Download files
Abstract
Permanently cold environments are populated by a diversity of microorganisms that possess cold-adapted enzymes with potential biotechnological applications. Advances in molecular techniques have enabled bioprospecting of novel enzymes from extreme environments without tedious cultivation efforts. Cloning and sequencing of the metagenomic DNA from the water column of the meromictic Antarctic lake, Ace Lake, enabled enzyme identification by sequence-based searches and functional screening for enzyme activity. The sequence-based search for hydrolases using profile Hidden Markov Models indicated the presence of uncharacterized subtilases, lipases and glycoside hydrolases in the metagenomic dataset. Information from the bioinformatic analysis was utilized to perform targeted functional screening of clones containing the candidate hydrolase genes. Alongside this targeted approach, ~20000 clones from the Ace Lake metagenomic library were randomly screened via agar-based assay. As both functional screening approaches yielded very low positive identifications, the capacity of the sequence-based screening to detect bona fide hydrolases was further tested by cloning selected subtilase genes and overexpressing them in Escherichia coli. One of the overexpressed subtilase genes showed proteolytic activity when tested with N-succinyl-Ala-Ala-Pro-Phe-ρ-nitroanilide. Finally, in order to get a bigger picture of the abundance of subtilase and other peptidase genes in Ace Lake, taxonomic affiliation and relative abundance of all candidate peptidases were assessed. The Ace Lake peptidases composition was also compared to peptidases in Organic Lake and Southern Ocean metagenomic datasets. The analysis indicated an abundance of diverse metallopeptidases and serine peptidases in all three datasets. Peptidases related to protein and membrane regulatory control and chaperones with peptidase activity were the most abundant groups, which are linked to microbial survival as a response to environmental stress. In conclusion, this study revealed the advantage of cultivation-independent methods for bioprospecting enzymes from cold environments, and established a foundation for future functional studies of other enzymes.