IDENTIFICATION AND CHARACTERIZATION OF NOVEL Β-GALACTOSIDASES FROM A SEQUENCE-BASED METAGENOME ANALYSIS OF STABILIZATION PONDS

EBERHARDT MF; IRAZOQUI JM; AMADIO AF

Salta

Congreso; Reunión anual SAIB; 2019

SAIB

Whey, a by-product of the dairy industry, especially of cheese production, is composed of lactose (4.5?5 % w/v), proteins (0.6?0.8 % w/v), lipids (0.4?0.5?5 w/v), and mineral salts (8?10 % of dried extract). It represents an environmental problem because of the high BOD, COD and the large volume of production. The worldwide production of whey is currently estimated at nearly 180?190 million tons/year. Alternatively, it can be used as an attractive raw material for value-added products through physicochemical or enzymatic treatments. For example, Galactooligosaccharides (GOS) are non-digestible carbohydrates derived from lactose, composed of galactose monomers and a terminal glucose unit, varying in chain length and type of linkage. It has been demonstrated to have prebiotic properties, related to their impact on the composition and activity of the intestinal microbiota. GOS can be produced from lactose by the transgalactosylation activity of β-galactosidases (EC 3.2.1.23). Here, we report the identification, production, and characterization of novel genes encoding the putative lactose-modifying enzyme β-galactosidase from wholegenome shotgun metagenome from stabilization ponds of two small dairy companies in the central region of Santa Fe. The genes were identified via the conservation of catalytic domains, compared against the CAZy database. All hits corresponding to families with β-galactosidases activity (GH1, GH2, GH35, and GH42) were selected, using 70% identity and 70% sequence coverage as thresholds. These enzymes were taxonomically classified using the closest BLAST hit against the nr database. 189 candidate genes were found for GH1, GH2, GH35, and GH42 families. All sequences identified corresponded to bacteria. Candidates per family differ significantly: GH1 is the most represented with 100 candidates, while GH2 has 60, GH42 has 8 and GH35 only 6. Twelve candidates were selected for cloning and expression in heterologous systems. Candidate genes were amplified and cloned into plasmid vectors for inducible protein expression. Five putative β-galactosidase proteins were efficiently expressed. Clones allowed Escherichia coli DH5α to use lactose as a sole carbon source and hydrolyze 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) on agar plates. Four affinity-purified enzymes cleaved efficiently ortho-Nitrophenyl-β-galactoside (ONPG) substrate at physiological pH (5?7) with optimal temperatures between 30?37 °C. In conclusion, we successfully identified and characterized a novel group of mesophilic βgalactosidases from diary stabilization ponds metagenomes. Optimization of expression in heterologous systems, purification, and hydrolysis reaction conditions would allow evaluating its use for the production of lactose-free products or whey value-added by-products, such as GOS.