Reduced Enzyme Dynamics upon Multipoint Covalent Immobilization Leads to Stability-Activity Tradeoff

The successful incorporation of enzymes into materials through multipoint covalent immobilization (MPCI) has served as the foundation for numerous advances in diverse fields, including biocatalysis, biosensing, and chemical weapons defense. Despite this success, a mechanistic understanding of the impact of this approach on enzyme stability has remained elusive, which is critical for realizing the full potential of MPCI. Here, we showed that the stabilization of lipase upon MPCI to polymer brush surfaces resulted from the rigidification of the enzyme with an increase in the number of enzyme-brush attachments. This was evident by a 10-fold decrease in the rates of enzyme unfolding and re-folding as well as a reduction of the intrinsic fluctuations of the folded and unfolded states, which was measured by single-molecule (SM) Forster Resonance Energy Transfer imaging. Moreover, our results illuminated an important tradeoff between stability and activity as a function of this decrease in structural dynamics of...