Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.

Petroleum-based plastics are durable and accumulate in all ecological niches. Knowledge on enzymatic degradation is sparse. Today, less than 50 verified plastics-active enzymes are known. First examples of enzymes acting on the polymers polyethylene terephthalate (PET) and polyurethane (PUR) have been reported together with a detailed biochemical and structural description. Furthermore, very few polyamide (PA) oligomer active enzymes are known. In this article, the current known enzymes acting on the synthetic polymers PET and PUR are briefly summarized, their published activity data were collected and integrated into a comprehensive open access database. The Plastics-Active Enzymes Database (PAZy) represents an inventory of known and experimentally verified enzymes that act on synthetic fossil fuel-based polymers. Almost 3000 homologs of PET-active enzymes were identified by profile hidden Markov models. Over 2000 homologs of PUR-active enzymes were identified by BLAST. Based on multiple sequence alignments, conservation analysis identified the most conserved amino acids, and sequence motifs for PET-and PUR-active enzymes were derived.

Global economies depend on the use of fossil‐fuel‐based polymers with 360–400 million metric tons of synthetic polymers being produced per year. Unfortunately, an estimated 60% of the global production is disposed into the environment. Within this framework, microbiologists have tried to identify plastic‐active enzymes over the past decade. Until now, this research has largely failed to deliver functional biocatalysts acting on the commodity polymers such as polyethylene (PE), polypropylene (PP), polyvinylchloride (PVC), ether‐based polyurethane (PUR), polyamide (PA), polystyrene (PS) and synthetic rubber (SR). However, few enzymes are known to act on low‐density and low‐crystalline (amorphous) polyethylene terephthalate (PET) and ester‐based PUR. These above‐mentioned polymers represent >95% of all synthetic plastics produced. Therefore, the main challenge microbiologists are currently facing is in finding polymer‐active enzymes targeting the majority of fossil‐fuel‐based plastics. However, identifying plastic‐active enzymes either to implement them in biotechnological processes or to understand their potential role in nature is an emerging research field. The application of these enzymes is still in its infancy. Here, we summarize the current knowledge on microbial plastic‐active enzymes, their global distribution and potential impact on plastic degradation in industrial processes and nature. We further outline major challenges in finding novel plastic‐active enzymes, optimizing known ones by synthetic approaches and problems arising through falsely annotated and unfiltered use of database entries. Finally, we highlight potential biotechnological applications and possible re‐ and upcycling concepts using microorganisms.

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.