Identification and characterization of a novel β-galactosidase active at low temperatures from the Antarctic fungus Tetracladium sp., expressed in Saccharomyces cerevisiae

Identification and characterization of a novel β-galactosidase active at low temperatures from the Antarctic fungus Tetracladium sp., expressed in Saccharomyces cerevisiae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification and characterization of a novel β-galactosidase active at low temperatures from the Antarctic fungus Tetracladium sp ., expressed in Saccharomyces cerevisiae Fernando Gutierrez, Jennifer Alcaino, Victor Cifuentes, Marcelo Baeza This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7074902/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 9 You are reading this latest preprint version Abstract Background β-Galactosidases are widely used in the dairy industry to produce lactose-free milk and prebiotics such as galacto-oligosaccharides and lactulose. Since commercial β-galactosidases have optimal activity at 35 to 70 ºC, β-galactosidases that are highly active at lower temperatures are desirable to reduce production costs and minimize microbial contamination in industrial processes. Potential sources of cold-active β-galactosidases are microorganisms living in cold environments such as Antarctica. The aim of this work was to identify genes encoding β-galactosidases from Antarctic fungi and express them in Saccharomyces cerevisiae for their characterization. Results By searching 16 ORFeomes from eight Antarctic fungi, an ORF encoding β-galactosidase was identified in Tetracladium sp. (Tspgal), and the gene structure was determined in the corresponding genome. Phylogenetic analyses indicate that this is a novel β-galactosidase closely related to β-galactosidases from saprophytic fungi. The closest β-galactosidase with a known 3D structure was from Cellvibrio japonicus , which differed from that from Tetracladium sp. mainly in unstructured regions, with most of the active site residues conserved. The Tspgal expressed in S. cerevisiae showed maximum activity from 25 ºC to 40 ºC and from pH 5.5 to pH 7.0 (maximum at 35 ºC and pH 6.0). At pH 6.0, the recombinant enzyme retained 25% and 36% of its activity at 10ºC and 50ºC, respectively. The thermal enzymatic inactivation of the recombinant β-galactosidase correlated with its thermal protein unfolding, a behavior similar to that observed for mesophilic enzymes. Tspbgal hydrolyzed lactose optimally at pH 5.0 at 35°C, retaining about 80% of its activity at pH 6.0 and 7.0, conditions that coincide with the pH of whey, a major dairy byproduct and potential source of value‑added products derived from lactose. Conclusions A novel β -galactosidase was identified in the ORFeome of the Antarctic fungus Tetracladium sp., which was successfully expressed in S. cerevisiae exhibiting structural and thermal stability properties comparable to mesophilic enzymes. The recombinant enzyme exhibited high activity at 25–35 ºC and retained 25% of its maximum activity at 10 ºC, an attractive trait for reducing energy costs and minimizing microbial contamination in milk treatments. β-galactosidase Tetracladium sp. psychrophiles protein flexibility dairy industry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background β-galactosidases (EC 3.2.1.23) are enzymes that catalyze the hydrolysis of β-galactosidic bonds in oligosaccharides and transgalactosylation reactions. These enzymes have attracted increasing biotechnological interest, particularly in the dairy industry, where they are used to produce low-lactose and lactose-free milk. In addition, these enzymes could be used to hydrolyze lactose in whey, a by-product of the cheese industry, to produce various valuable products, such as galactose-containing compounds (including galacto-oligosaccharides, which are used as prebiotics), ethanol, syrup sweeteners, and lactic acid [ 1 – 3 ]. Currently, most β-galactosidases used in the food and pharmaceutical industries derive from fungi and yeasts, with Kluyveromyces sp. and Aspergillus sp. being the main sources [ 4 ]. Commercial β-galactosidases generally exhibit optimal activity at temperatures between 35ºC and 70ºC. However, identifying β-galactosidases with novel properties, such as high activity at lower temperatures, which can help reduce production costs and minimize microbial contamination in industrial processes, remains an active area of research. Potential sources of cold-active β-galactosidases are microorganisms living in environments with consistently low temperatures, such as polar regions, high mountains, and deep-sea habitats [ 5 , 6 ]. As can be seen in Table 1 , the majority of β-galactosidase-producing microorganisms correspond to bacteria from cold environments, with reported optimal enzymatic temperatures (Topt) ranging from 15 ºC to 45 ºC, but some with reported Topt as high as 60 ºC (Marinomonas sp. BSi20414, isolated from the Arctic Ocean). The β-galactosidases reported from fungi generally have an elevated Topt (50ºC or higher) but, in some cases, maintain high activity at lower temperatures (30–35ºC), such as those from Penicillium chrysogenum NCAIM 00237 and Cladosporium tenuissimum URM 7803. Table 1 Microorganisms producing ß-galactosidases with reported optimal temperature for enzymatic activity (T opt ) Microorganism Geographical origin T opt (ºC) Ref. Bacteria Alkalilactibacillus ikkensis Arctic 20–30 [ 7 ] Alteromonas sp. L82 Deep-sea water, Mariana Trench 25–45 [ 8 ] Alteromonas sp. ML117 marine 30 [ 9 ] Alteromonas sp. ML52 Deep-sea water, Mariana Trench 35 [ 10 ] Arthrobacter psychrolactophilus strain F2 - 10 [ 11 ] Arthrobacter sp. 32cB Antarctica 28 [ 12 ] Arthrobacter sp. ON14 Great Wall Station, Antarctica 15 [ 13 ] Arthrobacter strain Pennsylvania farmland 20 [ 14 ] Exiguobacterium antarcticum B7 Antarctica 30 [ 15 ] Halorubrum lacusprofundi Antarctica 50 [ 16 ] Marinomonas sp. BSi20414 Canada Basin, Arctic Ocean 60 [ 17 ] Micrococcus antarcticus Antarctica 25 [ 18 ] Pseudoalteromonas haloplanktis Antarctica 45 [ 19 ] Rahnella sp. R3 Tianshan Mountains 35 [ 20 ] Thermothielavioides terrestris North Vietnam 60 [ 21 ] Fungi/yeast Aspergillus terreus India 40 [ 22 ] Cladosporium tenuissimum URM 7803 Brazil 35–50 [ 23 ] Aspergillus niger Brazilian biome Cerrado 50 [ 24 ] Aspergillus lacticoffeatus Indonesia 50–60 [ 25 ] Aspergillus awamori (MTCC 548) India 55–60 [ 26 ] Penicillium chrysogenum NCAIM 00237 - 30 [ 27 ] The performance of an enzyme with respect to temperature is closely related to its structural properties, and it has been described that cold-active enzymes generally exhibit increased local and/or global flexibility compared to their mesophilic counterparts. A high structural flexibility of a protein can be achieved by several factors, such as a lower content of secondary structures, longer and more abundant hydrophilic loops, a larger hydrophobic surface, a smaller hydrophobic core, and a lower number of ionic-electrostatic interactions, hydrogen bonds, and salt bridges [ 28 – 31 ]. Reduced proline content in bacterial cold-active proteins [ 32 , 33 ] has been proposed as an adaptation to mitigate the negative effect of proline isomerization on protein folding [ 34 ]. Structural properties of the active site, such as larger opening catalytic sites and longer linkers that can adopt different conformations to facilitate substrate accessibility, have also been proposed as key factors in the increased activity of cold-active enzymes at low temperatures [ 35 – 37 ]. It is important to note that no single "structural strategy" or adaptation is common to all cold-active enzymes, and each may exhibit a particular combination of the above characteristics. This work used 16 ORFeomes corresponding to eight Antarctic fungi to search for potential genes encoding β-galactosidases. A putative coding sequence for β-galactosidase was found in Tetracladium sp. and expressed in Saccharomyces cerevisiae . The recombinant β-galactosidase was characterized in terms of its structural properties and hydrolytic activity under various conditions. Methods Strains, plasmids, and culture conditions Escherichia coli DH5α (F-, Φ80d lacZ △ M15, △ ( lacZYA -argF) U169 , deoR , recA1 , endA1 , hsdR17 (rk-,mk+), phoA , s upE44 , λ-, thi-1 , gyrA96 , relA1 ) was routinely cultured in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) supplemented with 0.2% glucose at 37°C. S. cerevisiae INVSc1 (MATa, his3D1, leu2, trp1-289, ura3-52) was grown in YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% bactopeptone) supplemented with 1% glucose at 30°C, with orbital shaking at 170 r.p.m. The SC medium (0.85% yeast nitrogen base without amino acids and with ammonium sulfate, 0.5% casamino acids, 0.4% NaOH, 2% glycerol, 1% succinic acid, 2% glucose, 0.005% histidine, 0.01% leucine, 0.01% uracil, 0.01% adenine hemisulfate) was used for S. cerevisiae transformant selection. Agar was added at a final concentration of 1.5% for semi-solid media. Plasmid pUC57 was used for cloning experiments in E. coli , and transformants were selected on LB-agar plates supplemented with 100 µg/ml ampicillin. For molecular cloning and gene expression in S. cerevisiae , plasmid pYES3/CT was used. Standard molecular and biochemical methods Standard methods such as plasmid DNA and protein extractions, agarose and polyacrylamide gel electrophoresis, conventional PCR, and restriction digestions were performed as previously described [ 38 ] unless otherwise noted. T4 DNA ligase, PfuUltraII Fusion HS DNA polymerase, restriction endonucleases, DNase I, RNase A, and T4 polynucleotide kinase were purchased from Agilent Technologies, Thermo Scientific, and Life Technologies and were used according to the manufacturer's instructions. Transformations were performed by electroporation using a GenePulser XcellTM (BioRad, Hercules, CA, USA) equipment with settings of 1.8 kV, 25 µF, 200 Ω for E. coli , and 1.5 kV, 25 µF, 200 Ω for S. cerevisiae . When required, sample absorbance at different wavelengths was measured using Jasco UV-Vis spectrophotometer V-630 and Epoch 2 Microplate Spectrophotometer (Biotek, Winooski, VT, USA). Prediction of sequences encoding β-galactosidases The protein sequences of β-galactosidases were downloaded from the UniProt database and used to build a local database using Geneious Prime software. The translated ORF sequences, predicted from the transcriptomes of eight Antarctic yeasts [ 39 ], were compared to the local database using BLASTp with the "Bin into 'hashit' vs. 'no hit'" option in the Geneious software. The "hit sequences" were then selected and compared by BLASTp to the UniProt database for annotation, applying a similarity threshold of ≥ 30%, using the Blosum62 cost matrix. Sequences annotated as related to β-galactosidase were selected for further analysis. The prediction of domains, families, or functional sites was performed using the InterProScan server ( https://www.ebi.ac.uk/interpro/search/sequence/ ). The DeepLoc-2.0 web server ( https://services.healthtech.dtu.dk/services/DeepLoc-2.0/ ) was used to predict subcellular localization. Maximum likelihood evolutionary analysis Protein sequences were aligned using MAFFT [ 40 ] and trimmed using trimAl [ 41 ]. The phylogeny was inferred using IQ-TREE [ 42 ] with automatic model selection (MFP), and branch support was assessed using 1,000 ultrafast bootstrap replicates [ 43 ]. 3D structural modeling and comparison The 3D protein models were constructed using the SWISS-MODEL server ( https://swissmodel.expasy.org ) with the best ortholog identified as a template considering the following parameters: coverage ≥ 50%, similarity ≥ 30%, Global Model Quality Estimate (GMQE) ≥ 0.8. The quality of the models was assessed using VERIFY 3D [ 44 , 45 ] available at UCLA-DOE LAB-SAVES v6.1 ( https://saves.mbi.ucla.edu/ ). Protein structural properties related to flexibility, such as the number of hydrogen bonds, salt bridges, apolar solvent-accessible surface area (apoSASA), and content of secondary structures [ 46 ], were calculated in each model. The properties and parameters used in the ChimeraX software [ 47 , 48 ] for these calculations were: apoSASA (radius = 1.4 Å, peak density = 2), hydrogen bonds, and salt bridges (radius = 0.075 Å, dashes = 6, distance tolerance = 0.4 Å, angle tolerance = 20º). The secondary structure content was determined from PDB files using the Pfeature web server ( https://webs.iiitd.edu.in/raghava/pfeature/sec.php ). The percentage of residues in regions predicted to be rigid (0), flexible (1), or very flexible (2) was calculated using MEDUSA [ 49 ]. Comparative superimposition analysis of protein 3D structures was performed using the Dali server ( http://ekhidna2.biocenter.helsinki.fi/dali/ ; [ 50 ]). Cloning, expression, and purification The ORF sequence of the selected β-galactosidase was modified in silico according to the codon usage of S. cerevisiae and then synthesized by Gene Universal ( https://www.geneuniversal.com/ ), including cloning into the vector pUC57 (named pUC57Bgal1) and transformed into E. coli DH5α by electroporation. The pUC57Bgal1 plasmid was purified from E. coli culture and used as a template in a PCR reaction using primers Bgal1KpnI (5'-GTACggtaccATGGCTTCTTCTGATAAAAACTTCCCT-3') and Bgal1ApaI (5'-GTACgggcccTTCATCAGCTTCCAAAGAGTAAAC-3'), which contained restriction sites for KpnI and ApaI (indicated in lower case), respectively. The PCR products were resolved on a 1% agarose gel. The amplicon band of the expected length (1,709 bp) was purified from the gel using the Gene Clean protocol [ 51 ], ligated into the pYES3/CT vector, and transformed by electroporation into E. coli DH5α. The recombinant plasmid pYES3/CTbgal1 was purified from E. coli cultures and transformed into S. cerevisiae by electroporation. Transformants were selected on SC agar plates after incubation at 30 ºC for 16 h and verified by PCR using primers Gal1 (5’-AATATACCTCTATACTTTAACGTC-3’) and CYC1 (5’-GCGTGAATGTAAGCGTGAC3’). A S. cerevisiae transformant clone was cultured in 50 ml SC medium for 48 h at 30°C. The culture was centrifuged at 5,230 g for 10 min, the cell pellet was suspended in 50 ml sterile distilled water, and centrifuged at 5,230 g for 5 min. The cell pellet was then inoculated into SC medium supplemented with 2% galactose to achieve an OD 600nm of 0.4, incubated at 30°C, and culture aliquots of 50 ml were collected after 8, 24, and 48 h. These aliquots were centrifuged at 5,230 g for 10 min, the supernatant was discarded, and the cell pellet was stored at -50 ºC until further processing. Five ml of the cell pellet was suspended in one volume of 10 mM Tris-HCl, 0.5 M NaCl, and 10 mM imidazole, pH 8.0, and one ml of cell suspension was aliquoted into lysis tubes. Two hundred µl of 0.5 mm diameter glass beads (Biospec, Bartlesville, OK, USA) were added to each tube, and the cells were disrupted using a Mini-Beadbeater-16 (Biospec, Bartlesville, OK, USA) with 7 cycles of agitation for 3 min, followed by 3 min of incubation on ice. The supernatant obtained after centrifugation of the sample at 19,745 g for 15 min was mixed with 100 ml of 10 mM Tris-HCl, 0.5M NaCl, 10 mM imidazole, pH 8.0, and then loaded into a HisTrap FF crude column (GE Healthcare, Chicago, IL, USA). Samples were eluted using an Akta prime plus equipment (GE Healthcare, Chicago, IL, USA) with a mobile phase of 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, at a flow rate of 1 ml/min and an imidazole gradient from 10 to 500 mM. One ml aliquots were collected for subsequent analyses. Enzyme activity assays Enzyme assays were performed under different temperatures and pH conditions. The pH was adjusted using two sets of buffers: i) 0.1 M sodium acetate buffer (pH 5.0 and 5.5); 100 mM phosphate buffer (pH 6.0 to 7.0), containing 10 mM KCl, 1 mM MgSO 4 ; ii) phosphate-citrate buffer (pH 5.0 to 7.0). For the β-galactosidase activity determination [ 52 ], 7.5 µl of protein sample (25 µg/ml) was mixed with 200 µl of the appropriate pH buffer, and 70 µl of 4 mg/ml o-nitrophenyl-β-D-galactopyranoside (ONPG) was added. The mixture was incubated at different temperatures, and the reaction was stopped at different times by adding 500 µl of 1.0 M Na 2 CO 3 . The release of o-nitrophenol from ONPG was measured by absorbance at 420 nm. For lactose hydrolysis determination, a solution of 5% lactose was used, the reaction was stopped by incubation at 100 ºC for 10 min, and the release of glucose was quantified using the Glucose Assay Kit abcam (Cambridge, UK), according to the manufacturer’s instructions. For the α-Glucosidase activity determination [ 53 ], 7.5 µl of protein sample (25 µg/ml) was mixed with 50 µl of buffer pH 5.0, pH 6.0, or pH 7.0 and 17.5 µl of 1.5 mg/ml of 4-nitrophenyl-α-D-glucopyranoside (PNPG). The reaction mixture was incubated at 35°C for 1 h and then stopped by adding 125 µl of 1 M NaHCO 3 . The release of pNP was determined by absorbance at 405 nm. For the β-Glucuronidase [ 54 ] and β-Glucosidase [ 55 ] activity determinations, 25 µl of enzyme sample was mixed with 150 µl of buffer pH 5.0, pH 6.0, or pH 7.0, and 25 µl of 1 mg/ml 4-methylumbelliferyl-β-D-glucuronide (4MBG) or 4-methylumbelliferyl-β-D-glucoside (4MBDG), respectively. The reaction was incubated at 35°C for 1 h and stopped by adding 1.3 ml of 0.1 M glycine-NaOH buffer pH 10.4. The reaction was then exposed to UV light using a CUV40A transilluminator (ClinX, Shanghai, China) to observe fluorescence emission, indicating the enzymatic release of 4-methylumbelliferone. Protein thermal unfolding kinetics Thermal protein unfolding kinetics were assessed as previously described [ 56 ] using a CFX96 Real-Time System (BioRad, Hercules, CA, USA) in 96-well PCR microplates. In each well, 13 µl of the appropriate buffer at the desired pH, 10 µl protein sample (1 µg/µl), and 2 µl 125X concentrated SYPRO Orange were added. The temperature was increased from 5 ºC to 95 ºC with a ramp of 1 ºC per min. The excitation wavelength was set to 470 nm, and the emission was registered at 569 nm. Results Prediction of β-galactosidases in the ORFeomes of Antarctic fungi and structure of the putative gene and enzyme. By mining the transcriptomes of eight Antarctic fungi for ORFs encoding β-galactosidases, a putative ORF of 1,692 nt was identified in the transcriptome of Tetracladium sp. and mapped to the corresponding genome to determine the gene structure. The encoding gene tspbgal spans 1,884 nt (NCBI accession number PQ310115) and consists of four exons, resulting in a coding sequence of 1,692 nt (Fig. 1 A). A strong Kozak sequence was identified, including the translation start codon ATG. Tspbgal was predicted to be a cytoplasmic protein (70% probability), and the predicted domains were glycosyl hydrolase family 42, a transglycosylase, and a domain of unknown function 5597 (DUF5597). Multiple alignment and phylogenetic analyses were performed with Tspbgal and 89 related β-galactosidases, with sequences from 558 to 601 residues identified through BLASTp searches in the NCBI Protein Reference and Swiss-Prot databases. Tspbgal grouped with β-galactosidases from the species Baudoinia panamericana, Cyphellophora europaea, Exophiala dermatitidis, Capronia coronata , and Pseudogymnoascus sp., but with low bootstrap support (Figure S1 ). A three-dimensional model of Tspbgal was constructed by homology modeling using Cellvibrio japonicus Ueda107 tetrameric β-galactosidase (Bgl35A, PDB ID: 4D1I) as the template, the best ortholog found in the Swiss Model Server. Superposition analysis of the monomers from the predicted model of Tspbgal and Bgl35A using the Dali server aligned 492 C-alpha atoms showed a sequence identity of 34%, a Z-score of 61.1, and an r.m.s.d. value of 1.1 Å. Several structural differences (labeled SD1 to SD8 in Fig. 1 B) were observed by superimposing the three-dimensional structures of the monomers from the crystal structure of Bgl35A and the predicted model for Tspbgal. Four loops in Tspbgal (SD1, SD2, SD3, and SD8) were 4, 21, 11, and 18 residues longer than the corresponding loops in Bgl35A, respectively. Loops SD5 and SD7 in Tspbgal were 13 and 8 residues shorter than their counterparts in Bgl35A, respectively. The helix SD4 and the helix-loop-helix SD6 structures observed in Bgl35A were present as loops in Tspbgal and were shorter by 3 and 13 residues, respectively. To analyze the active sites, the crystal structure of Bgl35A was soaked with the iminosugar 1-deoxygalactonojirimycin (1,5-dideoxy-1,5-imino-D-galactitol, DGJ) [ 57 ]. Residues N67, K134, N135, and N204, which coordinate DGJ in the active site cavity of Bgl35A, are conserved in Tspbgal, corresponding to residues N36, K104, N105, and N179, respectively. Residue N383 of Bgl35A, which interacts with O6 of DGJ at a calculated distance of 3.2 Å, is absent in Tspbgal. However, other nearby residues in Tspbgal may potentially form hydrogen bonds with O6 of DGJ. The best candidate is residue Y334, which might form a hydrogen bond with DGJ of 4.3 Å. Additionally, residues Q35 and R358 from Tspbgal, located in a region that is predicted to be flexible, may form hydrogen bonds with O6 of DGJ of 7.8 Å and 8.4 Å, respectively, also positioning them as candidates to coordinate O6 of DGJ. The proposed catalytic residues E205 and E349 in Bgl35A are structurally conserved in Tspbgal, corresponding to residues E180 and E356, respectively. The conservation of these residues can be observed in other 89 fungal β-galactosidases related to Tspbgal, as shown in Fig. 2 and Figure S2 , including the putative active site residues of Tspbgal that are conserved relative to Bgl35A, as well as residue Y334 in Tspbgal, which likely coordinates O6 of DGJ. Purification and enzymatic characterization of recombinant Tspbgal As described in the Materials and Methods section, the S. cerevisiae INVSc1 strain was transformed with the pYES3/CTbgal1 vector by electroporation, and transformants were selected by PCR with the Gal1/CYC1 primer pair (amplicon size ~ 2,000 bp; see Figure S3 A). Tspbgal expression was induced, culture aliquots were collected at 8, 24, and 48 h post-induction, proteins were extracted from cell pellets, and analyzed by SDS-PAGE and for β-galactosidase activity. A protein band of the expected size (63kDa) was observed in the SDS-PAGE, and β-galactosidase activity was detected in all protein samples (Figure S3 B). Recombinant Tspbgal was purified by His-tagged affinity chromatography on nickel columns using an imidazole gradient (Fig. 3 A). A protein with the expected molecular weight (63kDa) eluted in fractions between 8 and 16 min, corresponding to the fractions displaying β-galactosidase activity (Fig. 3 B). The activity of the purified β-galactosidase was evaluated at temperatures ranging from 10°C to 60°C and pH levels ranging from 5.0 to 7.0, with and without a buffer containing MgSO₄. The enzyme activity was higher at temperatures between 35ºC and 40ºC and pH levels from 5.5 to 6.6, with the highest activity at 35ºC and pH 6.0 (Fig. 3 C). The performance of β-galactosidase activity across pH levels was not affected in buffers with or without MgSO₄. The enzyme maintained at least 60% of its maximum activity at temperatures ranging from 25ºC to 40ºC, and pH levels between 5.0 and 7.0 (Fig. 3 D). It also retained 33% of its maximum activity at 10ºC and pH 6.0. The enzyme showed high stability at 35ºC and pH 6.0, retaining 87% of its maximum activity after 4 h and 67% after 24 h at these conditions (Fig. 3 E). The specificity of recombinant Tspbgal was evaluated in assays performed using PNPG (for α-glucosidase activity), 4MBG (for β-glucuronidase activity), and 4MBDG (for β-glucosidase activity) as substrates at 35ºC and pH 5.0, 6.0, and 7.0. No activity was detected for any of these substrates under these conditions (data not shown). The ability of Tspbgal to hydrolyze lactose was tested using a 5% solution of lactose at 35°C and a pH of 6.0, conditions that yield the maximum enzyme activity. As shown in Fig. 4 , the hydrolysis of approximately 40–50% of the lactose was achieved after 3–4.5 h of incubation, and nearly 70% after 11.5 h. When the lactose hydrolysis was tested at different temperatures at pH 6.0, the Tspbgal retained a residual activity of 52% and 59% at 22 ºC and 45 ºC, respectively. The highest lactose hydrolysis by Tspbgal was observed at pH 5.0 at 35°C, with residual activity of around 80% at pH 6.0 and 7.0. Thermal unfolding of Tspbgal and relationship between the structural properties of fungal β-galactosidases and their optimal activity temperature. The thermal unfolding of Tspbgal was assessed at pH levels ranging from 5.0 to 9.0. A variation in the thermal unfolding curves was observed, with lower thermal stability occurring at pH values above 7.0 (see Fig. 5 A). The highest melting temperatures (Tm) were observed at pH 5.5 (Tm = 49 ± 0.2°C) and pH 6.0 (Tm = 49 ± 0.4°C), which were slightly lower at pH 5.0 (Tm = 46 ± 0.1°C) and pH 6.6 (Tm = 47 ± 0.2°C). The lowest Tm values were observed at pH 8.0 to 9.0, ranging from 35 ºC to 38 ºC. The β-galactosidase activity was evaluated at pH levels from 5.0 to 7.0 and temperatures from 10 ºC to 60 ºC and expressed as a percentage with respect to the maximum activity. As shown in Fig. 5 B, the effect of temperature on enzyme activity correlated with its effect on protein structure, since maximum enzyme activity was observed under conditions when still no protein unfolding was detected (35 ºC to 40 ºC), and the interpolated 50% of enzyme activity corresponded with 50% of protein unfolding. The structural properties associated with protein flexibility that have been proposed to enhance enzyme activity at lower temperatures were determined in 47 β-galactosidases, for which data on their optimal temperature for enzyme activity (OTEA) and the optimal temperature for growth (OTG) of the producer organism were available, and analyzed by principal component analysis (PCA). As shown in Fig. 6 A, the two principal components explain 53.7% of the variance, and the OTG was the parameter most related to OTEA. Some positive relations to OTEA were observed regarding structural protein properties, including solvent accessible surface area (SASA), the percentage of residues classified as medium flexible (Med1), and the percentage of α-helix (Alpha). Negative relations were observed for the number of hydrogen bonds (Hbond) and salt bridges (SaltBrid), apolar solvent accessible surface area (apoSASA), and the percentage of β-sheet. Spearman's correlation was calculated for each parameter versus OTEA (see Fig. 6 B). Positive correlations with a p-value of ≤ 0.05 were found for OTG (0.6) and the percentage of α-helix (0.4). Negative correlations were found for apoSASA (-0.4) and the percentage of β-sheet (-0.4). Discussion The search of the ORFeomes from eight Antarctic fungi revealed an ORF encoding a β-galactosidase in the Tetracladium sp. ORFeome. Genome mapping defined a 1,884-nt gene consisting of four exons with a 1,692-nt coding sequence. The predicted 564-residue β-galactosidase from Tetracladium sp. showed low sequence identity with other described β-galactosidases; its closest relatives were found in the genera Hyaloscypha , Scytalidium , Hyphodiscus , Helotiales , Mollisia , and Cadophora . These are all mycorrhizal and saprophytic filamentous fungi [ 58 – 60 ], and one species, Cadophora malorum , has also been isolated from a cold environment, specifically from lakes on the Antarctic Peninsula [ 61 ]. The best template for three-dimensional structure modeling of the β-galactosidase from Tetracladium sp. was the GH35 β-galactosidase Bgl35A from C. japonicus . The two β-galactosidases structurally differed primarily in their unstructured loops. Four residues were conserved in the active site of both enzymes. It is likely that the role of residue N383 in the β-galactosidases from C. japonicus in their interaction with DGJ is fulfilled by residue Y334 in the β-galactosidase from Tetracladium sp. These residues were conserved across the 89 fungal β-galactosidases more closely related to that from Tetracladium sp. In general, considering the results obtained with ONPG and lactose as substrates, the recombinant Tetracladium sp. β-galactosidase exhibits higher activity at temperatures between 25 and 40°C and pH between 5.5 and 7.0, and notably retains 25% of its activity at 10 ºC. These are promising properties for its potential application in the treatment of milk to reduce lactose content at lower temperatures, helping to reduce energy costs and minimize contamination. On the other hand, the pH range where high activity was observed matches that of whey. Whey is a byproduct of the dairy industry, which, depending on the treatment, can be acidic (pH 5.0) or sweet (pH 6.0–7.0), and has been proposed to be used for the production of whey protein hydrolysates and other value‑added products [ 1 ]. The typical whey composition contains 4.5–5% w/v lactose, which can be used for biofuel production. For example, the combined use of β-galactosidase from Kluyveromyces marxianus and S. cerevisiae was used to produce 28.9 g/L ethanol during fermentation at 35°C [ 62 ]. Furthermore, enzymes with β-galactosidase activity can also carry out transgalactosylations to produce prebiotics, such as galactooligosaccharides, which can be used to support the growth of beneficial microorganisms in the human gastrointestinal tract [ 63 – 65 ]. It has been reported that the activity of β-galactosidases is influenced by the presence of various ions. For example, enzymes from Kluyveromyces lactis and Kluyveromyces fragilis require Mn²⁺, Na⁺, and Mg²⁺ for optimal activity [ 3 ], while the enzyme from Arthrobacter oxydans SB showed improved activity in the presence of Mn²⁺ or Fe²⁺ [ 66 ]. In this work, the β-galactosidase from Tetracladium sp. activity was similar in the presence or absence of Mg 2+ . Regarding psychrophilic enzymes, most are inactivated by temperature before their structure unfolds, which reveals the heat lability of the active site. This is different in mesophilic enzymes, where thermal inactivation correlates with protein unfolding [ 35 ]. As Tetracladium sp. is a cold-adapted fungus that was isolated from Antarctic soil, it was expected that the behavior of its β-galactosidase would resemble that of psychrozymes. However, the enzyme inactivation by temperature coincides with protein unfolding. Therefore, Tetracladium sp. β-galactosidase is structurally more like enzymes from mesophiles, such as C. japonicus , which has optimal activity at pH 6.5 and thermal stability between 35°C and 45°C, with a rapid decrease in activity at 55°C and above [ 67 ]. Conclusions A novel β-galactosidase was identified in the Antarctic fungus Tetracladium sp., which was successfully expressed in S. cerevisiae . The recombinant enzyme showed structural and thermal stability properties similar to mesophilic enzymes but with improved performance at temperatures below 35 ºC, a property desirable in the dairy industry to reduce production costs and microbial contamination. Abbreviations apoSASA: apolar solvent-accessible surface area ONPG: o-nitrophenyl-β-D-galactopyranoside PNPG: 4-nitrophenyl-α-D-glucopyranoside 4MBG: 4-methylumbelliferyl-β-D-glucuronide 4MBDG: 4-methylumbelliferyl-β-D-glucoside DGJ: 1,5-dideoxy-1,5-imino-D-galactitol Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All of the data generated and used in this work are included in the manuscript and are available as supplementary material. Competing interests The authors declare that they have no competing interests Funding This research was funded by Grant Fondecyt 1230427 from the Agencia Nacional de Investigacion y Desarrollo de Chile. Authors' contributions MB, JA, and VC designed the study and discussed the results. FG performed the experiments, protein structural modeling, and characterization. FG and MB performed the bioinformatics and biostatistical analyses. FG, JA, and MB drafted the manuscript. Acknowledgments The authors would like to thank Salvador Barahona and Dionisia Sepulveda for their technical assistance. References Zotta T, Solieri L, Iacumin L, Picozzi C, Gullo M: Valorization of cheese whey using microbial fermentations. Appl Microbiol Biotechnol. 2020; 104 :2749-2764. 10.1007/s00253-020-10408-2 Movahedpour A et al.: β-Galactosidase: From its source and applications to its recombinant form. 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Supplementary Files Supplementarymaterialslegends.docx figs1.pdf figS2.jpg figS3.jpg Cite Share Download PDF Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 25 Aug, 2025 Reviews received at journal 23 Aug, 2025 Reviews received at journal 25 Jul, 2025 Reviewers agreed at journal 17 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers invited by journal 16 Jul, 2025 Editor assigned by journal 11 Jul, 2025 Submission checks completed at journal 11 Jul, 2025 First submitted to journal 08 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7074902","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486967358,"identity":"9c5a563e-0e0d-47c1-b11d-cf06c8b60fa8","order_by":0,"name":"Fernando Gutierrez","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"","lastName":"Gutierrez","suffix":""},{"id":486967359,"identity":"084b9ea0-3d12-42cc-8d47-5b91c572eae7","order_by":1,"name":"Jennifer Alcaino","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Alcaino","suffix":""},{"id":486967360,"identity":"dae0a20f-44fd-4bdd-a436-80877ae0855c","order_by":2,"name":"Victor Cifuentes","email":"","orcid":"","institution":"Universidad de Chile","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"","lastName":"Cifuentes","suffix":""},{"id":486967361,"identity":"f39aae6a-68f3-48ee-893d-8049af829f3c","order_by":3,"name":"Marcelo Baeza","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYDACCTBpA2QwNh4AsmSI1ZIG0tIA0sJDrJbDYAZxWnRnNz98+KPmfGL/7OaGAx8Y7AhrMbtzzNhA4tjtxBl3DjYcnMGQTISWGwlmEgZstxM3SCQ2HOZhOECMlvTvPxL+nYNo+UOclhwzhoNtByBaGIjScudMsWRjX7LxjBuJDQd7DIjxy+32jR9/fLOT7Z+R/vDBjwo7OYJa0IABqRpGwSgYBaNgFGAFAKSKQxiial2+AAAAAElFTkSuQmCC","orcid":"","institution":"Universidad de Chile","correspondingAuthor":true,"prefix":"","firstName":"Marcelo","middleName":"","lastName":"Baeza","suffix":""}],"badges":[],"createdAt":"2025-07-08 12:38:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7074902/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7074902/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02850-6","type":"published","date":"2025-10-14T15:57:25+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87063022,"identity":"38bb201c-7e3a-449e-a3c6-58a8a9423c8c","added_by":"auto","created_at":"2025-07-18 17:38:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":299864,"visible":true,"origin":"","legend":"\u003cp\u003eGene and protein structure of β-galactosidase from \u003cem\u003eTetracladium\u003c/em\u003esp. A) Scheme of the protein functional domains (above) and gene exon-intron structure (below). The translation start codon in the Kozak sequence is indicated. B) Superposition of the crystal structure of β-galactosidase from \u003cem\u003eC.\u003c/em\u003e \u003cem\u003ejaponicus \u003c/em\u003e(cyan) and the predicted model of the β-galactosidase from \u003cem\u003eTetracladium\u003c/em\u003esp. (brown). The iminosugar 1-deoxygalactonojirimycin (DGJ), is shown in pink. The major structural differences between both structures are labeled SD1 to SD8 and colored in blue for the enzyme from \u003cem\u003eTetracladium \u003c/em\u003esp. and yellow for the one from \u003cem\u003eC. japonicus.\u003c/em\u003e Zoom of the active site (discontinuous circle) indicating the conserved residues in both models and showing those corresponding to \u003cem\u003eC. japonicus\u003c/em\u003e in parentheses. The non-conserved residue in \u003cem\u003eC. japonicus\u003c/em\u003e is written in red, and the putative alternative residues in \u003cem\u003eTetracladium\u003c/em\u003e sp. are in blue, including the length of the predicted hydrogen bond with DGJ.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/b0c6772cd4ca229c5d01c67c.png"},{"id":87063019,"identity":"6acbce56-0520-43d3-8f75-59de3137520b","added_by":"auto","created_at":"2025-07-18 17:38:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":480927,"visible":true,"origin":"","legend":"\u003cp\u003eSequence alignment of fungal β-galactosidases. Only one representative from each genus is shown, along with the regions containing conserved residues. The full alignment is shown in Figure S2. Conserved predicted domains are shown at the top. Residues in the active site, as shown in Figure 1B, are indicated by blue boxes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/18cbe05884591c60eb53ac68.png"},{"id":87063027,"identity":"c91e8124-3f7b-47de-acca-4fee6658402b","added_by":"auto","created_at":"2025-07-18 17:38:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":153082,"visible":true,"origin":"","legend":"\u003cp\u003ePurification and characterization of recombinant β-galactosidase. The intracellular protein extract from recombinant \u003cem\u003eS. cerevisiae\u003c/em\u003e at 48 h post-induction was loaded onto a HisTrap FF crude column. 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0 was used as the mobile phase at 1 ml/min with an imidazole gradient from 10 to 500 mM, and 1 ml fractions were collected. A) Protein elution profile measured at 280 nm (blue) and imidazole gradient (red). B) SDS-PAGE analysis of the collected fractions, along with colorimetric detection of β-galactosidase activity (yellow; bottom). The arrow indicates the protein band with the expected relative molecular weight (63 kDa). St, protein standard; R, crude protein extract; 5 to 16, elution fractions 5 to 16 min. C) Recombinant β-galactosidase activity on ONPG at different temperatures and pH adjusted with two sets of buffers: sodium acetate and phosphate containing 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e (AP) and phosphate citrate (PC). D) Percentage of activity relative to the maximum activity shown in C, represented by circle size and color. E) Stability assay conducted over time; the relative enzyme activities were assessed after incubation at 35ºC and pH 6.0 from 0 to 24 h (curves of two independent experiments with three replicates each). In panels C and E, the average of triplicates is shown, and the bars represent the standard deviation.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/fda5d31001be95f87a32725c.png"},{"id":87063057,"identity":"9430eb23-277f-4ec1-b000-a45bd62b1a85","added_by":"auto","created_at":"2025-07-18 17:38:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135149,"visible":true,"origin":"","legend":"\u003cp\u003eLactose hydrolysis by Tspbgal. Glucose release was quantified and used to calculate the percentage of lactose hydrolysis. Assays were performed on 5% lactose solution at 35ºC and pH 6.0. The residual lactose hydrolysis activity at pH 6.0 at different temperatures is shown in the top inset, and at 35ºC and different pH in the bottom inset. Values represent the mean of three independent experiments, and error bars indicate the standard deviation.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/0190c8fb98200decb603f35d.png"},{"id":87063018,"identity":"6bd06a0c-06fa-44a4-96b6-e3e72be70cf6","added_by":"auto","created_at":"2025-07-18 17:38:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":149897,"visible":true,"origin":"","legend":"\u003cp\u003eThermal unfolding and activity of Tspbgal at different pH levels. A) Assays were performed in 96-well microplates, each well containing 13 μl of the appropriate pH buffer, 10 μl of protein sample (1 μg/μl), and 2 μl of 125X conc SYPRO Orange. The temperature ramp was from 5 °C to 95 °C with 1 °C increments every 60 seconds. Excitation and emission wavelengths were 470 nm and 569 nm, respectively. Relative unfolding was calculated as the percentage of fluorescence at each point relative to the maximum fluorescence detected in each curve. The melting temperature at each pH is shown in the inserted box. B) Comparison of the percentage of enzyme activity (act) and protein unfolding (unf) at different temperatures and pH. Values represent the mean of three independent experiments, and error bars indicate the standard deviation.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/61eaf7d2f0fd10db19d07608.png"},{"id":87063028,"identity":"b59019ea-24e0-4473-a38a-a89884ca471c","added_by":"auto","created_at":"2025-07-18 17:38:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":180069,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between the structural properties of β-galactosidases and their optimal temperature for activity. A) Principal component analysis (PCA) of data from 47 β-galactosidases, and B) data dispersion of each parameter versus temperature of the enzyme. Spearman's correlations between parameters and optimal temperature for activity with a p-value ≤0.05 are indicated in parentheses. OTEA, optimal temperature for enzyme activity; OTG, optimal growth temperature; Alpha, α-helix; Beta, β-sheet; Med1, % of residues classified as medium flexibility by MEDUSA; Med2, % of residues classified as high flexibility by MEDUSA; Hbond, number of hydrogen bonds; SaltBrid, number of salt bridges; SASA, solvent accessible surface area; apoSASA, apolar solvent accessible surface area.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/14911c7fac4245b18eeddc73.png"},{"id":93956126,"identity":"cbb2d2ae-3890-4e39-a34d-c58ac989e400","added_by":"auto","created_at":"2025-10-20 16:10:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2443000,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/07b68bec-2fe5-4470-bcf1-d6aebb053ce5.pdf"},{"id":87062998,"identity":"3e786148-6289-49ad-91be-ab2f7d3a2294","added_by":"auto","created_at":"2025-07-18 17:38:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14366,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialslegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/b5fd48c5d089471bb286bcac.docx"},{"id":87063045,"identity":"7df767cc-942a-490c-873d-33429e6713ce","added_by":"auto","created_at":"2025-07-18 17:38:21","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10583,"visible":true,"origin":"","legend":"","description":"","filename":"figs1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/0c19b137b3be9f5b7ab18948.pdf"},{"id":87063011,"identity":"eeadc82c-447f-4dd7-b5f6-6e7c85842c76","added_by":"auto","created_at":"2025-07-18 17:38:18","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8733975,"visible":true,"origin":"","legend":"","description":"","filename":"figS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/3bbe7c3608548abb934a88eb.jpg"},{"id":87063017,"identity":"216e6c66-4be7-4f12-b1ca-3716d7bb566c","added_by":"auto","created_at":"2025-07-18 17:38:19","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1313230,"visible":true,"origin":"","legend":"","description":"","filename":"figS3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7074902/v1/e0b4c58d29b89ae038e212ae.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eIdentification and characterization of a novel β-galactosidase active at low temperatures from the Antarctic fungus \u003cem\u003eTetracladium sp\u003c/em\u003e., expressed in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eβ-galactosidases (EC 3.2.1.23) are enzymes that catalyze the hydrolysis of β-galactosidic bonds in oligosaccharides and transgalactosylation reactions. These enzymes have attracted increasing biotechnological interest, particularly in the dairy industry, where they are used to produce low-lactose and lactose-free milk. In addition, these enzymes could be used to hydrolyze lactose in whey, a by-product of the cheese industry, to produce various valuable products, such as galactose-containing compounds (including galacto-oligosaccharides, which are used as prebiotics), ethanol, syrup sweeteners, and lactic acid [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, most β-galactosidases used in the food and pharmaceutical industries derive from fungi and yeasts, with \u003cem\u003eKluyveromyces\u003c/em\u003e sp. and \u003cem\u003eAspergillus\u003c/em\u003e sp. being the main sources [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Commercial β-galactosidases generally exhibit optimal activity at temperatures between 35ºC and 70ºC. However, identifying β-galactosidases with novel properties, such as high activity at lower temperatures, which can help reduce production costs and minimize microbial contamination in industrial processes, remains an active area of research. Potential sources of cold-active β-galactosidases are microorganisms living in environments with consistently low temperatures, such as polar regions, high mountains, and deep-sea habitats [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As can be seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the majority of β-galactosidase-producing microorganisms correspond to bacteria from cold environments, with reported optimal enzymatic temperatures (Topt) ranging from 15 ºC to 45 ºC, but some with reported Topt as high as 60 ºC (Marinomonas sp. BSi20414, isolated from the Arctic Ocean). The β-galactosidases reported from fungi generally have an elevated Topt (50ºC or higher) but, in some cases, maintain high activity at lower temperatures (30–35ºC), such as those from \u003cem\u003ePenicillium chrysogenum\u003c/em\u003e NCAIM 00237 and \u003cem\u003eCladosporium tenuissimum\u003c/em\u003e URM 7803.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMicroorganisms producing ß-galactosidases with reported optimal temperature for enzymatic activity (T\u003csub\u003eopt\u003c/sub\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMicroorganism\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGeographical origin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eT\u003csub\u003eopt\u003c/sub\u003e (ºC)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eRef.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBacteria\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAlkalilactibacillus ikkensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArctic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20–30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAlteromonas\u003c/em\u003e sp. L82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDeep-sea water, Mariana Trench\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25–45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAlteromonas\u003c/em\u003e sp. ML117\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003emarine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAlteromonas\u003c/em\u003e sp. ML52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDeep-sea water, Mariana Trench\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eArthrobacter psychrolactophilus\u003c/em\u003e strain F2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eArthrobacter\u003c/em\u003e sp. 32cB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAntarctica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eArthrobacter\u003c/em\u003e sp. ON14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGreat Wall Station, Antarctica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eArthrobacter\u003c/em\u003e strain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePennsylvania farmland\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eExiguobacterium antarcticum B7\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAntarctica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eHalorubrum lacusprofundi\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAntarctica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMarinomonas\u003c/em\u003e sp. BSi20414\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCanada Basin, Arctic Ocean\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMicrococcus antarcticus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAntarctica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudoalteromonas haloplanktis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAntarctica\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eRahnella\u003c/em\u003e sp. R3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTianshan Mountains\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eThermothielavioides terrestris\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNorth Vietnam\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFungi/yeast\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAspergillus terreus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIndia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCladosporium tenuissimum\u003c/em\u003e URM 7803\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBrazil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35–50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAspergillus niger\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBrazilian biome Cerrado\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAspergillus lacticoffeatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIndonesia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50–60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAspergillus awamori\u003c/em\u003e (MTCC 548)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIndia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55–60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePenicillium chrysogenum\u003c/em\u003e NCAIM 00237\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe performance of an enzyme with respect to temperature is closely related to its structural properties, and it has been described that cold-active enzymes generally exhibit increased local and/or global flexibility compared to their mesophilic counterparts. A high structural flexibility of a protein can be achieved by several factors, such as a lower content of secondary structures, longer and more abundant hydrophilic loops, a larger hydrophobic surface, a smaller hydrophobic core, and a lower number of ionic-electrostatic interactions, hydrogen bonds, and salt bridges [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e–\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Reduced proline content in bacterial cold-active proteins [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] has been proposed as an adaptation to mitigate the negative effect of proline isomerization on protein folding [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Structural properties of the active site, such as larger opening catalytic sites and longer linkers that can adopt different conformations to facilitate substrate accessibility, have also been proposed as key factors in the increased activity of cold-active enzymes at low temperatures [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e–\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It is important to note that no single \"structural strategy\" or adaptation is common to all cold-active enzymes, and each may exhibit a particular combination of the above characteristics.\u003c/p\u003e\u003cp\u003eThis work used 16 ORFeomes corresponding to eight Antarctic fungi to search for potential genes encoding β-galactosidases. A putative coding sequence for β-galactosidase was found in \u003cem\u003eTetracladium\u003c/em\u003e sp. and expressed in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. The recombinant β-galactosidase was characterized in terms of its structural properties and hydrolytic activity under various conditions.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eStrains, plasmids, and culture conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e DH5α (F-, Φ80d\u003cem\u003elacZ\u003c/em\u003e△ M15, △ (\u003cem\u003elacZYA\u003c/em\u003e-argF) \u003cem\u003eU169\u003c/em\u003e, \u003cem\u003edeoR\u003c/em\u003e, \u003cem\u003erecA1\u003c/em\u003e, \u003cem\u003eendA1\u003c/em\u003e, \u003cem\u003ehsdR17\u003c/em\u003e (rk-,mk+), \u003cem\u003ephoA\u003c/em\u003e, s\u003cem\u003eupE44\u003c/em\u003e, λ-, \u003cem\u003ethi-1\u003c/em\u003e,\u003cem\u003egyrA96\u003c/em\u003e, \u003cem\u003erelA1\u003c/em\u003e) was routinely cultured in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) supplemented with 0.2% glucose at 37°C. \u003cem\u003eS. cerevisiae\u003c/em\u003e INVSc1 (MATa, his3D1, leu2, trp1-289, ura3-52) was grown in YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% bactopeptone) supplemented with 1% glucose at 30°C, with orbital shaking at 170 r.p.m. The SC medium (0.85% yeast nitrogen base without amino acids and with ammonium sulfate, 0.5% casamino acids, 0.4% NaOH, 2% glycerol, 1% succinic acid, 2% glucose, 0.005% histidine, 0.01% leucine, 0.01% uracil, 0.01% adenine hemisulfate) was used for \u003cem\u003eS. cerevisiae\u003c/em\u003e transformant selection. Agar was added at a final concentration of 1.5% for semi-solid media. Plasmid pUC57 was used for cloning experiments in \u003cem\u003eE. coli\u003c/em\u003e, and transformants were selected on LB-agar plates supplemented with 100 µg/ml ampicillin. For molecular cloning and gene expression in \u003cem\u003eS. cerevisiae\u003c/em\u003e, plasmid pYES3/CT was used.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStandard molecular and biochemical methods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStandard methods such as plasmid DNA and protein extractions, agarose and polyacrylamide gel electrophoresis, conventional PCR, and restriction digestions were performed as previously described [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] unless otherwise noted. T4 DNA ligase, PfuUltraII Fusion HS DNA polymerase, restriction endonucleases, DNase I, RNase A, and T4 polynucleotide kinase were purchased from Agilent Technologies, Thermo Scientific, and Life Technologies and were used according to the manufacturer's instructions. Transformations were performed by electroporation using a GenePulser XcellTM (BioRad, Hercules, CA, USA) equipment with settings of 1.8 kV, 25 µF, 200 Ω for \u003cem\u003eE. coli\u003c/em\u003e, and 1.5 kV, 25 µF, 200 Ω for \u003cem\u003eS. cerevisiae\u003c/em\u003e. When required, sample absorbance at different wavelengths was measured using Jasco UV-Vis spectrophotometer V-630 and Epoch 2 Microplate Spectrophotometer (Biotek, Winooski, VT, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePrediction of sequences encoding β-galactosidases\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protein sequences of β-galactosidases were downloaded from the UniProt database and used to build a local database using Geneious Prime software. The translated ORF sequences, predicted from the transcriptomes of eight Antarctic yeasts [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], were compared to the local database using BLASTp with the \"Bin into 'hashit' vs. 'no hit'\" option in the Geneious software. The \"hit sequences\" were then selected and compared by BLASTp to the UniProt database for annotation, applying a similarity threshold of ≥ 30%, using the Blosum62 cost matrix. Sequences annotated as related to β-galactosidase were selected for further analysis. The prediction of domains, families, or functional sites was performed using the InterProScan server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/search/sequence/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/search/sequence/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The DeepLoc-2.0 web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/DeepLoc-2.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/DeepLoc-2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict subcellular localization.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMaximum likelihood evolutionary analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eProtein sequences were aligned using MAFFT [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and trimmed using trimAl [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The phylogeny was inferred using IQ-TREE [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] with automatic model selection (MFP), and branch support was assessed using 1,000 ultrafast bootstrap replicates [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003e3D structural modeling and comparison\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe 3D protein models were constructed using the SWISS-MODEL server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the best ortholog identified as a template considering the following parameters: coverage ≥ 50%, similarity ≥ 30%, Global Model Quality Estimate (GMQE) ≥ 0.8. The quality of the models was assessed using VERIFY 3D [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] available at UCLA-DOE LAB-SAVES v6.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://saves.mbi.ucla.edu/\u003c/span\u003e\u003cspan address=\"https://saves.mbi.ucla.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eProtein structural properties related to flexibility, such as the number of hydrogen bonds, salt bridges, apolar solvent-accessible surface area (apoSASA), and content of secondary structures [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], were calculated in each model. The properties and parameters used in the ChimeraX software [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] for these calculations were: apoSASA (radius = 1.4 Å, peak density = 2), hydrogen bonds, and salt bridges (radius = 0.075 Å, dashes = 6, distance tolerance = 0.4 Å, angle tolerance = 20º). The secondary structure content was determined from PDB files using the Pfeature web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://webs.iiitd.edu.in/raghava/pfeature/sec.php\u003c/span\u003e\u003cspan address=\"https://webs.iiitd.edu.in/raghava/pfeature/sec.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The percentage of residues in regions predicted to be rigid (0), flexible (1), or very flexible (2) was calculated using MEDUSA [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Comparative superimposition analysis of protein 3D structures was performed using the Dali server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ekhidna2.biocenter.helsinki.fi/dali/\u003c/span\u003e\u003cspan address=\"http://ekhidna2.biocenter.helsinki.fi/dali/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCloning, expression, and purification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ORF sequence of the selected β-galactosidase was modified in silico according to the codon usage of \u003cem\u003eS. cerevisiae\u003c/em\u003e and then synthesized by Gene Universal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneuniversal.com/\u003c/span\u003e\u003cspan address=\"https://www.geneuniversal.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), including cloning into the vector pUC57 (named pUC57Bgal1) and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α by electroporation. The pUC57Bgal1 plasmid was purified from \u003cem\u003eE. coli\u003c/em\u003e culture and used as a template in a PCR reaction using primers Bgal1KpnI (5'-GTACggtaccATGGCTTCTTCTGATAAAAACTTCCCT-3') and Bgal1ApaI (5'-GTACgggcccTTCATCAGCTTCCAAAGAGTAAAC-3'), which contained restriction sites for \u003cem\u003eKpnI\u003c/em\u003e and \u003cem\u003eApaI\u003c/em\u003e (indicated in lower case), respectively. The PCR products were resolved on a 1% agarose gel. The amplicon band of the expected length (1,709 bp) was purified from the gel using the Gene Clean protocol [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], ligated into the pYES3/CT vector, and transformed by electroporation into \u003cem\u003eE. coli\u003c/em\u003e DH5α. The recombinant plasmid pYES3/CTbgal1 was purified from \u003cem\u003eE. coli\u003c/em\u003e cultures and transformed into \u003cem\u003eS. cerevisiae\u003c/em\u003e by electroporation. Transformants were selected on SC agar plates after incubation at 30 ºC for 16 h and verified by PCR using primers Gal1 (5’-AATATACCTCTATACTTTAACGTC-3’) and CYC1 (5’-GCGTGAATGTAAGCGTGAC3’). A \u003cem\u003eS. cerevisiae\u003c/em\u003e transformant clone was cultured in 50 ml SC medium for 48 h at 30°C. The culture was centrifuged at 5,230 g for 10 min, the cell pellet was suspended in 50 ml sterile distilled water, and centrifuged at 5,230 g for 5 min. The cell pellet was then inoculated into SC medium supplemented with 2% galactose to achieve an OD\u003csub\u003e600nm\u003c/sub\u003e of 0.4, incubated at 30°C, and culture aliquots of 50 ml were collected after 8, 24, and 48 h. These aliquots were centrifuged at 5,230 g for 10 min, the supernatant was discarded, and the cell pellet was stored at -50 ºC until further processing. Five ml of the cell pellet was suspended in one volume of 10 mM Tris-HCl, 0.5 M NaCl, and 10 mM imidazole, pH 8.0, and one ml of cell suspension was aliquoted into lysis tubes. Two hundred µl of 0.5 mm diameter glass beads (Biospec, Bartlesville, OK, USA) were added to each tube, and the cells were disrupted using a Mini-Beadbeater-16 (Biospec, Bartlesville, OK, USA) with 7 cycles of agitation for 3 min, followed by 3 min of incubation on ice. The supernatant obtained after centrifugation of the sample at 19,745 g for 15 min was mixed with 100 ml of 10 mM Tris-HCl, 0.5M NaCl, 10 mM imidazole, pH 8.0, and then loaded into a HisTrap FF crude column (GE Healthcare, Chicago, IL, USA). Samples were eluted using an Akta prime plus equipment (GE Healthcare, Chicago, IL, USA) with a mobile phase of 20 mM Tris-HCl, 0.5 M NaCl, pH 8.0, at a flow rate of 1 ml/min and an imidazole gradient from 10 to 500 mM. One ml aliquots were collected for subsequent analyses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEnzyme activity assays\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEnzyme assays were performed under different temperatures and pH conditions. The pH was adjusted using two sets of buffers: i) 0.1 M sodium acetate buffer (pH 5.0 and 5.5); 100 mM phosphate buffer (pH 6.0 to 7.0), containing 10 mM KCl, 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e; ii) phosphate-citrate buffer (pH 5.0 to 7.0).\u003c/p\u003e\u003cp\u003eFor the β-galactosidase activity determination [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], 7.5 µl of protein sample (25 µg/ml) was mixed with 200 µl of the appropriate pH buffer, and 70 µl of 4 mg/ml o-nitrophenyl-β-D-galactopyranoside (ONPG) was added. The mixture was incubated at different temperatures, and the reaction was stopped at different times by adding 500 µl of 1.0 M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The release of o-nitrophenol from ONPG was measured by absorbance at 420 nm. For lactose hydrolysis determination, a solution of 5% lactose was used, the reaction was stopped by incubation at 100 ºC for 10 min, and the release of glucose was quantified using the Glucose Assay Kit abcam (Cambridge, UK), according to the manufacturer’s instructions.\u003c/p\u003e\u003cp\u003eFor the α-Glucosidase activity determination [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], 7.5 µl of protein sample (25 µg/ml) was mixed with 50 µl of buffer pH 5.0, pH 6.0, or pH 7.0 and 17.5 µl of 1.5 mg/ml of 4-nitrophenyl-α-D-glucopyranoside (PNPG). The reaction mixture was incubated at 35°C for 1 h and then stopped by adding 125 µl of 1 M NaHCO\u003csub\u003e3\u003c/sub\u003e. The release of pNP was determined by absorbance at 405 nm.\u003c/p\u003e\u003cp\u003eFor the β-Glucuronidase [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and β-Glucosidase [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] activity determinations, 25 µl of enzyme sample was mixed with 150 µl of buffer pH 5.0, pH 6.0, or pH 7.0, and 25 µl of 1 mg/ml 4-methylumbelliferyl-β-D-glucuronide (4MBG) or 4-methylumbelliferyl-β-D-glucoside (4MBDG), respectively. The reaction was incubated at 35°C for 1 h and stopped by adding 1.3 ml of 0.1 M glycine-NaOH buffer pH 10.4. The reaction was then exposed to UV light using a CUV40A transilluminator (ClinX, Shanghai, China) to observe fluorescence emission, indicating the enzymatic release of 4-methylumbelliferone.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein thermal unfolding kinetics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThermal protein unfolding kinetics were assessed as previously described [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] using a CFX96 Real-Time System (BioRad, Hercules, CA, USA) in 96-well PCR microplates. In each well, 13 µl of the appropriate buffer at the desired pH, 10 µl protein sample (1 µg/µl), and 2 µl 125X concentrated SYPRO Orange were added. The temperature was increased from 5 ºC to 95 ºC with a ramp of 1 ºC per min. The excitation wavelength was set to 470 nm, and the emission was registered at 569 nm.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePrediction of β-galactosidases in the ORFeomes of Antarctic fungi and structure of the putative gene and enzyme.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBy mining the transcriptomes of eight Antarctic fungi for ORFs encoding β-galactosidases, a putative ORF of 1,692 nt was identified in the transcriptome of \u003cem\u003eTetracladium\u003c/em\u003e sp. and mapped to the corresponding genome to determine the gene structure. The encoding gene \u003cem\u003etspbgal\u003c/em\u003e spans 1,884 nt (NCBI accession number PQ310115) and consists of four exons, resulting in a coding sequence of 1,692 nt (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A strong Kozak sequence was identified, including the translation start codon ATG. Tspbgal was predicted to be a cytoplasmic protein (70% probability), and the predicted domains were glycosyl hydrolase family 42, a transglycosylase, and a domain of unknown function 5597 (DUF5597).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMultiple alignment and phylogenetic analyses were performed with Tspbgal and 89 related β-galactosidases, with sequences from 558 to 601 residues identified through BLASTp searches in the NCBI Protein Reference and Swiss-Prot databases. Tspbgal grouped with β-galactosidases from the species \u003cem\u003eBaudoinia panamericana, Cyphellophora europaea, Exophiala dermatitidis, Capronia coronata\u003c/em\u003e, and \u003cem\u003ePseudogymnoascus\u003c/em\u003e sp., but with low bootstrap support (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA three-dimensional model of Tspbgal was constructed by homology modeling using \u003cem\u003eCellvibrio japonicus\u003c/em\u003e Ueda107 tetrameric β-galactosidase (Bgl35A, PDB ID: 4D1I) as the template, the best ortholog found in the Swiss Model Server. Superposition analysis of the monomers from the predicted model of Tspbgal and Bgl35A using the Dali server aligned 492 C-alpha atoms showed a sequence identity of 34%, a Z-score of 61.1, and an r.m.s.d. value of 1.1 \u0026Aring;. Several structural differences (labeled SD1 to SD8 in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) were observed by superimposing the three-dimensional structures of the monomers from the crystal structure of Bgl35A and the predicted model for Tspbgal. Four loops in Tspbgal (SD1, SD2, SD3, and SD8) were 4, 21, 11, and 18 residues longer than the corresponding loops in Bgl35A, respectively. Loops SD5 and SD7 in Tspbgal were 13 and 8 residues shorter than their counterparts in Bgl35A, respectively. The helix SD4 and the helix-loop-helix SD6 structures observed in Bgl35A were present as loops in Tspbgal and were shorter by 3 and 13 residues, respectively. To analyze the active sites, the crystal structure of Bgl35A was soaked with the iminosugar 1-deoxygalactonojirimycin (1,5-dideoxy-1,5-imino-D-galactitol, DGJ) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Residues N67, K134, N135, and N204, which coordinate DGJ in the active site cavity of Bgl35A, are conserved in Tspbgal, corresponding to residues N36, K104, N105, and N179, respectively. Residue N383 of Bgl35A, which interacts with O6 of DGJ at a calculated distance of 3.2 \u0026Aring;, is absent in Tspbgal. However, other nearby residues in Tspbgal may potentially form hydrogen bonds with O6 of DGJ. The best candidate is residue Y334, which might form a hydrogen bond with DGJ of 4.3 \u0026Aring;. Additionally, residues Q35 and R358 from Tspbgal, located in a region that is predicted to be flexible, may form hydrogen bonds with O6 of DGJ of 7.8 \u0026Aring; and 8.4 \u0026Aring;, respectively, also positioning them as candidates to coordinate O6 of DGJ. The proposed catalytic residues E205 and E349 in Bgl35A are structurally conserved in Tspbgal, corresponding to residues E180 and E356, respectively. The conservation of these residues can be observed in other 89 fungal β-galactosidases related to Tspbgal, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, including the putative active site residues of Tspbgal that are conserved relative to Bgl35A, as well as residue Y334 in Tspbgal, which likely coordinates O6 of DGJ.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePurification and enzymatic characterization of recombinant Tspbgal\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs described in the Materials and Methods section, the \u003cem\u003eS. cerevisiae\u003c/em\u003e INVSc1 strain was transformed with the pYES3/CTbgal1 vector by electroporation, and transformants were selected by PCR with the Gal1/CYC1 primer pair (amplicon size\u0026thinsp;~\u0026thinsp;2,000 bp; see Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Tspbgal expression was induced, culture aliquots were collected at 8, 24, and 48 h post-induction, proteins were extracted from cell pellets, and analyzed by SDS-PAGE and for β-galactosidase activity. A protein band of the expected size (63kDa) was observed in the SDS-PAGE, and β-galactosidase activity was detected in all protein samples (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). Recombinant Tspbgal was purified by His-tagged affinity chromatography on nickel columns using an imidazole gradient (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A protein with the expected molecular weight (63kDa) eluted in fractions between 8 and 16 min, corresponding to the fractions displaying β-galactosidase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The activity of the purified β-galactosidase was evaluated at temperatures ranging from 10\u0026deg;C to 60\u0026deg;C and pH levels ranging from 5.0 to 7.0, with and without a buffer containing MgSO₄. The enzyme activity was higher at temperatures between 35\u0026ordm;C and 40\u0026ordm;C and pH levels from 5.5 to 6.6, with the highest activity at 35\u0026ordm;C and pH 6.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The performance of β-galactosidase activity across pH levels was not affected in buffers with or without MgSO₄. The enzyme maintained at least 60% of its maximum activity at temperatures ranging from 25\u0026ordm;C to 40\u0026ordm;C, and pH levels between 5.0 and 7.0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). It also retained 33% of its maximum activity at 10\u0026ordm;C and pH 6.0. The enzyme showed high stability at 35\u0026ordm;C and pH 6.0, retaining 87% of its maximum activity after 4 h and 67% after 24 h at these conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe specificity of recombinant Tspbgal was evaluated in assays performed using PNPG (for α-glucosidase activity), 4MBG (for β-glucuronidase activity), and 4MBDG (for β-glucosidase activity) as substrates at 35\u0026ordm;C and pH 5.0, 6.0, and 7.0. No activity was detected for any of these substrates under these conditions (data not shown).\u003c/p\u003e\u003cp\u003eThe ability of Tspbgal to hydrolyze lactose was tested using a 5% solution of lactose at 35\u0026deg;C and a pH of 6.0, conditions that yield the maximum enzyme activity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the hydrolysis of approximately 40\u0026ndash;50% of the lactose was achieved after 3\u0026ndash;4.5 h of incubation, and nearly 70% after 11.5 h. When the lactose hydrolysis was tested at different temperatures at pH 6.0, the Tspbgal retained a residual activity of 52% and 59% at 22 \u0026ordm;C and 45 \u0026ordm;C, respectively. The highest lactose hydrolysis by Tspbgal was observed at pH 5.0 at 35\u0026deg;C, with residual activity of around 80% at pH 6.0 and 7.0.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThermal unfolding of Tspbgal and relationship between the structural properties of fungal β-galactosidases and their optimal activity temperature.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe thermal unfolding of Tspbgal was assessed at pH levels ranging from 5.0 to 9.0. A variation in the thermal unfolding curves was observed, with lower thermal stability occurring at pH values above 7.0 (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The highest melting temperatures (Tm) were observed at pH 5.5 (Tm\u0026thinsp;=\u0026thinsp;49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C) and pH 6.0 (Tm\u0026thinsp;=\u0026thinsp;49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u0026deg;C), which were slightly lower at pH 5.0 (Tm\u0026thinsp;=\u0026thinsp;46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C) and pH 6.6 (Tm\u0026thinsp;=\u0026thinsp;47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C). The lowest Tm values were observed at pH 8.0 to 9.0, ranging from 35 \u0026ordm;C to 38 \u0026ordm;C. The β-galactosidase activity was evaluated at pH levels from 5.0 to 7.0 and temperatures from 10 \u0026ordm;C to 60 \u0026ordm;C and expressed as a percentage with respect to the maximum activity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the effect of temperature on enzyme activity correlated with its effect on protein structure, since maximum enzyme activity was observed under conditions when still no protein unfolding was detected (35 \u0026ordm;C to 40 \u0026ordm;C), and the interpolated 50% of enzyme activity corresponded with 50% of protein unfolding.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe structural properties associated with protein flexibility that have been proposed to enhance enzyme activity at lower temperatures were determined in 47 β-galactosidases, for which data on their optimal temperature for enzyme activity (OTEA) and the optimal temperature for growth (OTG) of the producer organism were available, and analyzed by principal component analysis (PCA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the two principal components explain 53.7% of the variance, and the OTG was the parameter most related to OTEA. Some positive relations to OTEA were observed regarding structural protein properties, including solvent accessible surface area (SASA), the percentage of residues classified as medium flexible (Med1), and the percentage of α-helix (Alpha). Negative relations were observed for the number of hydrogen bonds (Hbond) and salt bridges (SaltBrid), apolar solvent accessible surface area (apoSASA), and the percentage of β-sheet. Spearman's correlation was calculated for each parameter versus OTEA (see Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Positive correlations with a p-value of \u0026le;\u0026thinsp;0.05 were found for OTG (0.6) and the percentage of α-helix (0.4). Negative correlations were found for apoSASA (-0.4) and the percentage of β-sheet (-0.4).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe search of the ORFeomes from eight Antarctic fungi revealed an ORF encoding a β-galactosidase in the \u003cem\u003eTetracladium\u003c/em\u003e sp. ORFeome. Genome mapping defined a 1,884-nt gene consisting of four exons with a 1,692-nt coding sequence. The predicted 564-residue β-galactosidase from \u003cem\u003eTetracladium\u003c/em\u003e sp. showed low sequence identity with other described β-galactosidases; its closest relatives were found in the genera \u003cem\u003eHyaloscypha\u003c/em\u003e, \u003cem\u003eScytalidium\u003c/em\u003e, \u003cem\u003eHyphodiscus\u003c/em\u003e, \u003cem\u003eHelotiales\u003c/em\u003e, \u003cem\u003eMollisia\u003c/em\u003e, and \u003cem\u003eCadophora\u003c/em\u003e. These are all mycorrhizal and saprophytic filamentous fungi [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], and one species, \u003cem\u003eCadophora malorum\u003c/em\u003e, has also been isolated from a cold environment, specifically from lakes on the Antarctic Peninsula [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The best template for three-dimensional structure modeling of the β-galactosidase from \u003cem\u003eTetracladium\u003c/em\u003e sp. was the GH35 β-galactosidase Bgl35A from \u003cem\u003eC. japonicus\u003c/em\u003e. The two β-galactosidases structurally differed primarily in their unstructured loops. Four residues were conserved in the active site of both enzymes. It is likely that the role of residue N383 in the β-galactosidases from \u003cem\u003eC. japonicus\u003c/em\u003e in their interaction with DGJ is fulfilled by residue Y334 in the β-galactosidase from \u003cem\u003eTetracladium\u003c/em\u003e sp. These residues were conserved across the 89 fungal β-galactosidases more closely related to that from \u003cem\u003eTetracladium\u003c/em\u003e sp.\u003c/p\u003e\u003cp\u003eIn general, considering the results obtained with ONPG and lactose as substrates, the recombinant \u003cem\u003eTetracladium\u003c/em\u003e sp. β-galactosidase exhibits higher activity at temperatures between 25 and 40\u0026deg;C and pH between 5.5 and 7.0, and notably retains 25% of its activity at 10 \u0026ordm;C. These are promising properties for its potential application in the treatment of milk to reduce lactose content at lower temperatures, helping to reduce energy costs and minimize contamination. On the other hand, the pH range where high activity was observed matches that of whey. Whey is a byproduct of the dairy industry, which, depending on the treatment, can be acidic (pH 5.0) or sweet (pH 6.0\u0026ndash;7.0), and has been proposed to be used for the production of whey protein hydrolysates and other value‑added products [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The typical whey composition contains 4.5\u0026ndash;5% w/v lactose, which can be used for biofuel production. For example, the combined use of β-galactosidase from \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e was used to produce 28.9 g/L ethanol during fermentation at 35\u0026deg;C [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Furthermore, enzymes with β-galactosidase activity can also carry out transgalactosylations to produce prebiotics, such as galactooligosaccharides, which can be used to support the growth of beneficial microorganisms in the human gastrointestinal tract [\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIt has been reported that the activity of β-galactosidases is influenced by the presence of various ions. For example, enzymes from \u003cem\u003eKluyveromyces lactis\u003c/em\u003e and \u003cem\u003eKluyveromyces fragilis\u003c/em\u003e require Mn\u0026sup2;⁺, Na⁺, and Mg\u0026sup2;⁺ for optimal activity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], while the enzyme from \u003cem\u003eArthrobacter oxydans\u003c/em\u003e SB showed improved activity in the presence of Mn\u0026sup2;⁺ or Fe\u0026sup2;⁺ [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In this work, the β-galactosidase from \u003cem\u003eTetracladium\u003c/em\u003e sp. activity was similar in the presence or absence of Mg\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRegarding psychrophilic enzymes, most are inactivated by temperature before their structure unfolds, which reveals the heat lability of the active site. This is different in mesophilic enzymes, where thermal inactivation correlates with protein unfolding [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. As \u003cem\u003eTetracladium\u003c/em\u003e sp. is a cold-adapted fungus that was isolated from Antarctic soil, it was expected that the behavior of its β-galactosidase would resemble that of psychrozymes. However, the enzyme inactivation by temperature coincides with protein unfolding. Therefore, \u003cem\u003eTetracladium\u003c/em\u003e sp. β-galactosidase is structurally more like enzymes from mesophiles, such as \u003cem\u003eC. japonicus\u003c/em\u003e, which has optimal activity at pH 6.5 and thermal stability between 35\u0026deg;C and 45\u0026deg;C, with a rapid decrease in activity at 55\u0026deg;C and above [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA novel β-galactosidase was identified in the Antarctic fungus \u003cem\u003eTetracladium\u003c/em\u003e sp., which was successfully expressed in \u003cem\u003eS. cerevisiae\u003c/em\u003e. The recombinant enzyme showed structural and thermal stability properties similar to mesophilic enzymes but with improved performance at temperatures below 35 \u0026ordm;C, a property desirable in the dairy industry to reduce production costs and microbial contamination.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eapoSASA: apolar solvent-accessible surface area\u003c/p\u003e\n\u003cp\u003eONPG: o-nitrophenyl-\u0026beta;-D-galactopyranoside\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePNPG: 4-nitrophenyl-\u0026alpha;-D-glucopyranoside\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4MBG: 4-methylumbelliferyl-\u0026beta;-D-glucuronide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4MBDG: 4-methylumbelliferyl-\u0026beta;-D-glucoside\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDGJ: 1,5-dideoxy-1,5-imino-D-galactitol\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the data generated and used in this work are included in the manuscript and are available as supplementary material.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Grant Fondecyt 1230427 from the Agencia Nacional de Investigacion y Desarrollo de Chile.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMB, JA, and VC designed the study and discussed the results. FG performed the experiments, protein structural modeling, and characterization. FG and MB performed the bioinformatics and biostatistical analyses. 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\u003cem\u003e108\u003c/em\u003e:354. 10.1007/s00253-024-13117-2\u003c/li\u003e\n\u003cli\u003eMano MCR, Neri-Numa IA, da Silva JB, Paulino BN, Pessoa MG, Pastore GM: Oligosaccharide biotechnology: an approach of prebiotic revolution on the industry. \u003cem\u003eAppl Microbiol Biotechnol.\u003c/em\u003e 2018; \u003cem\u003e102\u003c/em\u003e:17-37. 10.1007/s00253-017-8564-2\u003c/li\u003e\n\u003cli\u003eWang H, Yang R, Hua X, Zhao W, Zhang W: Enzymatic production of lactulose and 1-lactulose: current state and perspectives. \u003cem\u003eAppl Microbiol Biotechnol.\u003c/em\u003e 2013; \u003cem\u003e97\u003c/em\u003e:6167-6180. 10.1007/s00253-013-4998-3\u003c/li\u003e\n\u003cli\u003eBanerjee G, Ray A, Hasan KN: Is divalent magnesium cation the best cofactor for bacterial \u0026beta;-galactosidase. \u003cem\u003eJournal of Biosciences.\u003c/em\u003e 2018; \u003cem\u003e43\u003c/em\u003e:941-945. 10.1007/s12038-018-9814-x\u003c/li\u003e\n\u003cli\u003eLarsbrink J et al.: Structural and enzymatic characterization of a glycoside hydrolase family 31 \u0026alpha;-xylosidase from \u003cem\u003eCellvibrio japonicus\u003c/em\u003e involved in xyloglucan saccharification. \u003cem\u003eBiochem J.\u003c/em\u003e 2011; \u003cem\u003e436\u003c/em\u003e:567-580. 10.1042/bj20110299\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"β-galactosidase, Tetracladium sp., psychrophiles, protein flexibility, dairy industry","lastPublishedDoi":"10.21203/rs.3.rs-7074902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7074902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eβ-Galactosidases are widely used in the dairy industry to produce lactose-free milk and prebiotics such as galacto-oligosaccharides and lactulose. Since commercial β-galactosidases have optimal activity at 35 to 70 \u0026ordm;C, β-galactosidases that are highly active at lower temperatures are desirable to reduce production costs and minimize microbial contamination in industrial processes. Potential sources of cold-active β-galactosidases are microorganisms living in cold environments such as Antarctica. The aim of this work was to identify genes encoding β-galactosidases from Antarctic fungi and express them in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e for their characterization.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBy searching 16 ORFeomes from eight Antarctic fungi, an ORF encoding β-galactosidase was identified in \u003cem\u003eTetracladium\u003c/em\u003e sp. (Tspgal), and the gene structure was determined in the corresponding genome. Phylogenetic analyses indicate that this is a novel β-galactosidase closely related to β-galactosidases from saprophytic fungi. The closest β-galactosidase with a known 3D structure was from \u003cem\u003eCellvibrio japonicus\u003c/em\u003e, which differed from that from \u003cem\u003eTetracladium\u003c/em\u003e sp. mainly in unstructured regions, with most of the active site residues conserved. The Tspgal expressed in \u003cem\u003eS. cerevisiae\u003c/em\u003e showed maximum activity from 25 \u0026ordm;C to 40 \u0026ordm;C and from pH 5.5 to pH 7.0 (maximum at 35 \u0026ordm;C and pH 6.0). At pH 6.0, the recombinant enzyme retained 25% and 36% of its activity at 10\u0026ordm;C and 50\u0026ordm;C, respectively. The thermal enzymatic inactivation of the recombinant β-galactosidase correlated with its thermal protein unfolding, a behavior similar to that observed for mesophilic enzymes. Tspbgal hydrolyzed lactose optimally at pH 5.0 at 35\u0026deg;C, retaining about 80% of its activity at pH 6.0 and 7.0, conditions that coincide with the pH of whey, a major dairy byproduct and potential source of value‑added products derived from lactose.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA novel β -galactosidase was identified in the ORFeome of the Antarctic fungus \u003cem\u003eTetracladium\u003c/em\u003e sp., which was successfully expressed in \u003cem\u003eS. cerevisiae\u003c/em\u003e exhibiting structural and thermal stability properties comparable to mesophilic enzymes. The recombinant enzyme exhibited high activity at 25\u0026ndash;35 \u0026ordm;C and retained 25% of its maximum activity at 10 \u0026ordm;C, an attractive trait for reducing energy costs and minimizing microbial contamination in milk treatments.\u003c/p\u003e","manuscriptTitle":"Identification and characterization of a novel β-galactosidase active at low temperatures from the Antarctic fungus Tetracladium sp., expressed in Saccharomyces cerevisiae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 17:38:08","doi":"10.21203/rs.3.rs-7074902/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-25T11:01:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-23T11:55:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T12:45:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45541840571273274510813181635721006417","date":"2025-07-17T14:46:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143056778027725700172829432345547702662","date":"2025-07-17T00:16:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-16T11:01:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-11T09:55:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-11T09:55:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2025-07-08T12:27:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66acd327-3bb4-4a88-b2ef-6461bb6bc069","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:05:55+00:00","versionOfRecord":{"articleIdentity":"rs-7074902","link":"https://doi.org/10.1186/s12934-025-02850-6","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2025-10-14 15:57:25","publishedOnDateReadable":"October 14th, 2025"},"versionCreatedAt":"2025-07-18 17:38:08","video":"","vorDoi":"10.1186/s12934-025-02850-6","vorDoiUrl":"https://doi.org/10.1186/s12934-025-02850-6","workflowStages":[]},"version":"v1","identity":"rs-7074902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7074902","identity":"rs-7074902","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}