β-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization

Area of Science:

• Biotechnology
• Environmental Microbiology
• Enzyme Engineering

Background:

• Dairy industry wastewater, particularly cheese whey, poses environmental challenges due to high organic load.
• Whey's composition, rich in lactose and proteins, presents an opportunity for producing value-added products.
• Beta-galactosidases are key enzymes for lactose modification, yielding valuable components like glucose, galactose, and galactooligosaccharides.

Purpose of the Study:

• To identify and characterize novel beta-galactosidase genes from the metagenome of dairy industry stabilization ponds.
• To explore the potential of these enzymes for the valorization of dairy by-products.

Main Methods:

• Whole-genome shotgun sequencing of dairy pond metagenomes.
• Bioinformatic analysis for identifying candidate beta-galactosidase genes based on conserved catalytic domains (GH1, GH2, GH42).
• Cloning, expression, and biochemical characterization of selected beta-galactosidase enzymes.

Main Results:

• 394 bacterial candidate beta-galactosidase genes were identified from the metagenome.
• Twelve candidate genes were selected, cloned, and expressed, resulting in six functional enzymes.
• Five expressed enzymes demonstrated efficient cleavage of ortho-nitrophenyl-beta-galactoside and lactose.
• One novel beta-galactosidase exhibited superior activity compared to previously reported enzymes from metagenomic studies.

Conclusions:

• Novel mesophilic beta-galactosidases were successfully identified, cloned, and expressed from dairy stabilization pond metagenomes.
• These enzymes represent a promising alternative for the efficient conversion of dairy by-products into value-added compounds.
• The findings contribute to sustainable dairy industry practices through waste stream valorization.