Extracellular proteases of lactic acid bacteria in targeted hydrolysis of plant proteins

Lactic acid bacteria (LAB) are a diverse group of bacteria that show remarkable adaptability to a variety of ecological niches. LAB have been utilized for centuries to prepare and preserve both animal-based and plant-based food products. In these nutrient-rich environments, LAB have developed demanding growth requirements, including those for several amino acids. Some LAB can utilize extracellular oligopeptides and proteins due to their adapted and highly regulated proteolytic system. Protein utilization is initiated by multi-domain extracellular proteases that are often attached to cell as cell envelope proteases (CEPs). CEPs are best characterized for their role in milk fermentation, wherein they hydrolyze milk proteins with different specificity and selectivity into oligopeptides that can be taken up by the cell and further degraded into amino acids. The underlying mechanisms of the proteolytic diversity of CEPs have yet to be discovered. Oligopeptides can possess a broad range of bioactivities, which are determined by their amino acid sequence. Bioactive peptides can be identified imbedded in a variety of food proteins where they are inactive until they become released through hydrolysis. Targeted hydrolysis of these identified bioactive peptides can be challenging to achieve and control because specific hydrolysis of the two peptide ends can be crucial for obtaining the desired peptide functionality without losing activity. A diverse set of highly specific proteases is required for targeted hydrolysis of bioactive peptides.

The overall aim of this PhD project is to identify and characterize extracellular proteases that can perform novel targetable proteolysis, resulting in the release of desired peptides as food ingredients from previously unexplored and protein-rich plant sources. In this PhD thesis, CEPs of LAB are explored as relevant and promising candidates to achieve the desired and diverse proteolytic activity needed for targeted release of bioactive oligopeptides. Side-streams from the potato starch industry contain accumulated potato proteins that imbed identified oligopeptides with emulsifying and antioxidant activity. Potato proteins are considered as a sustainable source for food-relevant bioactive peptides. Therefore, this PhD project investigates the use of CEPs in targeted hydrolysis of plant proteins.

In a literature study (Manuscript I), the extracellular proteolytic activity of traditional applied food microbes, including LAB, was reviewed in terms of their ability and potential application to hydrolyse plant proteins. The application potential of extracellular microbial proteases seems to be transferable from dairy and other animal-based food proteins to plant-based proteins. Few plant sources have been studied as targets for extracellular proteolysis of microbes. These plant proteins are commonly hydrolyzed to a limited extent. The proteolytic activity of microbes is rarely linked to their specific proteases, and vice versa.

This PhD thesis includes a comparative structure analysis with the purpose to investigate the prevalence and diversity of CEP homologs in LAB from different ecological niches (Manuscript II). CEP homologs were identified in LAB strains from various plant-based ecological niches. Together with well-known CEPs from dairy-derived and human-derived LAB strains, these CEPs were structurally characterized using AlphaFold modeling, a recent advance in protein structure prediction. Structural similarities of the multi-domain CEPs were found to be shared by LAB, regardless of species and habitat. The boundaries of the protein domains could be determined more precisely, revealing greater structural diversity of CEPs than previously known.

Furthermore, this PhD thesis discusses the need for protease assays using plant-based protein substrates. Two protease assays were developed with the purpose to analyze and screen CEP activity against potato protein. A bioassay used a chemically defined growth medium with high molecular potato protein as a nitrogen source to directly correlate LAB growth with CEP activity (Manuscript III). This growth medium meets the minimum requirements for lactococcal growth if the studied LAB strain has extracellular protease activity against the potato protein substrate. Lactococcus cremoris MG1363 was used as an isogenic background for expression of different CEPs, and proteolytic activity was further analyzed by the release of amino acids. A high-throughput biochemical assay was used to detect activity of purified proteases in real-time as an increase in fluorescent emission (Manuscript IV). The potato protein patatin was fluorescently labeled with an amine reactive dye, resulting in an efficient homo-FRET quenched protease substrate for analyzing patatin-specific hydrolysis. A proteomics and peptidomics approach identified several Lysine residues with different occupancies, indicating a heterogenic labeling pattern of patatin.

In conclusion, the results of this PhD project identify CEPs from plant-derived LAB strains, and their structural characterization contributes to a better understanding of the structural diversity of CEPs. Multiple domain architectures of CEPs are shared by LAB across ecological niches, indicating that CEPs can target various proteins. Nevertheless, the modest hydrolysis of potato proteins, like for other plant proteins, facilitated by dairy-associated CEPs suggests that CEP activity can be found among proteolytic LAB from environments with plant-based proteins. Targeted hydrolysis of potato proteins can be analyzed using the two presented assays, which may be applicable for future characterization of CEPs with novel proteolytic activities on plant-based proteins. The structural characterization of CEPs may support future bioengineering experiments aimed at analyzing and modulating the activity, stability and/or adhesion properties of CEPs.