Projects per year
Abstract
Food waste and industrial side streams with high protein contents are potential and widely unexplored sources for new food ingredients. In particular, peptides liberated by proteolysis may hold unidentified latent bioactivities. However, protease selection for this liberation process is extremely challenging, as liberation of target peptides will require specific proteolysis. Microbes are experts in spontaneous food fermentation why they may contain proteases with desired functionalities. Auxotrophic lactic acid bacteria (LAB) have adapted to diverse nutrient rich environments where they require peptide availability and uptake. Interestingly, extracellular proteases of some LABs generate sufficient peptide supply by hydrolyzing surrounding proteins, but they often display high substrate selectivity. For instance, extracellular proteases of Lactococcus lactis are highly selective towards certain caseins. Screening other microbes may reveal novel proteases with new desired specificities that may be substantiated further through bioengineering. Nevertheless, identification of proteolysis specific for certain proteins or peptide products will require efficient high-throughput screening assays to characterize new proteolytic activity.
Here, we present a fluorescence-based assay to measure proteolysis of the main potato protein, patatin, which was recently shown to embed emulsifying peptides. This patatin-specific assay detects protease activity with high sensitivity by measuring the increase in fluorescence emission. Emission increases due to the decreased homo-FRET (fluorescence resonance energy transfer), which results from proteolysis of extensively labelled patatin. The assay can easily be performed in a microplate format to achieve reproducible, high-throughput screening of proteolytic activity. We have validated the microplate format of the assay, which detects patatin-specific proteolytic activity efficiently by a variety of commercial proteases. By applying bottom-up/middle-down mass spectrometry-based proteomics, we have been able to map cleavage patterns for the assayed proteases. Additionally, we investigated the effect of protein labelling on proteolytic activity by comparing cleavage patterns of labelled and non-labelled patatin. We show that our approach combining high-throughput homo-FRET assays and mass spectrometry are useful to characterize specific activity and cleavage patterns of isolated proteases in vitro, which can easily be adapted for other proteases and substrates. Our vision is to adapt this assay for screening of cell cultures to facilitate characterization of e.g. membrane-bound extracellular protease activity.
Here, we present a fluorescence-based assay to measure proteolysis of the main potato protein, patatin, which was recently shown to embed emulsifying peptides. This patatin-specific assay detects protease activity with high sensitivity by measuring the increase in fluorescence emission. Emission increases due to the decreased homo-FRET (fluorescence resonance energy transfer), which results from proteolysis of extensively labelled patatin. The assay can easily be performed in a microplate format to achieve reproducible, high-throughput screening of proteolytic activity. We have validated the microplate format of the assay, which detects patatin-specific proteolytic activity efficiently by a variety of commercial proteases. By applying bottom-up/middle-down mass spectrometry-based proteomics, we have been able to map cleavage patterns for the assayed proteases. Additionally, we investigated the effect of protein labelling on proteolytic activity by comparing cleavage patterns of labelled and non-labelled patatin. We show that our approach combining high-throughput homo-FRET assays and mass spectrometry are useful to characterize specific activity and cleavage patterns of isolated proteases in vitro, which can easily be adapted for other proteases and substrates. Our vision is to adapt this assay for screening of cell cultures to facilitate characterization of e.g. membrane-bound extracellular protease activity.
Original language | English |
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Publication date | 28 Jan 2020 |
Publication status | Published - 28 Jan 2020 |
Event | Microbial Food and Feed Ingredients: Enzymes for food and feed applications - University of Copenhagen, Copenhagen, Denmark Duration: 15 Apr 2020 → 17 Apr 2020 https://miffi.org/ |
Conference
Conference | Microbial Food and Feed Ingredients |
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Location | University of Copenhagen |
Country/Territory | Denmark |
City | Copenhagen |
Period | 15/04/2020 → 17/04/2020 |
Internet address |
Fingerprint
Dive into the research topics of 'Characterizing patatin specific protease activity by high-throughput homo-FRET assay and mass spectrometry'. Together they form a unique fingerprint.Projects
- 1 Finished
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PROVIDE: Protein valorization through informatics, hydrolysis, and separation
Gregersen, S., Overgaard, M. T., Hansen, E. B., Bang-Berthelsen, I., Jacobsen, C., García Moreno, P. J., Marcatili, P. & Yesiltas, B.
01/09/2017 → 30/12/2022
Project: Research
Activities
- 1 Talks and presentations in private or public companies
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PROVIDE and the Potatoes
Simon Gregersen (Lecturer)
10 Feb 2021Activity: Talks and presentations › Talks and presentations in private or public companies
Research output
- 1 Journal article
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Emulsifier peptides derived from seaweed, methanotrophic bacteria, and potato proteins identified by quantitative proteomics and bioinformatics
Yesiltas, B., Gregersen Echers, S., Lægsgaard, L., Brinch, M. L., Olsen, T. H., Marcatili, P., Overgaard, M. T., Hansen, E. B., Jacobsen, C. & García-Moreno, P. J., 15 Nov 2021, In: Food Chemistry. 362, 12 p., 130217.Research output: Contribution to journal › Journal article › Research › peer-review
Open AccessFile22 Citations (Scopus)41 Downloads (Pure)