title: Expression, purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system.
abstract: In recent years, various studies in the field of industrial enzymes of biotechnology have gained importance due to increasing development in enzyme technology. The different areas where enzymes are used and their economic value of biotechnological products further increases their importance. There are hundreds of different types of cheese but each is made by coagulating milk using rennet to give curds. Today, researchers have begun to develop alternative systems in the cheese industry related to milk-clotting enzymes. In this study, the nucleic acid sequence encoding the optimized chymosin enzyme was used and cloned by Not I and Mlu I restriction enzymes into pTOLT vector system. Then using this construct, the enzyme as a fusion with Tol-A-III protein was produced in Escherichia coli BL21 (DE3) cells. After disrupting the E. coli cell and separating from the constituents by high speed centrifugation, the enzyme was purified by affinity chromatography and fractions were analyzed by SDS-PAGE. Purified enzyme has shown its activity. Optimum temperature and pH of CHY-Tol-A-III protein were 40°C and 6.5, respectively.
authors.affiliation: a Department of Bioengineering, Faculty of Engineering , Karamanoğlu Mehmetbey University , Karaman , Turkey.
authors.forename: Yakup
authors.initials: Y
authors.lastname: Ulusu
authors.affiliation: b Department of Chemistry, Faculty of Art and Science , Gaziosmanpaşa University , Tokat , Turkey.
authors.forename: Sema Bilgin
authors.initials: SB
authors.lastname: Şentürk
authors.affiliation: c Department of Bioengineering, Faculty of Engineering , Gaziosmanpaşa University , Tokat , Turkey.
authors.forename: Hülya
authors.initials: H
authors.lastname: Kuduğ
authors.affiliation: c Department of Bioengineering, Faculty of Engineering , Gaziosmanpaşa University , Tokat , Turkey.
authors.forename: İsa
authors.initials: İ
authors.lastname: Gökçe
mesh_terms: D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D012085:Chymosin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D010957:Plasmids; D059748:Proteolysis; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
chemical_list: D012085:Chymosin
keywords: Chymosin; Escherichia coli; TOL-A-III; inducible pTOLT system; recombinant protein production; rennet