Expression, purification, and characterization of bovine chymosin enzyme using an inducible pTOLT system
| Autor: | Sema Bilgin Şentürk, Hülya Kuduğ, Yakup Ulusu, İsa Gökçe |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Proteolysis Biology medicine.disease_cause Biochemistry Chromatography Affinity 03 medical and health sciences Affinity chromatography Sequence Homology Nucleic Acid medicine Animals Chymosin Cloning Molecular Polyacrylamide gel electrophoresis Escherichia coli chemistry.chemical_classification Base Sequence medicine.diagnostic_test Temperature 0402 animal and dairy science 04 agricultural and veterinary sciences General Medicine Hydrogen-Ion Concentration 040201 dairy & animal science Restriction enzyme 030104 developmental biology Enzyme chemistry bacteria Electrophoresis Polyacrylamide Gel Rennet Plasmids Biotechnology |
| Zdroj: | Preparative Biochemistry & Biotechnology. 46:596-601 |
| ISSN: | 1532-2297 1082-6068 |
| DOI: | 10.1080/10826068.2015.1085399 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|