Identification and characterisation of a novel polyesterase from Coniochaeta pulveracea CAB 683 and its synergistic potential in bioplastic hydrolysis

The efficient end-of-life management of biodegradable plastics remains a major challenge for sustainable polymer adoption, particularly for heterogeneous blends that are difficult to depolymerise. Here, we report the identification and functional characterisation of CPEst2, a novel polyester-degrading lipase discovered through targeted proteome mining of the South African white-rot fungus Coniochaeta pulveracea CAB 683. A profile Hidden Markov Model (HMM), generated from functionally validated fungal carboxyl ester hydrolases, was used to screen the fungal proteome, leading to the identification of two putative polyesterases, CPEst1 and CPEst2. Heterologous expression in Saccharomyces cerevisiae confirmed exclusive extracellular production and polyesterase activity of CPEst2, a predicted 32.6 kDa α/β-hydrolase containing a conserved catalytic triad (S171-D225-H284). Structural modelling revealed high similarity to the Gibberella zeae lipase GZEL and suggested a putative "lockhole-hinge-lockpin" activation mechanism. Biochemical characterisation demonstrated broad substrate specificity toward short- and medium-chain p-nitrophenyl esters, with optimal activity at pH 7.0 and 40 °C. CPEst2 completely hydrolysed poly(ε-caprolactone) (PCL) nanoparticle emulsions within 60 min and caused 30% mass loss in PCL films after 168 h, releasing 0.94 g/L 6-hydroxyhexanoic acid. FT-IR, DSC, GPC, HPLC, and SEM analyses confirmed extensive polymer degradation. Importantly, CPEst2 acted synergistically with a recombinant cutinase-like enzyme (CLE) on commercial thermoplastic starch-containing bags, significantly enhancing polymer weight loss and monomer release. These findings highlight CPEst2 as a promising biocatalyst for sustainable enzymatic depolymerisation and valorisation of heterogeneous bioplastic materials.