Novel proteases from sequence-based metagenomics of dairy industries stabilization ponds

IRAZOQUI, JOSE MATÍAS; EBERHARDT MA. FLORENCIA; AMADIO, ARIEL

Congreso; Congreso Conjunto SAIB-SAMIGE 2020; 2020

SAIB-SAMIGE

Whey, a by-product of a dairy industry, especially cheese production, is compose 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 and COD and large volume of production. Alternatively, it can be used as an attractive raw material for value-added products through physico-chemical or enzymatic treatments. A common product obtained from whey is whey protein concentrate (WPC), using membrane technologies. However, WPC could be transform in whey protein hydrolysates (WPH) using proteases which have shown more activities than WPC and has increased value. Most commercial proteases mix are composed of the serin-proteases families S01 and S08, from a limited number of microorganisms. In this study, we present the identification, cloning and expression of proteases using a sequence-based metagenomics approach. Shortly, we sequence the metagenome from stabilization ponds from two small dairy industries in Santa Fe province, and then the predicted genes were compared to the S01 and S08 proteases families using HMMer profiles obtained from public databases. We obtained a total of 186 S01 candidate proteins and 177 S08 candidates. These sequences were taxonomically classified using the best hit of BLAST against the nr database. When compared to the phyla level, the taxonomic distribution of both proteases families are similar, being Proteobacteria, Firmicutes, and Bacteroidetes the most abundant. Based on the family and taxonomic diversity, 10 candidates were selected for cloning and expression in heterologous systems. Up to now, 3 putative proteases were successfully expressed in Escherichia coli BL21 strains. All enzymes belonged to the S01 families, one from Bacteroidetes, one from Firmicutes and the last one from the recently reported Patescibacteria phylum.In conclusion, we were able to identify and express enzymes using a sequence-based metagenomics approach. Even though further analysis is necesary to functionally characterize the enzymes and to obtain the peptide profile that each one produces, this study represents a first step in the production of novel enzymes to obtain WPH from cheese whey.