Thermophilic carboxylesterases from hydrothermal vents of the volcanic island of Ischia active on synthetic and biobased polymers and mycotoxins

Hydrothermal vents have a widespread geographical distribution and are of high interest for investigating microbial communities and robust enzymes for 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 (Geobacillus and Brevibacillus spp.), Proteobacteria and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate (PHB) and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus spp., and to some extent, Fontimonas and Schleiferia spp. The screening at 37-70°C of metagenomic fosmid library from above enrichment cultures resulted in identification and successful production in Escherichia coli of three hydrolases (IS10, IS11 and IS12), all derived from yet uncultured Chloroflexota and showing low sequence identity (33-56%) to characterized enzymes. Enzymes exhibited maximal esterase activity at temperatures 70-90°C, with IS11 showing the highest thermostability (90% activity after 20 min incubation at 80°C). IS10 and IS12 were highly substrate-promiscuous and hydrolysed all 51 monoester substrates tested. Enzymes were active with polyesters (PLA and polyethylene terephthalate model substrate, 3PET) and mycotoxin T-2 (IS12). IS10 and IS12 had a classical α/β hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in the hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of the N-terminal β-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 includes catalytic residues Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain encloses the catalytic cleft like a lid contributing to substrate binding. Thus, this study has identified novel thermotolerant carboxylesterases with a broad substrate range including polyesters and mycotoxins for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications including recycling of synthetic and biobased polyesters increasingly used in textiles, fibres, coatings and adhesives. Here, we have discovered three novel thermotolerant carboxylesterases (IS10, IS11 and IS12) from high-temperature enrichment cultures from the Ischia hydrothermal vents incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest “effective volumes” among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous compared to IS10 and IS12, IS11 had a higher thermostability with high temperature optimum (80-90 °C) for activity, hydrolysed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity in these enzymes makes them attractive candidates for further optimisation and potential application in plastics recycling.


INTRODUCTION
pharmaceutical, cosmetic, detergent, food, textile, paper and biodiesel industries (22,23). Most 117 of known carboxylesterases belong to the large protein superfamilies of α/β hydrolases and β- 118 lactamases and have been classified into 16 families based on sequence analysis (22,24,25). A 119 significant number of these enzymes have been characterised both biochemically and 120 structurally, because they are of high interest for biotechnological applications (22,23,26). 121 Screening of metagenome gene libraries and genome mining has greatly expanded the number 122 of novel carboxylesterases including enzymes active against aryl esters or polymeric esters 123 (polyesterases) (21)(22)(23)26,27). However, the increasing demand for environmentally friendly 124 industrial processes has stimulated research on the discovery of new enzymes and their 125 application as biocatalysts to meet the challenges of a circular bioeconomy (28,29). The global 126 enzyme market is expected to grow from $8.18 billion in 2015 to $17.50 billion by 2024 (28). 127 However, the majority of known enzymes are originated from mesophilic organisms, which 128 have limited stability under harsh industrial conditions including high temperatures, extreme 129 pH, solvents, and salts (30,31). Thus, the discovery of robust enzymes including 130 carboxylesterases and engineering of more active variants represent the key challenges for the 131 development of future biocatalytic processes. Extremophilic microorganisms are an attractive 132 source of industrial biocatalysts, because they evolved robust enzymes that function under 133 extreme conditions (high/low temperatures, high/low pH, salts) (14,26,30,32). In addition, 134 extremophilic enzymes found in one environment are typically also tolerant to other extreme 135 conditions making them attractive biocatalysts for various applications including 136 depolymerization of natural and synthetic polymers (32)(33)(34)(35). 137 Hydrothermal vents are extreme environments located in tectonically active sites, which 138 are classified as terrestrial and marine (deep-sea and shallow-sea) systems (36). Hydrothermal Asn162, as well as numerous hydrophobic residues potentially involved in substrate binding. 155 Structural models of IS10 and IS12 revealed classical α/β hydrolase domains with a catalytic   processed and analysed as previously described (38).

206
All statistical analysis was conducted using R programming environment (39) (Table S2) and T-2 mycotoxin (Merck Life Science S.L.U., Madrid, Spain) was 301 assayed at 37ºC using a pH indicator assay with Phenol Red and monitored at 550 nm (46).

302
The reaction products of enzymatic degradation of T-2 mycotoxin were analysed using 303 reversed phase chromatography on a Waters 600 HPLC system equipped with a Zorbax Eclipse 304 Plus C18 column (Agilent, 4.6 x 100 mm, 3.5 μm, 40ºC) and a light scattering detector (ELSD).

307
Polyester depolymerization activity of purified proteins against 3PET (bis(benzoyloxyethyl) 308 terephthalate) was measured using 1.5% agarose plates containing 0.2% of emulsified and refinement statistics for this structure are summarized in Table S3.  (PHB), polylactic acid (PLA) and commercial polyester blend (Table S1). After two weeks of  motifs linked to esterase activity in lipolytic families previously described ( Fig. S2 and S3). S-x-G around the active site serine (22), clustering together with representatives of family IV 454 ( Fig. S2 and S3).

455
The protein IS11 contained a β-lactamase domain (PF00144) and the consensus tetrapeptide S-456 x-x-K, perfectly conserved among all penicillin-binding enzymes and β-lactamases, 457 surrounding the active serine Ser68. In addition, Lys71 and Tyr160 were also conserved as part 458 of the catalytic triad of family VIII esterases, which groups enzymes with homology to class C 459 β-lactamases and penicillin-binding proteins ( Fig. S2 and S3). hexanoate, whereas IS12 was most active with p-NP-acetate and αN-propionate ( Figure 2). All 467 enzymes were active within a broad pH range (pH 6.0-10.0) with maximal activities at pH 9

468
(data not shown). The purified metagenomic carboxylesterases exhibited saturation kinetics 469 with model esterase substrates at optimal pH (9.0) and 30 ºC ( to be more active against the tested model substrates compared to IS11.

475
Since the selected carboxylesterases originated from thermophilic environments, we 476 investigated the effect of temperature on the activity (temperature profiles) and thermostability 477 of purified carboxylesterases using p-NP-butyrate as substrate (Fig. 3). All enzymes showed 478 significant activity at 20ºC, but reaction rates increased 5-10 times at higher temperatures with

521
The purified metagenomic esterases were also tested for hydrolytic activity against the T- against T-2 was confirmed using HPLC, which also revealed the formation of different reaction 532 products (Fig. 5). IS10 produced HT-2 as the main product, whereas HT-2 was present as the 533 minor product in the reaction mixture with IS12, which produced mostly the T-2 triol as the 534 main product (Fig. 5). Since the T-2 triol is known to be less toxic than T-2 and HT-2 (64), 535 IS12 might represent a promising candidate for the biodetoxification of T-2 and HT-2.

579
The C-terminal domain of IS11 represents a typical lipocalin fold with one α-helix and an 580 eight-stranded antiparallel β-barrel containing a hydrophobic core (Fig. 10). Lipocalins are a 581 diverse family of small individual proteins or domains (160-180 aa), which bind various 582 hydrophobic molecules (e.g. fatty acids) in a binding pocket located inside the barrel (71).

583
Although lipocalins are very divergent in their sequences and functions, their structures exhibit 584 remarkable similarity. The lipocalin α-helix of IS11 closes off the top of the β-barrel, whose 585 interior represents a ligand-binding site coated mostly with hydrophobic residues (Fig. 10) participates in substrate binding, rather than in the oligomerization.

601
High-quality structural models of IS10 and IS12 proteins constructed using the Phyre2 602 server (Fig. S8) revealed the presence of a core domain with a classical α/β hydrolase fold and 603 an all-helical domain, as well as a serine hydrolase catalytic triad (Ser155, His280, and Asp250 604 in both proteins) (Fig. S9). The putative catalytic nucleophile Ser155 is located on the classical 605 nucleophilic elbow, a short sharp turn between a β-strand and α-helix. It is located at the bottom 606 of the active site, which is mostly covered by the all-helical lid domain (Fig. S9)           .7 x 10 4 a Reaction conditions were as indicated in Materials and Methods (pH 9.0, 30ºC). Results are mean ± SD of three independent experiments. αN = α-naphthyl, pNP = p-nitrophenyl.