De novo design of a polycarbonate hydrolase.

Abstract

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha /\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha /\beta $-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. With the help of Rosetta an active site catalysing the hydrolysis of polycarbonate is introduced into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in Escherichia coli. Thermal analysis suggests that the design has a melting temperature of >95$^{\circ }$C. Activity toward polycarbonate was confirmed using atomic force spectroscopy (AFM), proving the successful design of a PCase.