During the “National Antarctic Research Program” (PNRA16_00105 – A1) in November 2017, artificial structures in stainless steel mounting plastic (polyvinyl chloride, PVC and polyethylene, PE) panels were deployed, through holes in the pack-ice, at −5 and − 20 meters (m) at four different stations of Terra Nova Bay: Road Bay (RB, Lat 74° 41.473’ S, Long 164° 07.125′E), Punta Stocchino (PTS, Lat 74° 41.651’S Long 164° 07.303′E), Tethys Bay (TB, Lat 74° 41.417’S Long 164° 06.303′E), and Amorphous Glacier (AG, Lat 74° 41.234’S Long 164° 02.135′E; Figure 1). Microbial biofilms growing on the panels and samples of surrounding seawater were collected three and 12  months after immersion (Caruso, 2020). Contextually to the sampling, the main physical–chemical parameters of the water (temperature, salinity, dissolved oxygen, pH, and fluorescence) were recorded at each station using a SeaBird 9/11 plus multiparametric probe (Caroppo et al., 2022). At the Zucchelli laboratory¹, microbial biofilms were scraped using a sterile scalpel taking care to avoid contamination. They were in part serially diluted in sterile marine water and inoculated (0.1 mL) on Marine Agar plates (Conda Laboratories, Madrid, Spain), incubated at 4–5°C for 15 days minimum for culturable heterotrophic bacteria (Cappello et al., 2021), and in part stored at −20°C for shipment to and further analyses in microbiological labs in Italy (see below). Seawater samples received the same treatment above, i.e., plating on Marine Agar or storage and delivery to microbiology labs in Italy.

At the arrival in Italy, 2.4 g of frozen biofilm samples were defrosted at 5°C, resuspended in sterile physiological solution (0.9% w/v NaCl) at a concentration of 2 mL per gram of biofilm and inoculated, either directly or serially diluted (10⁻³, 10⁻⁶ and 10⁻⁹), on 16 selective solid culture media (specific media and relative composition are reported in Supplementary Table SA). Before plating, part of the resuspended samples was subjected to a heat pre-treatment of 5 min at 55°C to enrich in spore-forming microorganisms. All media were added with nalidixic acid (25 mg/L; Merck KGaA, Darmstadt, Germany) and cycloheximide (50 mg/L; Merck KGaA) as selective agents to inhibit fast-growing Gram-negative bacteria and fungi, respectively. All the colonies isolated from biofilms and seawater samples plated on Marine Agar plates at Zucchelli station (see above) were also streaked in the above reported conditions.

To test the effect of temperature on microbial isolates, isolation plates were incubated at three temperatures: 4, 20 and 28°C for 1 month. Colonies were morphologically dereplicated, i.e., only one isolate was selected among those showing similar morphology considering color, size, shape, consistency and sporification observed at the stereomicroscope (Bel Photonics). Microscopic observation (Zeiss Primo Star, 40x magnification) followed to distinguish unicellular cocci or rods from filamentous microbes, multicellular structures, spores etc. The strains were also briefly phenotypically characterized according to the standard procedures in use at the CNR-ISP laboratory (Messina, Italy), including Gram staining, oxidase production, colony pigmentation, glucose fermentation and production of proteolytic, glycolytic and phosphatase enzymes (for detailed protocols, see Caruso et al., 2022; Supplementary Materials).

Microbial isolates were also tested for growth in liquid cultures. All liquid cultivation media were always added with 2% w/v artificial sea salt (Haquoss, Aquarialand, Torino, Italy), unless otherwise stated. Working Cell Banks (WCBs) were prepared by suspending microbial cells either in Luria Bertani (LB) broth, added with sterile glycerol at a final concentration of 15% v/v (unicellular bacteria), or in Tryptic Soy Broth (TSB; Conda Laboratories; filamentous actinomycetes) or in Shallow Stationary Culture medium (SSC) (filamentous fungi). SSC in g/L: 10 glucose, 0.198 ammonium tartrate, 2 KH₂PO₄, 0.5 MgSO₄·7 H₂O, 0.1 CaCl₂·2 H₂O, 1 thiamine, added with 1 mL/L trace elements solution after sterilization [in g/L: 15 sodium nitro-acetate, 3 MgSO₄·7 H₂O, 0.5 MnSO₄·H₂O, 0.1 NaCl, 0.1 FeSO₄·7 H₂O, 0.05 CuSO₄·5 H₂O, 0.1 CoSO₄, 0.082 CaCl₂·2 H₂O, 0.1 ZnSO₄·7 H₂O, 0.01 AlK(SO₄)₂·12 H₂O, 0.01 H₂BO₃, 0.01 NaMoO₄] (Hong et al., 2016). WCBs were stored at −80°C. For unicellular bacteria, 0.1 mL of the WCB was inoculated in 10 mL LB broth in 50-mL sterile tubes, incubated at 20°C and under agitation at 180 revolutions per minute (rpm) for 24 h. 1 mL of the enrichment culture (1% inoculum) was transferred into 500-mL baffled Erlenmeyer flasks containing 100 mL of LB broth and incubated at 20°C and 180 rpm for 24 h. In the case of filamentous actinomycetes, 0.5 mL of the WCB was inoculated in 20 mL TSB in 100-mL Erlenmeyer flasks, incubated at 20°C and 180 rpm for 72 h. 5 mL of enrichment culture (5% inoculum) was transferred into 500-mL baffled Erlenmeyer flasks containing 100 mL of TSB, incubated at 20°C and 180 rpm for 120–144 h. For fungi, 0.5 mL of the WCB was inoculated in 20 mL SSC in 100-mL Erlenmeyer flasks, incubated at 20°C and 180 rpm for 96 h. 5 mL of enrichment culture (5% inoculum) was transferred into 500-mL baffled Erlenmeyer flasks containing 100 mL of SSC, incubated at 20°C and 180 rpm for 168–192 h.

As primary screening, isolates were grown on Mannitol Agar Medium (MAM) (in g/L: 20 mannitol, 2 KNO₃, 2 MgSO₄·7 H₂O, 2 Na₂HPO₄, 15 agar) added with 2% w/v artificial sea salt and supplemented with ABTS (5 mM; Merck KGaA) or with azure B (25 mg/L; Merck KGaA). The plates were incubated at 20°C for 2 weeks. Colonies were selected as positive hits by the shifting toward green of plates color in the ABTS assay and by the appearance of a clear degradation halo in azure B-added agar. Secondary screening was conducted on microbial cultures grown in liquid (100 mL) as described above, after separation by centrifugation at 3,220 g for 30 min at 4°C of the biomass (pellet) from the supernatants. Enzyme activity assay was performed on both the supernatant of all the positive strains selected from the primary screening, and on their crude extracts prepared by sonication for 8 cycles of 30 s each on ice, followed by centrifugation at 38,724 g for 45 min at 4°C. Enzymatic activities, assayed using 0.05 mL samples from intracellular and extracellular fractions, were determined in 96-well plates, by using the automated liquid handling system epMotion 5075 (Eppendorf, Hamburg, Germany), on the following substrates (purchased from Merck KGaA): 1 mM ABTS (420 nm, ε_420nm = 36.0 mM⁻¹ cm⁻¹), 1 mM 2,6-dimethoxy phenol (2,6-DMP, 468 nm, ε_468nm = 49.6 mM⁻¹ cm⁻¹), or 10 mM 1,2-dihydroxy benzene (catechol, 410 nm, ε_410nm = 22.6 mM⁻¹ cm⁻¹) in 50 mM sodium acetate, pH 5.0, and 0.05 mM azure B (at 600 nm), 0.05 mM Remazol Brilliant Blue R (RBBR, at 600 nm), or 0.05 mM Reactive Black 5 (RB5, at 600 nm) in 50 mM sodium malonate, pH 4.5, added of 0.1 mM H₂O₂. The 96-well plates were incubated under shaking at 20°C for different times (5 min, 1, 5 and 24 h) and the absorbance at the specific wavelengths was recorded using a microtiter plate reader (Sunrise, Tecan, Switzerland). Controls with only the substrate and the corresponding buffer as negative controls were included. Each sample was evaluated in triplicate. The samples whose activity exceeded the control value were selected and the enzymatic activities were assayed spectrophotometrically at 20°C by monitoring 5 mM ABTS oxidation in 50 mM sodium acetate, pH 5.0 at 420 nm for 5 min.

Enzymatic activity on ABTS, 2,6-DMP and catechol was expressed as mU/mL, using the following equation:

where one laccase activity unit (U) is defined as the amount of enzyme required to oxidize 1 μmol of ABTS, 2,6-DMP or catechol per min at 20°C, ∆Abs/min is the registered absorbance increase per minute, ε is the molar extinction coefficient of the substrate, and Vr and Vs are the total reaction volume and the volume of sample (enzyme) used, respectively. The values were normalized to mU/mg protein, using the total protein concentration of the samples quantified through the Biuret method (Gornall et al., 1949). Decolorization activities on azure B, RBBR and RB5 were expressed as percentage of decolorization, using the following equation:

where Abs_t is the absorbance read at a given time, while Abs_i is the initial basal absorbance of the sample.

M68 was isolated from Marine Agar plates prepared by inoculating a seawater sample collected from station RB at −20 m and phenotypically characterized following the methods described in (Caruso et al., 2022). Salt tolerance of M68 strain was tested either in liquid or in solid culture. 1% of enriched cultures was transferred into 500-mL Erlenmeyer flasks containing 100-mL of LB broth added with increasing NaCl concentrations (from 340 to 5,130 mM), and then incubated at 20°C and under agitation at 180 rpm for 24–48 h. The strain was considered tolerant to the salt concentration when the OD₆₀₀ value reached at least 0.3 in 48 h (in comparison to a sample grown in standard LB broth). Meanwhile, the growth optimum for NaCl was defined in liquid medium comparing the 48 h growth in absence and in presence of different concentrations of salt. Growth curves at different temperatures (4, 20 and 30°C) were recorded as by OD_600nm detection; the optimal temperature was assumed to be when the highest OD₆₀₀ increase was observed. In parallel, M68 strain was grown on LB agar plates added with increasing NaCl concentrations (from 340 to 5,130 mM), and incubated at different temperatures for 24–48 h. Growth was considered as positive when colonies become visible on the culture media within a time interval of 48 h.

M68 strain was grown in LB broth added with 2% w/v artificial sea salt: cells were harvested by centrifugation at 13,030 g for 15 min at 4°C and 500 mg pellet was first washed with 10.3% w/v saccharose and, after centrifugation, resuspended in 0.45 mL of TE buffer (50 mM Tris–HCl, pH 8.0 and 20 mM EDTA), added with 0.007 mL of RNAse (100 mg/mL). Following incubation at 37°C for 30 min, 0.05 mL of 5 M NaCl and 0.12 mL of 10% w/v SDS were added. The samples were incubated at 65°C for 80 min, then following 0.24 mL of 5 M potassium acetate addition, they were incubated at −20°C for 10 min. After centrifugation at 13,030 g for 15 min at room temperature (RT), 0.6 mL of supernatant were collected and mixed with 0.75 mL of 100% 2-propanol. The DNA pellet obtained by centrifugation at 13,030 g for 15 min at RT was washed in 70% v/v ethanol, air-dried, and finally resuspended in sterile deionized water. Genomic DNA extraction was verified by electrophoresis on 0.8% w/v agarose gel with Atlas ClearSight DNA Stain (Bioatlas, Tartu, Estonia). Molecular markers were from SibEnzyme (Novosibirsk, Russia). Genomic DNA concentration and its purity were determined at NanoDrop® UV–Vis spectrophotometer (ND-1000, Thermo Fisher Scientific, Waltham, Massachusetts, United States) and samples were kept at −20°C. 16S rDNA was sequenced by BMR Genomics (Padova, Italy) and sequence was deposited at GenBank (accession number OP804337).

M68 bacterial strain was grown in 500-mL Erlenmeyer flasks containing 100 mL LB broth added with 2% w/v artificial sea salt and 2 mM CuSO₄ (final concentration). Cells were collected after 24 h by centrifugation at 23,419 g for 45 min at 4°C. Cell extracts were obtained by sonication for 8 cycles of 30 s each on ice, followed by centrifugation at 38,724 g for 45 min at 4°C. The obtained supernatant was added with 30% w/v - corresponding to 176  g/L - (NH₄)₂SO₄, and centrifuged at 38,724 g for 45 min at 4°C. The supernatant obtained after precipitation (SNP30) was used for the following kinetic studies.

The kinetic parameters were determined at 20°C in the presence of different concentrations of ABTS (from 0.125 to 10 mM) or 2,6-DMP (from 0.5 to 30 mM) in 50 mM sodium acetate, pH 5.0. The specific activity was expressed as mU per mg of total proteins. The kinetic data were fitted to the classical Michaelis–Menten equation.

Temperature dependence of enzymatic activity was determined by measuring 5 mM ABTS oxidation at 20°C in 50 mM sodium acetate, pH 5.0 in a 10–80°C temperature range, after pre-incubating buffer and substrate for 45 min at the selected temperature values. Enzyme preparation stability was measured at −20, 4, 20 and 37°C by incubating samples for 24 h and then determining the residual activity on 5 mM ABTS in 50 mM sodium acetate, pH 5.0 at 20°C.

pH dependence of enzymatic activity was determined by measuring 5 mM ABTS oxidation at 20°C in 12 mM multicomponent buffer (15 mM H₃PO₄, 15 mM Tris, 15 mM Na₂CO₃, 250 mM KCl), in the 3.0–9.0 pH range (Harris et al., 2001). Enzymatic activity was also assayed in 50 mM citrate–phosphate buffer, pH 5.0. The enzymatic stability at different pH values, was evaluated by incubating samples for up to 24 h in 50 mM sodium acetate, pH 5.0 or in 12 mM multicomponent buffer, pH 3.0, 4.0 and 5.0 at 20°C, and then determining the residual activity at different times on 5 mM ABTS in 50 mM sodium acetate, pH 5.0 at 20°C.

The effects of NaCl (0–1,000 mM), DMSO (0–40%) or Tween-80 (0–10%) on the enzymatic activity were assessed on 1 mM or 5 mM ABTS in 50 mM sodium acetate, pH 5.0 at 20°C.

Experiments were performed in triplicate and data were analyzed for statistical significance using one-way ANOVA followed by a Tukey’s multiple comparison test using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). Significance was assessed at p < 0.05.

Native-PAGE analysis was performed at 4°C on a 7.5% w/v acrylamide-resolving gel without SDS. Two different staining procedures were employed: (a) total protein composition was visualized by incubating the gel in fixing solution (40% v/v ethanol, 10% v/v acetic acid) for 30 min at RT and staining it with colloidal Coomassie staining solution (10% v/v orthophosphoric acid, 10% w/v ammonium sulphate, 0.12% w/v brilliant blue G, 20% v/v methanol) at 4°C overnight; (b) laccase activity was detected by incubating the gel at RT in 50 mM sodium acetate, pH 5.0, containing 10 mM ABTS. The protein band visualized by activity assay and the corresponding band on the Coomassie-stained gel were excised and conserved at 4°C.

The excised protein band from the Coomassie-stained gel was subjected to destaining, reduction, derivatization, and digestion with trypsin (1:10), and then analyzed with nLC-MS/MS. MS/MS analysis was carried out by an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Sci., Waltham, MA, United States), as previously described (Maffioli et al., 2020). Database search was performed using the Sequest search engine of Proteome Discoverer 1.4 (Thermo Fisher Sci.) against the proteome of Halomonas from Uniprot and NCBI sequence databases (released 12 December 2020). Only peptides with high Xcorr ≥ 1.5 and medium FDR confidence (FDR 0.05 relaxed) were kept for the identification. The minimum required peptide length was set to 6 amino acids with carbamidomethylation as fixed modification, Met oxidation and Arg/Gln deamidation as variable modifications. Multiple sequence alignment of the proteins identified through nLC-MS/MS was obtained using ClustalW in Uniprot and the Blastp tool (Align Sequences Protein BLAST) in NCBI (Eberini et al., 2002).

A total of 186 morphologically diverse microbes was isolated from the biofilm and seawater samples collected from the Terra Nova Bay. One hundred and forty-six microorganisms were isolated from biofilms grown on PVC and PE artificial panels deployed at the four sampling sites (RB, PTS, TB, and AG; Figure 1), and 40 from the surrounding seawater. The sampling campaign and the physical–chemical parameters of the sampling sites were recently described in Caroppo et al. (2022). Herein, samples were plated - in some cases after a heat pre-treatment to enrich in spore forming microorganisms and/or being serially diluted - on a variety of different isolation media (either with or without adding 2% w/v artificial sea salt) and incubated at three different temperatures, i.e., 4, 20 and 28°C, to capture as much as possible the cultivable diversity. All the selected microbes grew well on the media enriched with artificial sea salt, indicating that they are halotolerant, with only 28% of them strictly requiring salt addition to grow, being therefore considered obligate halophiles. For the microbial isolation, we used a salt concentration (2% w/v) that is slightly lower than that of the Antarctic Ocean, ranging from 3 to 3.5% w/v. This was done in order to account for the expected saline gradients that were present in the sampling area: at the RB site due to its proximity to the wastewater treatment plant of Mario Zucchelli research station and at the AG site, where the meltwater from the glacier is entering the sea (Caruso, 2020; Caroppo et al., 2022; Caruso et al., 2022). The majority of the isolates grew well at the three different temperatures used for plate incubation, showing that they tended to be psychrotolerant rather than psychrophilic. Only 8% grew better at 4°C in solid media, although their growth was detectable and generally abundant also at 20 and 28°C. By microscopical observation, 28 microorganisms were considered filamentous actinomycetes and 10 filamentous fungi (on the basis of the mycelium conformation, hyphae morphology and size, and in some cases spores’ presence), while the remaining ones generically resembled cocci and bacilli. Geographical distribution, sampling depth, sample origin and morphology-based classification of the 146 biofilm microbial isolates and of the 40 seawater microbial isolates are reported in Supplementary Figures S1, S2, respectively.

All the 186 microbial isolates were primarily screened for their ability to produce laccase enzymes on MAM plates (Casciello et al., 2017), added with 2% w/v artificial sea salt and supplemented with the soluble mediator ABTS (Yang et al., 2017) or with the dye azure B (Casciello et al., 2017). MAM was previously reported as an adequate medium for such screenings since it is known to support the growth of different types of microorganisms, without inducing pigment production, which can interfere with colorimetric enzyme assays (Casciello et al., 2017). After a 2 week-long incubation at 20°C, 25 microorganisms (19 unicellular bacteria, 1 filamentous actinomycete and 5 filamentous fungi) were able to oxidize ABTS, and 20 microorganisms (13 unicellular bacteria, 2 filamentous actinomycetes and 5 filamentous fungi) were able to degrade the dye azure B (Supplementary Table SB). Among them, 15 isolates showed both the activities on MAM plates. Geographical distribution, sampling depth, sample origin and morphology-based classification of these 30 microbial isolates are reported in Supplementary Figure S3.

Following this primary screening, the selected active strains were grown in liquid media (differently designed according to the morphology-based classification of the isolates, see Materials and Methods) to test their laccase-like activity production, by assaying either acellular broths (for putative secreted enzymes) or crude extracts after cell lysis by sonication (for intracellular enzymes). A broad range of substrates, i.e., ABTS, 2,6-DMP, catechol, azure B, RBBR and RB5, was evaluated. Twenty-five isolates confirmed their ability to oxidize ABTS, albeit at different extent; in addition, few of them converted 2,6-DMP (6 isolates) and catechol (4 isolates), and/or degraded azure B (12 and 5 isolates, respectively). The five additional isolates oxidizing only azure B in solid assay, confirmed this unique activity in liquid. None of the tested strains showed any enzymatic activity on RBBR and RB5 as substrates (Supplementary Table SB).

The vast majority of these active microorganisms (21 isolates out of 30) originated from RB, which is the site located in front of the wastewater treatment plant discharge of the Mario Zucchelli station (Figure 1). Twenty-one were classified as cocci/bacilli on the basis of their microscopic morphology. In these unicellular bacteria, the laccase activity was prevalently detected intracellularly (Supplementary Table SB). The remaining active few actinomycetes (3) and fungi (6), instead secreted the oxidative enzymes, but their activity was low and poorly reproducible (Supplementary Table SB). Consequently, following our enzymatic activity-based screening approach, further investigations focused on the five most active isolates (M47, M61, M68, M70, and M73), which were unicellular bacteria isolated from seawater samples (Figures 2A,B). Almost all of these strains (M61, M68, M70, and M73) were isolated from RB samples, with the exception of M47 which was isolated from a TB sample. Details on a preliminary phenotypical characterization of the strains are reported in Supplementary Table SC. The intracellular enzyme preparations from these five selected isolates oxidized both ABTS and 2,6-DMP at 20°C, with a signal corresponding to the enzymatic activity being detected already after 1 h and increasing within the 24 h of incubation. For all the strains, the activity on ABTS was higher than on 2,6-DMP (Figures 2A,B). In this study, we focused on the laccase-activity produced by the M68 isolate, since it showed a significantly higher activity within the 24 h of incubation: 3.05 mU/mg of total proteins in the cellular extract on ABTS and 1.77 mU/mg on 2,6-DMP (Figures 2A,B). M68 did not show any activity either on catechol or azure B (Supplementary Table SB). The further investigation and in-depth taxonomical characterization of the other four positive strains will be considered in future works.

M68 strain was isolated from a seawater sample collected at −20 m in RB and it was found to be a Gram-negative, rod-shaped, low oxidase producing, glucose-sucrose-lactose not fermenting microorganism, producing orange pigmented colonies, with active proteolytic and phosphatase activities as assayed using fluorogenic substrates (Supplementary Table SC). This isolate grew without NaCl (both in solid and in liquid media) and tolerated up to 15% w/v (2.5 M) NaCl, with an optimal growth at 5% w/v (860 mM) NaCl. The sequencing of its 16S rDNA revealed that it belongs to the genus Halomonas, within the phylum Proteobacteria (γ-subclass). Specifically, a 16S rDNA sequence identity of 100% was found with two strains of Halomonas meridiana (strain PR51-13 and strain DSM 5425).

To further characterize the laccase-like intracellular enzyme activity detected in M68 strain (no activity was detected in the acellular broth), an optimization of the growing and production conditions was attempted by evaluating the enzyme activity at different growth temperatures (from 4 to 30°C), NaCl concentrations (from 340 to 5,130 mM) and cultivation times (4, 6, 8, 10, 12, and 24 h; Supplementary Figures S4A–C). Anyway, no significant improvement in enzyme activity was observed; thus, the original growing conditions were maintained (growth at 20°C at 2% w/v artificial sea salt for 24 h). 2 mM CuSO₄ was also added to the cultivation broth, since Cu²⁺ is a cofactor of laccases (Pollegioni et al., 2015) and its addition to cultivation medium might increase laccase production (Ausec et al., 2017; Casciello et al., 2017; Berini et al., 2018). Under these culture conditions, the enzymatic activity of the cellular extract on 5 mM ABTS increased of about six-times up to 3 mU/mg of total proteins in the cellular extract (3.15 ± 0.38), compared to the activity detected in the absence of CuSO₄ (0.55 mU/mg).

Following partial precipitation of the proteins in the cellular extract with ammonium sulphate at 30% w/v of saturation, the enzymatic activity was recovered in the supernatant (SNP30). Treatment at percentages of ammonium sulphate higher than 30% w/v led to enzyme inactivation. The total laccase activity of SNP30 (tested on 5 mM ABTS) increased from 265 to 670 mU and the specific activity reached a figure of 9.70 ± 0.96 mU/mg total proteins: the 2.5–3.1-fold increase in activity can result from both the removal of potentially interfering compounds or inhibitors and the positive effect of the increased ionic strength (see below). Notably, the laccase-like activity was not recovered after a dialysis step against 50 mM sodium acetate, pH 5.0, confirming the highest stability of the enzyme in the presence of salts. The SNP30 enzymatic preparation (that we start hereby to name Ant laccase) was therefore directly used for the kinetic studies.

Ant laccase was identified through nLC–MS/MS. SNP30 enzyme preparation was separated by electrophoresis in a native protein gel, which was then stained in parallel by a colloidal Coomassie stain (Supplementary Figure S5A) and a laccase activity-specific stain (Supplementary Figure S5B). In this last, the protein band responsible of laccase activity was identified and the corresponding band was excised from Coomassie-stained gel. nLC–MS/MS analyses on the band, briefly summarized in Table 1 and Supplementary Figure S6, clearly show that Ant laccase matches with three proteins correlated to a potential laccase-like activity: a copper resistance protein CopA from H. meridiana (ID A0A0D7UVK4, sequence coverage 30.64%), and two copper resistance system multicopper oxidases from two Halomonas species, i.e., Halomonas piezotolerans (ID WP_153843658.1, sequence coverage 24.38%) and Halomonas sp. FME66 (ID WP_193092891.1, sequence coverage 21.09%). Alignment of these three protein sequences using ClustalW from Uniprot database yielded a sequence identity of 96.7%, confirming that they belong to the same very well conserved family of the copper resistance system multicopper oxidase. This result was further confirmed by searching for similar or identical proteins in the NCBI Halomonas sequence database that allowed to identify at least 100 sequences with an identity ranging from 100 to 82.55%.

Kinetic parameters of Ant laccase preparation were determined on the non-phenolic ABTS and the phenolic 2,6-DMP as substrates. In all cases, the dependence of the activity values on the substrate concentration followed a Michaelis–Menten kinetic (Figures 3A,B). The highest maximal activity was observed on ABTS as substrate (13.99 ± 0.41 mU/mg protein) that also showed a lower K_m value (approx. 2 mM), while the activity determined on 2,6-DMP was ca. 10-times lower (1.37 ± 0.03 mU/mg protein). The enzymatic preparation did not possess any dye-decolorizing activity (i.e., on azure B, RBBR and RB5) and no activity was detected on catechol as substrate, confirming what observed during the primary and secondary screening (Supplementary Table SB).

The activity of the Ant laccase preparation (SNP30) from Halomonas sp. M68 on ABTS was determined at different temperature and pH values. The optimum temperature for enzyme activity was at around 40°C, with activity values increasing of more than two-fold compared to that at 20°C. Nevertheless, nearly 90% of the enzyme activity measured in standard conditions (20°C), and more than 40% of that measured at 40°C, were preserved even at 10°C (Figure 4A): the enzyme maintained at least 50% of its maximum activity in the 10–60°C temperature range. A significant decrease in enzyme activity was observed only at >60°C. The Ant laccase preparation was quite stable: no significant loss of activity was apparent after 24 h incubation at −20, 4 and 20°C, and when incubated for 24 h at 37°C, ca. 65% of its initial activity was maintained (Supplementary Figure S7).

The highest activity of the Ant enzyme preparation was detected at acidic pH values (Figure 4B), highlighting a strong dependence of the enzyme activity on buffer composition even at optimal pH (Figure 4C). Ant laccase activity strongly decreased at pH 7.0 (reaching approx. 20% of that measured in standard conditions, at pH 5.0) and the enzyme was completely inactive at pH >8.0. The preparation possessed a good stability in the 3.0–6.0 pH range following incubation for 24 h at 20°C, showing the highest residual activity at pH 5.0–6.0 (Figure 4D).

High salt concentrations can affect the activity of laccases thus hampering various putative biotechnological applications. The Ant enzyme preparation well tolerated high NaCl concentrations, up to 1 M, while still maintaining ca. 80% of the activity measured in the absence of the sodium salt (Figure 5A). Additionally, to verify the potential for using the Ant laccase in processes requiring solvents and surfactants, the effects of DMSO and Tween-80 on its enzymatic activity were evaluated. Compared to the standard activity assayed in plain buffer, the activity of the enzyme preparation halved in the presence of 10% v/v DMSO or of 5% v/v Tween-80 (Figures 5B,C, respectively).