Cell-envelope proteinases of lactic acid bacteria catalyse the first step in the proteolytic system. They break down proteins from the surrounding media into polypeptides, which are transported via the cells peptide transport system into the cell for further processing. To date, research suggests that changes in the physicochemical properties of residues within the active site region of cell-envelope proteinases are responsible for differences in experimentally determined proteolytic profiles.

PrtP’s are a type of cell-envelope proteinase and are ideal templates for engineering tailored substrate specificity. I have focused on the PIII-type PrtP from Lactococcus cremoris, which has multiple domains, including an enzymatically active protease domain (PR domain). This research investigates whether there are non-conserved residues, particularly within the PR domain, that can be targeted to modify substrate specificity or catalytic activity. This is desirable because it will identify residues that can be targeted in protein engineering tailored to specific substrates. For example, to hydrolyse proteins that comprise plant-based milks.

Using multiple sequence alignments and visualization of AlphaFold models, four residues within the PR domain that may be important for substrate specificity or catalytic activity are identified. Surprisingly, during this analysis an extended duplicate region within the PIII-type enzymes cell wall spanning domain was identified that may be a defining characteristic of PIII-type PrtPs. Given that the PIII-type PrtP from Lactococcus cremoris has recently been suggested as a promising template for site directed engineering of bespoke CEP-based constructs this work undertook bioinformatic analysis of this protein and attempted to express and purify constructs of this protein. Limitations including fragmentation of the construct and aggregation prior to size exclusion chromatography resulted in being unable to purify useful amounts of the constructs for use in characterization.

In conclusion, potentially promising residues of PIII-type PrtP from Lactococcus cremoris that could be used for substrate tailored protein engineering were identified, as well as confirming the catalytic site residues. This information can be used by others to engineer PIII-type PrtP enzymes by substituting these residues to alter the catalytic activity and/or substrate specificity of the protein. This could have commercial utility by developing active constructs with specific substrate specificity that can be used, for example, in manufacturing of dairy alternatives, which had a market value of over US$2.4 Billion in 2023. Other important applications are as diverse as enrichment of foods via the generation of bioactive compounds and the development of enzymes that can break down waste for use in bioremediation efforts.