What question did this study set out to answer?

This research aims to enhance the thermal stability of enzymes for increased microbial production of chemicals by developing enzyme complexes with thermostable chaperone tags.

June 1, 2026

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

Key Points

This research aims to enhance the thermal stability of enzymes for increased microbial production of chemicals by developing enzyme complexes with thermostable chaperone tags.
Developed enzyme complexes via SpyTag/SpyCatcher conjugation of target enzymes and thermostable chaperone tags in vitro.
Assessed the stability of the enzyme complexes through zeta potential alterations and thermal stability tests.
Maximum increase in ΔT5030 by 7.7 °C for PGI-tagged enzymes.
PGI demonstrated greater stability for multimeric proteins compared to other chaperone tags.
Nitrile hydratase with PGI tag achieved 99% acrylamide yield at 42 °C versus 50% for wild-type.

Abstract

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 ΔT5030 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.

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