A versatile Bacillus esterase reveals a carboxylesterase blueprint for PET intermediate degradation.

The biodegradation of polyethylene terephthalate (PET), and its derived intermediate breakdown products, represents a core focus of multidisciplinary research. While Ideonella sakaiensis PETase (IsPETase) and leaf-compost cutinase (LCC) efficiently depolymerize PET, many other PET-active hydrolases display stronger activity toward soluble PET intermediates such as bis- and mono(2-hydroxyethyl) terephthalate (BHET and MHET). Here, we describe a versatile carboxylesterase derived from Bacillus halotolerans (BhEstB). BhEstB is structurally related to BsEstB from B. subtilis but divergent from canonical PETases and most characterized MHETases, including IsMHETase, while clustering more closely with carboxylesterases such as TfCa. BhEstB displayed broad esterase activity toward pNP-esters, with maximum activity at pH 8 and 20-30 °C. Although it showed modest overall activity on the bulk polymer, BhEstB significantly outperformed the engineered variant LCCICCG and the recently published archaeal feruloyl esterase PET46 in turning over BHET and MHET. BhEstB exhibits remarkably high activity toward BHET and MHET, achieving complete conversion of 50 mM BHET within 1 h at 30 °C (kcat = 15.57 ± 1.25 s⁻1, Km = 6.54 ± 1.01 mM, corresponding to a catalytic efficiency of 2.38 × 103 M⁻1 s⁻1) and subsequent efficient turnover of 39.8 mM MHET (77% (± 0.7%)) into terephthalic acid (TPA) within 24 h. These results identify BhEstB as one of the most efficient BHET-hydrolyzing enzymes characterized to date. Collectively, these findings establish BhEstB as a versatile BHET/MHET active Bacillus carboxylesterase with modest activity toward commercial semi-crystalline PET powder, contributing to the expanding diversity of PET-intermediate-degrading enzymes. KEYPOINTS: • BhEstB exhibits structural divergence from canonical PET esterases. • BhEstB efficiently degrades significant amounts of BHET and MHET at 30 °C. • BhEstB demonstrates moderate activity on PET powder at elevated temperatures.