TY - JOUR

T1 - Characterization of gluten-degrading prolyl endoprotease from Thermococcus kodakarensis

AU - Shetty, Radhakrishna

AU - Bang-Berthelsen, Claus Heiner

AU - Ciurkot, Klaudia Weronika

AU - Vestergaard, Mike

AU - Hägglund, Per Mårten

AU - Prakash, Harishchandra S.

AU - Hobley, Timothy John

N1 - Publisher Copyright: © 2022 The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.

PY - 2021/12/1

Y1 - 2021/12/1

N2 - 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 characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km 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 (α-gliadin) and SQQQFPQPQQPFPQQP (γ-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 new food processing possibilities and further the understanding of proline-specific protease diversity.

AB - 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 characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km 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 (α-gliadin) and SQQQFPQPQQPFPQQP (γ-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 new food processing possibilities and further the understanding of proline-specific protease diversity.

KW - hordein

KW - immunogenic peptide

KW - PEP

KW - prolyl endoprotease

U2 - 10.1093/femsle/fnac006

DO - 10.1093/femsle/fnac006

M3 - Journal article

C2 - 35038331

AN - SCOPUS:85124576698

VL - 368

JO - F E M S Microbiology Letters

JF - F E M S Microbiology Letters

SN - 0378-1097

IS - 21-24

M1 - 368

ER -