1. Experimental Preliminary Study for Production of Recombinant Subtilisin Enzyme by pET28b Cloning Vector.
- Author
-
Gedikli, Fatma, Can, Oznur, and Bilgin, Sema
- Subjects
BIOTECHNOLOGY ,PROTEINS ,BACILLUS (Bacteria) ,RESEARCH funding ,POLYMERASE chain reaction ,ENZYMES ,BIOINFORMATICS ,GENE expression ,ESCHERICHIA coli ,RECOMBINANT proteins ,PROTEOLYTIC enzymes ,GENETIC techniques ,ELECTROPHORESIS ,SEQUENCE analysis ,GENETICS - Abstract
Purpose: The purpose of this study is to enable the efficient production of the industrial enzyme subtilisin, a serine protease with extensive applications in various industries such as detergents, food processing, textiles, and pharmaceuticals. By leveraging recombinant DNA technology to produce subtilisin locally, the study aims to decrease reliance on foreign imports and enhance the sustainability of enzyme supply chains. This initiative seeks not only to meet the growing demand for subtilisin but also to promote economic independence, reduce costs, and foster innovation within the local biotechnology sector. Material and Methods: Following bioinformatic calculations and processes for pET28 and subtilisin, the enzyme was produced. Results: The results confirm successful cloning and expression of the subtilisin gene in the pET28b vector, creating the recombinant construct pET28b-subt. Agarose gel electrophoresis verified the transformation, showing distinct bands for the pET28b backbone and subtilisin insert. The recombinant subtilisin was purified from lysed E. coli using Ni-NTA affinity chromatography, with SDS-PAGE analysis revealing a molecular mass of 41,646.82 Da (Figure 5, Figure 6). These findings demonstrate successful production of subtilisin, though further optimization is needed for industrial applications. Conclusion: Production has been carried out and the sds-page result supports this. After this, it was seen that it was necessary to focus on activity studies. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF