posted on 2021-06-03, 18:05authored byHéctor Sánchez-Morán, James S. Weltz, Daniel K. Schwartz, Joel L. Kaar
A long-standing
goal in the field of biotechnology is to develop
and understand design rules for the stabilization of enzymes upon
immobilization to materials. While immobilization has sometimes been
successful as a strategy to stabilize enzymes, the design of synthetic
materials that stabilize enzymes remains largely empirical. We sought
to overcome this challenge by investigating the mechanistic basis
for the stabilization of immobilized lipases on random copolymer brush
surfaces comprised of poly(ethylene glycol) methacrylate (PEGMA) and
sulfobetaine methacrylate (SBMA), which represent novel heterogeneous
supports for immobilized enzymes. Using several related but structurally
diverse lipases, including Bacillus subtilis lipase A (LipA), Rhizomucor miehei lipase, Candida rugosa lipase, and Candida antarctica lipase B (CALB), we showed that
the stability of each lipase at elevated temperatures was strongly
dependent on the fraction of PEGMA in the brush layer. This dependence
was explained by developing and applying a new algorithm to quantify
protein surface hydrophobicity, which involved using unsupervised
cluster analysis to identify clusters of hydrophobic atoms. Characterization
of the lipases showed that the optimal brush composition correlated
with the free energy of solvation per enzyme surface area, which ranged
from −17.1 kJ/mol·nm2 for LipA to −11.8
kJ/mol·nm2 for CALB. Additionally, using this algorithm,
we found that hydrophobic patches consisting of aliphatic residues
had a higher free energy than patches consisting of aromatic residues.
By providing the basis for rationally tuning the interface between
enzymes and materials, this understanding will transform the use of
materials to reliably ruggedize enzymes under extreme conditions.