Characterisation of gluten-degrading prolyl endoprotease from Thermococcus kodakarensis

Radhakrishna Shetty, Claus Heiner Bang-Berthelsen, Klaudia Weronika Ciurkot, Mike Vestergaard, Per Mårten Hägglund, H.S. Prakash, Timothy John Hobley*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

There is increasing interest in gluten degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten degrading enzymes are of great interest. We have identified a new thermostable gluten degrading proline specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1 kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterise the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The VmaxKm and kcat values of the purified prolyl endoprotease were calculated to be 3.14 mM/s, 1.10 mM and 54 s-1 respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (alpha-gliadin) and SQQQFPQPQQPFPQQP (gamma-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up for new food processing possibilities and further the understanding of proline specific protease diversity.
Original languageEnglish
Article numberfnac006
JournalFEMS Microbiology Letters
Volume368
Issue number21-24
Number of pages7
ISSN0378-1097
DOIs
Publication statusPublished - 2021

Keywords

  • PEP
  • Hordein
  • Immunogenic peptide
  • Prolyl endoprotease

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