Biochemical and Molecular Characterization of Eel Fish Trypsin (Anguilla bicolor McClelland) as Potential Candidates Protease Enzyme

Trypsin is one alkaline protease type widely used in various industry fields. One type of potential fish trypsin source is Anguilla bicolor. This study aims to characterize biochemical and molecular characterization of eel fish trypsin (Anguilla bicolor McClelland) as a possible candidate protease enzyme. The method used in this research is experimental research consisting of biochemical and molecular characterization. Fish Trypsin Extract was isolated from the digestive organs and then crushed using an electric homogenizer. During the pulverization process, 50 mM Tris-HCl buffer was added at a ratio of 1: 8 (w/v). The supernatant was then collected and can be stored at -80°C to measure enzyme activity. The treatment was given to juveniles and adults with stadia of Anguilla bicolor. While the molecular method was carried out using In Silico analysis in the analysis of the diversity of trypsin sequences in various fish species, preparation of specific primers, and analysis of Whole Genome Sequencing diversity of different species of Anguilla Spp. After that, extraction of Anguilla bicolor DNA, optimization of primer annealing temperature, DNA amplification, fish trypsin DNA fragments using the Sanger and Nanopore methods, and analysis of sequencing and phylogenetic results. The result of the protein content of the trypsin extract in the juvenile stage of Anguilla bicolor had an average of 0.488 ± 0.004 g/dL, and the adult stage of Anguilla bicolor had an average of 1.778 ± 0.080 g/dL. The highest trypsin activity was obtained in the juvenile stadia, 0.529 ± 0.016 (U/mL), and in the adult stadia, 0.399 ± 0.009 (U/mL). Trypsin activity increases with increasing temperature used and reaches a maximum of 40ºC. The molecular character of the fish enzyme Anguilla bicolor shows that the sequence analyzed tend to be close to the Trypsinogen and Trypsin-like genes from Anguilla japonica, Anguilla anguilla, and Megalops cyprinoides.