The purification, characterization and potential biological applications of proline-spesific endopeptidases

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a cytosolic serine protease that hydrolyses proline containing small biological peptides. The members of prolyl oligopeptidase family play important roles in many physiological processes such as neurodegenerative diseases, maturation and degradation of peptide hormones and neuropeptides, learning and memory, DNA synthesis, cell differentiation and signal transduction. Thus the enzyme has been purified and characterized from various sources to elucidate the potential use as therapeutics. In this study recombinant Myxococcus xanthus Prolyl oligopeptidase expressed in E.coli was purified 224 fold, using metal-chelate affinity and gel permeation chromatography. The purified enzyme exhibited a monomeric molecular weight of 70 kDa, and an isoelectric point of 6.3. The optimum pH and temperature was estimated as 7.5 and 37°C, respectively. Enzyme activity was inhibited by various serin protease inhibitors such as Z-Pro-Prolinal and PMSF. Furthermore the substrate specificity (synthetic/natural) and the stability (thermal, pH and storage stability) of the purified enzyme were also investigated. The data showed that the Myxococcus xanthus Prolyl oligopeptidase is similar to Prolyl oligopeptidase isolated from other sources. The potential use of the enzyme was tested by the hydrolysis of the wheat gluten. For this purpose wheat gluten was subjected to enzymatic hydrolysis with Prolyl oligopeptidase, after optimizing the conditions. The resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and Prolyl oligopeptidase inhibition activities. Keywords: Serine protease, Prolyl oligopeptidase, Bioactive peptides, 2,4,6‐trinitrobenzene sulfonic acid.