Grafted-from Polymer Surface Display: A Platform for In Situ Engineering of Robust and Recoverable Biocatalysts

Protein-polymer conjugation and microbial surface display are two dominant strategies for enzyme engineering, yet they have evolved in isolation, restricted by distinct limitations in purification or stability. Here, we bridge this divide by introducing "grafted-from surface display," a platform that enables in situ atom transfer radical polymerization (ATRP) directly on surface-displayed proteins without prior and post purification. Using Escherichia coli displaying a cysteine-engineered benzaldehyde lyase (sBAL^S174C), we grew thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) polymers that significantly enhanced the surface-displayed enzyme's robustness, retaining >60% activity at 50 °C where unmodified variants lost substantial function. This architecture preserves cell viability and allows for the reversible, temperature-controlled recovery and purification of membrane-bound biocatalysts via simple centrifugation. In a multienzyme cascade with alcohol dehydrogenase, the conjugates achieved a benzoin yield of 15 mM and retained over 80% activity after four recycling cycles. This strategy establishes a facile framework for integrating synthetic polymer chemistry with cellular machinery, creating robust, self-recovering biocatalysts while eliminating the need for laborious protein isolation.