High-Throughput Screening Identifies Anionic Polymer Supports that Improve Enzyme Activity at Low pH and High Temperature

Elevated temperatures and nonoptimal pH can destabilize enzyme structure or change the protonation state of catalytic residues resulting in attenuated catalytic performance. Enzyme immobilization on polymer supports enables the fine-tuning of highly varied vicinal chemistries to improve enzyme performance by promoting correctly folded enzyme structure and adjusting the local microenvironment to more favorable conditions. Herein, we sought to investigate how multicomponent random copolymer brushes composed of monomers with anionic, cationic, neutral (zwitterionic, and mixed-charge), and aromatic properties stabilize covalently tethered lipase A from<i>Bacillus subtilis</i> at low pH and high temperature. Polymer brush compositions were screened using a high-throughput approach involving the combinatorial synthesis of random copolymer brushes and in situ characterization of immobilized lipase function. Although cationic supports provided a modest improvement over soluble lipase in maximum activity and thermal stability at low pH, more substantial enhancements in lipase stability were observed for anionic and neutral zwitterionic polymer supports, resulting in increases in temperature optima as great as 40 °C (from 40 to 80 °C) and an increase in maximum activity by more than 300%. These observations were counter to expectations regarding the role of surface charge on local pH and were attributed instead to the preservation of enzyme structure due to stabilizing electrostatic interactions between negatively charged polymer moieties and the net positively charged surface of lipase A. Our findings suggest that the stabilization of enzyme structure by charged polymers may offset unfavorable local changes in pH in certain situations, while in other situations these effects may be synergistic.