Research Article: Production of a novel promising recombinant trypsin from Fenneropenaeus merguiensis: Towards biochemical and structural characterizations

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 ۸۰۱ base pairs, was successfully isolated from the hepatopancreas of F. merguiensis. Sequence alignment of amino acids indicated a high degree of similarity (۹۲-۹۵%) with trypsins from F. chinensis, P. vannamei, and F. mondon. The purified recombinant trypsin exhibited a molecular weight of ۲۳ kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzymatic activity was observed at pH ۸ and ۵۰ °C, with the enzyme demonstrating stability within a pH range of ۷-۹ and retaining approximately ۵۰% of its initial activity across a temperature spectrum of ۴۰-۷۰ °C. The calculated half-life of the recombinant trypsin was ۲۱.۷۲ minutes at pH ۸ and ۵۰ °C. Kinetic parameters for casein as a substrate were determined, yielding Km, Vmax, kcat, and kcat/Km values of ۱۳۰.۴ μg/ml, ۰.۰۶۱۷ μg·min-¹.ml-¹, ۱۳۱.۸ min-¹, and ۱.۰۱ 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.

کلیدواژه ها:

Recombinant Trypsin ، Protease ، Banana shrimp ، E. coli Rosetta-gami