A PHA depolymerase from Cellulosimicrobium funkei: Discovery, biochemical characterisation and plastic degradation.

Polyhydroxyalkanoates are biodegradable microbial polyesters considered promising alternatives to petroleum-based plastics. However, the development of sustainable end-of-life strategies for polyhydroxyalkanoates requires the identification of efficient depolymerising enzymes. Here, an extracellular short-chain-length PHA depolymerase, termed CfPhaZ, was identified through metagenomic mining of polyester-enriched microbiomes. The enzyme was assigned to a Cellulosimicrobium funkei metagenome-assembled genome and expressed in Escherichia coli. CfPhaZ shows the canonical architecture of extracellular PHA depolymerases. Biophysical analyses demonstrated that the enzyme adopts a monomeric α/β-fold structure with a melting temperature of 54 °C. The hydrolytic activity of CfPhaZ toward poly(3-hydroxybutyrate) was validated by zymographic and spot-test assays and quantitatively characterised through direct HPLC-based quantification of 3-hydroxybutyrate release. CfPhaZ displayed optimal activity at pH 6.5 and 40 °C, while maintaining prolonged thermostability under mesophilic conditions. Fluorescence-based adsorption assays demonstrated a strong interaction between the enzyme and the substrate surface. Kinetic analyses were performed using both conventional and inverse Michaelis-Menten approaches, and a surface-area-based kinetic framework was implemented to better describe the heterogeneous nature of polymer hydrolysis. Furthermore, CfPhaZ efficiently degraded industrially relevant bioplastic films, reaching up to 40% conversion into soluble monomers. Overall, this study expands the current knowledge of extracellular PHA depolymerases from Gram-positive bacteria and highlights the potential of CfPhaZ for enzymatic recycling and upcycling of biodegradable plastics.