NOVEL PROTEASES FROM SEQUENCE-BASED METAGENOMICS OF DAIRY INDUSTRIES STABILIZATION PONDS

JOSÉ MATÍAS IRAZOQUI; MARÍA FLORENCIA EBERHARDT; ARIEL AMADIO

Congreso; SAIB-SAMIGE Joint Meeting 2020; 2022

SAIB-SAMIGE

Whey, a by-product of the dairy industry, especially 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 problembecause of the high BOD and COD and the large volume of production. Alternatively, it can be used as an attractive rawmaterial for value-added products through physico-chemical or enzymatic modifications. A common product obtained fromwhey is whey protein concentrate (WPC), which is produced using membrane technologies. However, WPC can be furthertreated to produce whey protein hydrolysates (WPH), which have shown higher digestibility than WPC and has an increasedvalue. Most commercial proteases mix are composed of the serin-proteases families S01 and S08, from a limited number ofmicroorganisms. In this study, we present the identification, cloning and expression of proteases using a sequence-basedmetagenomics approach. Shortly, we sequenced the metagenome from stabilization ponds from two small dairy industries inSanta Fe province, and then the predicted genes were compared to the S01 and S08 proteases families using HMMer profilesobtained from public databases. We obtained a total of 186 S01 candidate proteins and 177 S08 candidates. These sequenceswere taxonomically classified using the BLAST best-hit against the nr database. When compared at the phyla level, thetaxonomic distribution of both protease families is similar, being Proteobacteria, Firmicutes, and Bacteroidetes the mostabundant. Based on the family and taxonomic diversity, 10 candidates were selected for cloning and expression in heterologoussystems. Up to now, 3 putative proteases were successfully expressed in Escherichia coli BL21 strains. All enzymes belongedto the S01 families, one from Bacteroidetes, one from Firmicutes, and the last one from the recently reported Patescibacteriaphylum. In conclusion, we were able to identify and produce enzymes using a sequence-based metagenomics approach. Eventhough further analyses are due to functionally characterize the enzymes and to obtain the peptide profile that each oneproduces, this study represents a first step in the production of novel enzymes from metagenomes to modify whey derivedproducts.