A Promiscuous Bacillus Esterase Reveals a Carboxylesterase Blueprint for PET Intermediate Degradation

<title>Abstract</title> <p> The biodegradation of polyethylene terephthalate (PET), a major commodity polymer, has become a core focus of multidisciplinary research. To date, most of the 300 known PETases cluster into only a few evolutionary lineages. While the well-known <italic>Ideonella sakaiensis</italic> PETase ( <italic>Is</italic> PETase) is highly specific for the polymer, and leaf-compost cutinase (LCC) types sequentially cleave the polymer and its subsequent oligomers, most known PET-active enzymes actually prefer monomeric intermediates; thus, they have not evolved as true PETases <italic>sensu strictu</italic> . Here, we describe a novel PET-active enzyme derived from <italic>Bacillus halotolerans</italic> (BhEstB). BhEstB is a highly promiscuous <italic>para</italic> -nitrobenzyl ester hydrolase that is structurally related to BsEstB from <italic>B. subtilis</italic> but divergent from typical PETases, as it lacks the characteristic Trp-Met-Tyr/Phe motif known to facilitate PET binding. While its overall activity on the bulk polymer was only 21% of wild-type LCC (LCC ^wt ), BhEstB significantly outperformed the engineered variant LCC ^ICCG and the recently published archaeal feruloyl esterase PET46 in the turnover of bis- and mono(2-hydroxyethyl) terephthalate (BHET and MHET). BhEstB exhibits remarkably high activity toward BHET and MHET, degrading up to 50 mM of BHET and 38 mM of MHET at 30°C within 1 hour and 24 hours, respectively, with a K _cat of 15.5 +/-1.25 s ^-1 for BHET. These results establish BhEstB as one of the most efficient BHET-hydrolyzing enzymes characterized to date. From a phylogenetic viewpoint, BhEstB serves as a blueprint for an enzyme transitioning from monomeric intermediate processing toward true polymer activity, offering high potential for synergistic formulation in thermophilic plastic upcycling. </p>