Mansooreh Hooshiyar - Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
Gholam Hossein Ebrahimi Pour - Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
Vahid Khalaj - Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Mohammad Rostami-Nejad - Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran ۱۹۸۵۷۱۴۷۱۱, Iran
Kaveh Baghaii - Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran ۱۹۸۵۷۱۴۷۱۱, Iran

Background and Aim : Background: It appears for most patients with CD, that a lifelong gluten-free diet alone is required for CD treatment. There are a variety of fungal PEPs such as Aspergillus niger PEP, with therapeutic applications, which destroy internal pralines. Significantly, Aspergillus niger PEP reduces gluten peptides concentration in food and plays an important role in patients with CD. Aim: The aim of this study was to evaluate the expression of Aspergillus niger _ Proline-specific endoprotease in a ura۳ auxotroph strain of Saccharomyces cerevisiaeMethods : Methods: The AN-PEP amino acid sequence was synthesized.The plasmid Pyes۲ was utilized as expression vector. We were codon optimized the complete amino acid sequence of endoprotease AN-PEP according to S. cerevisiae codon usage. Saccharomyces cerevisiae auxotrophic was used as a host for the expression of the gene encoding endoprotease AN-PEP.Results : Results In this study, Aspergillus niger PEP gene was cloned in the PYes۲ expression vector and expressed in Saccharomyces cerevisiae. The Aspergillus niger PEP was expressed in secretary form and was confirmed by SDS-PAGE analysis and western blotting. The enzyme activity was determined using the Z-Gly-Pro-pNA substrate Results: The Aspergillus niger PEP the enzyme was expressed in the secretory form in The recombinant auxotroph strain of S. cerevisiae.Conclusion : Conclusion: The findings showed that the expression of the engineered protein may have potential applications in the production of gluten-free bread.

Keywords: Aspergillus niger _ Proline-specific endoprotease _ Expression _ Saccharomyces cerevisiae