Isolation, purification and kinetic characterization of prolyl endopeptidase from Titicum aestivum

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dc.contributor.advisorBradley, Gen
dc.contributor.authorAbrahams, Adriam Marken
dc.date.accessioned2026-08-12T23:25:36Z
dc.date.available2026-08-12T23:25:36Z
dc.date.issued2013en
dc.description.abstractPEP activity has been described in several locations and has mostly been linked to a variety of neurological disorders such as schizophrenia, amnaesia, depression as well as other disease states such as anorexia nervosa, bulimia nervosa and blood pressure regulation. The enzyme has also been previously isolated from a variety of archae, microorganisms and several eukaryotic species but no prolyl endopeptidases have been isolated from plants. Plants have high levels of proline and glutamine rich peptides in seeds. We therefore hypothesize plants must express PEPs during germination. Bioinformatics tools were used to identify known PEPs and putative plant PEPs. A global sequence alignment of putative plant PEPs and other known PEPs indicated that the active site amino acids Ser, His and Asp are conserved in putative plant PEP sequences. Furthermore, putative plant PEPs showed similar secondary structures to known PEPs and when a rice PEP was modelled onto porcine brain PEP structure, a high degree of similarity was found. Germination studies of wheat seed showed an increase of PEP activity over time with maximum PEP activity reached after 4 days of germination, which remained at this level until 9 days of germination, implying a function for PEP in plant seed germination. Wheat PEP was purified using ion exchange and gel filtration chromatography with a final yield of less than 1 percent and a relative purity (only 2 bands detected by SDS-PAGE). The purified wheat PEP had a molecular weight of approximately 55kDa, substrate specificity for chymotrypsin-like substrates (N-Suc-Ala-Ala-Pro-Phe-pNa, Km value of 0.58 mM, Kcat of 29.37 s–1; Kcat /Km 50813.14s–1 M–1); a pH optimum of 7.9; temperature optima of 37oC and a high sensitivity to temperature as indicated by loss of activity at temperatures above 40oC. Inhibition studies using E64, Leupeptin and PMSF confirmed that the wheat PEP is from the Serine protease family and is most likely a trypsin-like protease.en
dc.format113 leaves; 30 cmen
dc.formatpdfen
dc.identifier.citationAbrahams, Adriam Mark. 2013en
dc.identifier.othervital:11269en
dc.identifier.otherhttp://hdl.handle.net/10353/d1004356en
dc.identifier.otherEndopeptidasesen
dc.identifier.otherWheaten
dc.identifier.otherChemical kineticsen
dc.identifier.otherPharmacokineticsen
dc.identifier.urihttp://hdl.handle.net/20.500.11837/4530
dc.language.isoEnglishen
dc.publisherUniversity of Fort Hareen
dc.publisherFaculty of Science & Agricultureen
dc.rightsUniversity of Fort Hareen
dc.subjectEndopeptidasesen
dc.subjectWheaten
dc.subjectChemical kineticsen
dc.subjectPharmacokineticsen
dc.titleIsolation, purification and kinetic characterization of prolyl endopeptidase from Titicum aestivumen
dc.title.alternativeIsolation, purification and kinetic characterization of prolyl endopeptidase from Titicum aestivumen
dc.typeThesisen
dc.typeMastersen
dc.typeMSc (Biochemistry)en

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