the wild-type system. The ester bond between ReverseTag and ReverseCatcher demonstrates exceptional mechanical stability, withstanding forces over 2 nN, while remaining hydrolyzable under alkaline conditions with significant energy input. To leverage this for enzymatic catalysis, we introduced a T11S mutation into ReverseCatcher, allowing pH-controlled reversible bond formation and cleavage. Applying this strategy to exo -inulinase (EXINU) enables precise, pH-dependent enzyme activity control. This sustainable method for modulating enzyme-catalyzed reactions offers broad biotechnological potential.