Protein-engineered leaf and branch compost cutinase variants using computational screening and IsPETase homology

Abstract

Polyethylene terephthalate (PET) is a popular material used in plastic products, particularly in food-packaging. However, its waste is environmentally hazardous and current industrial recycling methods are energy-intensive and can cycle mechanically weaker plastic products over time. A green solution is to employ PET-hydrolyzing enzymes (PHE). Leaf and branch compost cutinase (LCC) has been identified as the best-performing naturally occurring PHE at temperatures above the glass transition temperature, Tg of PET while PETase, identified from Ideonella sakaiensis 201-F6 (IsPETase), has exhibited the best performance at moderate temperatures (< 45 °C). Using a high-throughput computational approach, we have screened for single-substitution variants for improved hydrolysis activity against PET using Rosetta interface energy scores as a target metric. Homology alignment of IsPETase was employed to generate an LCC variant that possessed a double cysteine mutation, characteristic of a unique disulfide bond in IsPETase, to study the impact on moderate temperature hydrolysis. A resulting point-mutation variant from our computational screening method and the IsPETase homology variant of LCC led to improved hydrolysis activity at 70 °C while the latter also possessed improved hydrolysis activity at moderate temperatures of 30 °C – 50 °C, demonstrating that a computational screening workflow and homology alignment between high-performing PHEs are promising strategies to improve hydrolysis activity.