Current Functional Metagenomic Approaches Only Expand the Existing Protease Sequence Space, but does not Presently Add Any Novelty to it

• Published in: Current microbiology 2015-01, Vol.70 (1), p.19-26
• Main Authors: Morris, Laura S, Marchesi, Julian R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bioinformatics; Biomedical and Life Sciences; Biotechnology; Ecosystems; EDTA (chelating agent); genes; Genomics; Life Sciences; Metagenomics; metalloproteinases; Microbiology; Molecular Sequence Data; Oxalobacteraceae - chemistry; Oxalobacteraceae - enzymology; Oxalobacteraceae - genetics; Peptide Hydrolases - chemistry; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; phenotype; Phylogeny; Proteases; Sequence Homology, Amino Acid; signal peptide; temperature; zinc
• ISSN: 0343-8651; 1432-0991
• DOI: 10.1007/s00284-014-0677-6

Proteases are a fundamental function in many organisms and thus many ecosystems and yet they are rarely obtained in functional metagenomic screens. Here, we have isolated an active protease gene (M1-2; 613 amino acids) which resided in a 38.4 kb fosmid clone that showed a classical protease-positive phenotype. It was classified as a zinc-dependent metalloprotease, with the closest annotated sequence as a neutral protease from Collimonas fungivorans (62 % similarity and 72 % homology). Further characterisation showed that its optimum temperature and pH were 42 °C and 8.0, respectively. Activity was inhibited by EDTA, but inhibition started to be reversed by excess Zn²⁺. A putative signal peptide was identified bioinformatically and this may be why this protease was successfully isolated using a functional metagenomic screen. Bioinformatic analysis shows that this does not represent a novel protease, but simply expands the current sequence space of known proteases.