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Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. Hightemperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical a/ b-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal b-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. [ABSTRACT FROM AUTHOR]

Amination of bulky ketones, particularly in (R)-configuration, is an attractive chemical conversion, however known ω-transaminases (ω-TAs) show insufficient performances. By applying two screening methods we discovered 10 amine transaminases from the class III ω-TAs family, 38-76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (S)-selectivity. We also report representatives from the class III ω-TAs capable of converting (R) and (S) amines and bulky ketones, and one converting amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (R)-amines. This configuration was also found to favor the utilization of putrescine as amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 contributes also to the preference of amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained = 50% of their activity in water-soluble solvents (up to 50% (v/v)). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (R)-amines for production of high value products, and screen those converting putrescine. [ABSTRACT FROM AUTHOR]

The shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (≤52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (≤356Umg-1) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities. [ABSTRACT FROM AUTHOR]

The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-β-glucanases, β-glucosidases, β-cellobiohydrolases, β-galactosidase, and β-xylosidases of known Gil families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of β-galactosidases/α-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active. site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Aiphaproteobacteria (Rhizobiales). [ABSTRACT FROM AUTHOR]