Thermostable Chaperone Tag Fusion In Vitro: Development of TST-TE Complexes for Enhanced Enzyme Thermal Stability

Enzyme thermostability often limits microbial production of high-value chemicals. We developed enzyme complexes by conjugating promising target enzymes (TEs) with Thermostable Chaperone Tags (TSTs) in vitro via SpyTag/SpyCatcher, aiming to improve thermal stability. All tested enzymes (PnUDH, BaxA, PAP, and CbFDH) exhibited improved thermostability. PGI was identified as a universal and efficient tag, with a maximum increase in Δ$T_{50}^{30}$ of 7.7 °C. TST conjugation enhances thermostability by increasing electrostatic repulsion to prevent aggregation, consistent with zeta potential alterations and stability data. Notably, PGI stabilized multimeric proteins more effectively. Additionally, SpyTag/SpyCatcher-mediated covalent conjugation generated complexes of varying sizes, demonstrating that larger complexes possess higher thermostability. Applying the PGI-based strategy to nitrile hydratase (NHase) enabled a 99% acrylamide yield at 42 °C, compared to 50% for the wild-type. This work developed a TST-based thermostabilization strategy that holds significant potential for industrial applications, offering a straightforward approach to enhance enzyme stability in practical settings.