Summary: Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate ( PET ) are of interest for several biotechnological applications like waste treatment, biocatalysis and sustainable polymer modifications. Recent studies suggest that a large variety of such enzymes are still to be identified and explored in a variety of microorganisms, including bacteria of the genus Pseudomonas . For activity‐based screening, methods have been established using agar plates which contain nanoparticles of polycaprolactone or PET prepared by solvent precipitation and evaporation. In this protocol article, we describe a straightforward agar plate‐based method using emulsifiable artificial polyesters as substrates, namely Impranil ^® DLN and liquid polycaprolactone diol ( PLD ). Thereby, the currently quite narrow set of screening substrates is expanded. We also suggest optional pre‐screening with short‐chain and middle‐chain‐length triglycerides as substrates to identify enzymes with lipolytic activity to be further tested for polyesterase activity. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the P. pertucinogena lineage originating from contaminated soils and diverse marine habitats.

Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.

Impranil ^® DLN‐SD is a poly(ester‐urethane) (PEU) that is widely used as coating material for textiles to fine‐tune and improve their properties. Since coatings increase the complexity of such plastic materials, they can pose a hindrance for sustainable end‐of‐life solutions of plastics using enzymes or microorganisms. In this study, we isolated Halopseudomonas formosensis FZJ due to its ability to grow on Impranil DLN‐SD and other PEUs as sole carbon sources. The isolated strain was exceptionally thermotolerant as it could degrade Impranil DLN‐SD at up to 50°C. We identified several putative extracellular hydrolases of which the polyester hydrolase Hfor _PE‐H showed substrate degradation of Impranil DLN‐SD and thus was purified and characterized in detail. Hfor _PE‐H showed moderate temperature stability (T _m = 53.9°C) and exhibited activity towards Impranil DLN‐SD as well as polyethylene terephthalate. Moreover, we revealed the enzymatic release of monomers from Impranil DLN‐SD by Hfor_ PE‐H using GC‐ToF‐MS and could decipher the associated metabolic pathways in H . formosensis FZJ. Overall, this study provides detailed insights into the microbial and enzymatic degradation of PEU coatings, thereby deepening our understanding of microbial coating degradation in both contained and natural environments. Moreover, the study highlights the relevance of the genus Halopseudomonas and especially the novel isolate and its enzymes for future bio‐upcycling processes of coated plastic materials.

Lubricants based on fatty acid ester (FAE) mixtures are widely used in the textile industry, e.g., in spin finishes applied during the production of synthetic fibres, or in sizes added to fibres before weaving. FAE lubricants can significantly impact the dyeing quality of a textile due to their hydrophobicity and must therefore be removed before dyeing. However, the solvents currently used for their removal pose an environmental risk, and biobased solutions are thus sought. A lipase-assisted pre-dyeing treatment for synthetic fibre textiles was developed in this study. Six lipases were tested for their ability to hydrolyse FAEs from a polyamide-with-elastane textile, and all were found to be active. The conditions for the washing of lipase-treated textiles were found to be crucial for the performance of the process. Among the possible lipid hydrolysis products of tripalmitin (selected as a model FAE), only palmitic acid removal improved during washing, in comparison with the original FAE. This improvement only occurred with washing solutions containing a monovalent base. A combination of lipase treatment and washing with a non-ionic surfactant and monovalent base was found to be effective in the removal of FAEs, with a performance similar to a current solvent-based pre-treatment process.

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 p NP-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 LCC ^ICCG 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 × 10 ³ 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.