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Background <p>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&#xa0;°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 <i>Saccharomyces cerevisiae</i> for their characterization.</p> Results <p>By searching 16 ORFeomes from eight Antarctic fungi, an ORF encoding β-galactosidase was identified in <i>Tetracladium</i> 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 <i>Cellvibrio japonicus</i>, which differed from that from <i>Tetracladium</i> sp. mainly in unstructured regions, with most of the active site residues conserved. The Tspgal expressed in <i>S. cerevisiae</i> showed maximum activity from 25&#xa0;°C to 40&#xa0;°C and from pH 5.5 to pH 7.0 (maximum at 35&#xa0;°C and pH 6.0). At pH 6.0, the recombinant enzyme retained 25% and 36% of its activity at 10&#xa0;°C and 50&#xa0;°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&#xa0;°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.</p> Conclusions <p>A novel β-galactosidase was identified in the ORFeome of the Antarctic fungus <i>Tetracladium</i> sp., which was successfully expressed in <i>S. cerevisiae</i> exhibiting structural and thermal stability properties comparable to mesophilic enzymes. The recombinant enzyme exhibited high activity at 25–35&#xa0;°C and retained 25% of its maximum activity at 10&#xa0;°C, an attractive trait for reducing energy costs and minimizing microbial contamination in milk treatments.</p>

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

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  • 发表日期：

    2025年10月14日

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  • Fernando Gutierrez,
  • Jennifer Alcaino,
  • Victor Cifuentes,
  • Marcelo Baeza

摘要

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 °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 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 °C to 40 °C and from pH 5.5 to pH 7.0 (maximum at 35 °C and pH 6.0). At pH 6.0, the recombinant enzyme retained 25% and 36% of its activity at 10 °C and 50 °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 °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 °C and retained 25% of its maximum activity at 10 °C, an attractive trait for reducing energy costs and minimizing microbial contamination in milk treatments.