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[EN] When bioprospecting for novel industrial enzymes, substrate promiscuity is a desirable property that increases the reusability of the enzyme. Among industrial enzymes, ester hydrolases have great relevance for which the demand has not ceased to increase. However, the search for new substrate promiscuous ester hydrolases is not trivial since the mechanism behind this property is greatly influenced by the active site's structural and physicochemical characteristics. These characteristics must be computed from the 3D structure, which is rarely available and expensive to measure, hence the need for a method that can predict promiscuity from sequence alone. Here we report such a method called EP-pred, an ensemble binary classifier, that combines three machine learning algorithms: SVM, KNN, and a Linear model. EP-pred has been evaluated against the Lipase Engineering Database together with a hidden Markov approach leading to a final set of ten sequences predicted to encode promiscuous esterases. Experimental results confirmed the validity of our method since all ten proteins were found to exhibit a broad substrate ambiguity., This study was conducted under the auspices of the FuturEnzyme and Oxipro Projects funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327 and 101000607. We also acknowledge financial support under Grants PID2020-112758RB-I00 (M.F.), PID2019-106370RB-I00 (V.G.) and PDC2021-121534-I00 (M.F.) and PID2019-106370RB-I00/AEI/10.13039/501100011033 (A.R.-M.), from the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (Digital Object Identifier 10.13039/501100011033), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (“NextGen-erationEU/PRTR”), and Grant 2020AEP061 (M.F.) from the Agencia Estatal CSIC.

[EN] Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and in silico metagenomic approaches, we retrieved 16 esterases from the α/β-hydrolase fold superfamily with the closest match from uncultured acidophilic Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium, and Ferrimicrobium), Acidiphilium, and other Proteobacteria inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in Escherichia coli. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33–68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30–65°C, retaining 20–61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U/g at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system., This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. We acknowledge financial support under Grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET, GA No. 604814) (MF), BIO2017-85522-R (MF), PID2020-112758RB-I00 (MF), PID2019-106370RB-I00 (VG), and PDC2021-121534-I00 (MF) from the Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (Doi: 10.13039/501100011033), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (“NextGenerationEU/PRTR”), and Grant 2020AEP061 (MF) from the Agencia Estatal CSIC. OG acknowledges the support of the Centre for Environmental Biotechnology Project funded by the European Regional Development Fund (ERDF) via the Welsh Government. SR thanks the Spanish Ministry of Science and Innovation for a Ph.D. fellowship (FPU19/00608).

Conventional piglets were inoculated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) through different routes, including intranasal, intratracheal, intramuscular and intravenous ones. Although piglets were not susceptible to SARS‐CoV‐2 and lacked lesions or viral RNA in tissues/swabs, seroconversion was observed in pigs inoculated parenterally (intramuscularly or intravenously).