Identification and characterization of R75estA, a halophilic polyester-degrading enzyme from a marine bacterium

Polyester-based plastics, particularly polyethylene terephthalate (PET), represent a major fraction of global plastic waste, and their enzymatic recycling has attracted increasing attention as a sustainable alternative to conventional thermomechanical and chemical methods. However, the repertoire of characterized polyester-degrading enzymes, and particularly of the recently proposed type III PETases, remains limited. Here, we report the identification and biochemical characterization of R75estA, a polyesterase from Stutzerimonas sp. R75, isolated from the Black Sea during bioprospecting for polyester-degrading bacteria. Phylogenetic analysis placed R75estA as the first characterized member of a clade of Stutzerimonas and Pseudomonas homologs, while AlphaFold-based structural analysis predicted a canonical α/β-hydrolase fold with sequence and active-site features shared with type III PETases such as PmC, HaloPETase1, and dsPETase05. Biochemical characterization of the purified recombinant enzyme showed a halophilic, alkaliphilic, and moderately thermoactive hydrolase, with optimal activity at 3 M NaCl, pH 8.0, and 50 °C. R75estA hydrolyzed p-nitrophenyl esters of varying chain lengths, olive oil, polycaprolactone, bis(2-hydroxyethyl) terephthalate (BHET), and PET microparticles. To our knowledge, R75estA is the first polyesterase characterized from the genus Stutzerimonas and from the Black Sea, expanding the known sequence, structural, and ecological diversity of type III PETases.