Production, purification and characterization of prolyl endopeptidase (PEP) enzyme, for gluten free food production

Gluten is a group of protein found in wheat, barley, rye and oats known as grains. Celiac is a systemic disease in which malabsorption in the small intestine is at the forefront and occurs with the intake of these vegetable proteins into the body. With the addition of gluten-containing foods to the diet, symptoms such as vomiting, fatty diarrhea, inability to gain weight and abdominal distension are observed. Gluten-free diet is the only treatment for celiac disease. Today, the use of bacterial and fungal oligopeptidases that break down gluten into non-toxic fragments is considered as a promising alternative therapy. Prolyl Endopeptidase (PEP) enzyme obtained from Aspergillus niger is an enzyme that completely digests gluten. In this study, it was aimed to produce recombinant A. niger Prolyl Endopeptidase (PEP) enzyme in E. coli expression system using the pET22b vector. E. coli BL21 (DE3) pLysE cells were used as the expression strain. The PEP enzyme, which was observed in a form of inclusion bodies, then it was refolded and the PEP protein was purified by affinity column chromatography. The activity test of the purified PEP enzyme was performed using gluten and benzyloxycarbonyl-glycine-proline-p-nitroanilide that is a synthetic substrate. As a result, produced PEP enzyme was obtained at a yield of 280 mg / L and it was capable to digest gluten. The PEP enzyme demonstrated its optimum activity at pH 6 and its optimum temperature was 30oC.