Rational mutagenesis reveals substrate-specific activity shifts in the Antarctic polyesterase MoPE.

Abstract

Despite rapid advances in biocatalytic polyester depolymerization, enzyme discovery and engineering remain largely focused on polyethylene terephthalate, leaving the molecular basis of activity towards increasingly used biodegradable polyesters poorly understood. Building on the broad substrate range of the polyesterase from the Antarctic bacterium Moraxella sp. TA144 (MoPE), we investigated the effect of protein engineering on the activity and degradation profile towards structurally distinct biodegradable polyesters, including poly(butylene adipate-co-terephthalate) (PBAT), polycaprolactone (PCL), and poly(3-hydroxybutyrate) (PHB). Guided by comparative structural analysis of benchmark polyesterases, 13 MoPE variants were designed and selected mutants were screened against the polyester substrates by quantifying soluble hydrolysis products. All expressed variants retained conventional esterolytic activity, although their relative activity ranking was not maintained against their performance on the polymeric substrates. Rather than universally improving depolymerization efficiency, the substitutions induced substrate-dependent shifts in product formation. In PBAT hydrolysis, Y214W altered the product profile towards the aliphatic fraction, increasing adipic acid release by 1.9-fold, whereas V122T and Y214W-F246I enhanced the formation of the PCL- and PHB-derived monomers by 15- and 2.2-fold, respectively. Molecular docking suggested that the observed activity shifts may be associated with mutation-induced changes in polymer accommodation within the catalytic cleft. Overall, this work demonstrates that rational mutagenesis can alter polyesterase activity against structurally distinct biodegradable polyesters, providing potential mechanistic insights to guide the development of specialized biocatalysts for targeted polyester degradation.