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Production of a novel promising recombinant trypsin from Fenneropenaeus merguiensis: Towards biochemical and structural characterizations | ||
| Iranian Journal of Fisheries Sciences | ||
| دوره 24، شماره 3، مرداد 2025، صفحه 473-504 اصل مقاله (1.73 M) | ||
| شناسه دیجیتال (DOI): 10.22092/ijfs.2025.133371 | ||
| نویسندگان | ||
| M. Eskandari Mehrabadi1؛ R. Hemmati* 1؛ A. Homaei2؛ Z. Salemi3؛ G. Rigi4 | ||
| 1Department of Biology, Faculty of Basic Sciences, Shahrekord University, Sharekord, Iran | ||
| 2Department of Marine Biology, Faculty of Marine Science and Technology, University of Hormozgan, Bandar Abbas, Iran | ||
| 3Department of Biochemistry, Arak University of Medical Sciences, Arak, Iran | ||
| 4Department of Genetics, Faculty of Basic Sciences, Shahrekord University, Sharekord, Iran | ||
| چکیده | ||
| This study presents the purification and characterization of novel recombinant trypsin derived from banana shrimp (Fenneropenaeus merguiensis) expressed in E. coli Rosetta-gami. The enzyme was purified using nickel sepharose chromatography and characterized through spectrophotometric kinetic and thermodynamic analyses, circular dichroism, and spectrofluorimetry. The cDNA encoding the putative trypsin, comprising 801 base pairs, was successfully isolated from the hepatopancreas of F. merguiensis. Sequence alignment of amino acids indicated a high degree of similarity (92-95%) with trypsins from F. chinensis, P. vannamei, and F. mondon. The purified recombinant trypsin exhibited a molecular weight of 23 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzymatic activity was observed at pH 8 and 50 °C, with the enzyme demonstrating stability within a pH range of 7-9 and retaining approximately 50% of its initial activity across a temperature spectrum of 40-70 °C. The calculated half-life of the recombinant trypsin was 21.72 minutes at pH 8 and 50 °C. Kinetic parameters for casein as a substrate were determined, yielding Km, Vmax, kcat, and kcat/Km values of 130.4 μg/ml, 0.0617 μg·min-¹.ml-¹, 131.8 min-¹, and 1.01 min-¹μg-¹, respectively. Traditional methods for extracting trypsin from marine sources are inefficient. However, by utilizing recombinant DNA technology, we have produced a trypsin derived F. merguiensis with unique properties, including thermal stability and alkaline pH tolerance, highlighting its suitability for biotechnological applications. | ||
| کلیدواژهها | ||
| Recombinant Trypsin؛ Protease؛ Banana shrimp؛ E. coli Rosetta-gami | ||
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