~ 573 ~ Journal of Entomology and Zoology Studies 2020; 8(4): 573-579 E-ISSN: 2320-7078 P-ISSN: 2349-6800 www.entomoljournal.com JEZS 2020; 8(4): 573-579 © 2020 JEZS Received: 20-05-2020 Accepted: 23-06-2020 Biswajit Mohanty College of Fisheries, Central Agricultural University (I), Lembucherra, Tripura, India Corresponding Author: Biswajit Mohanty College of Fisheries, Central Agricultural University (I), Lembucherra, Tripura, India Purification and characterization of trypsin from the visceral wastes of grass carp Ctenopharyngodon idella (Valenciennes, 1844) Biswajit Mohanty Abstract The proteolytic enzyme trypsin was purified from the gastrointestinal tract waste of grass carp Ctenopharyngodon idella (Valenciennes, 1844) with ammonium sulfate fractionation (ASF) followed by DEAE-cellulose chromatography, and Benzamidine Sepharose fast flow column affinity chromatography. Trypsin was purified 26.2-fold with an 11.1% yield. The purified enzyme was active between pH 9.0 and 11.0, and maximal activity of the enzyme was observed at pH 10.0. Highest activity was found at 60°C. The activity was reduced further after reaching the maximum activity point of temperature. The trypsin enzymatic activity was decreased by 40% and 60%, when incubated at 90°C for 30 min. The Km, Kcat, and catalytic efficiency values of purified trypsin were obtained is 0.062 mM and 19.23/s, and 310.16/s/mM. Degree of hydrolysis (DH) of the proteases on muscle protein increased with increase of enzyme concentrations. The enzyme activity was also further inhibited by SBTI, PMSF, and N-α-p-tosyl-L-lysine chloromethyl ketone. The molecular weight of the purified enzyme was obtained to be 20.2 kDa by SDS-PAGE. The study showed that trypsin from grass carp visceral waste of could find use in applications where maximum activity at moderate temperature is desired. Keywords: Grass carp; Trypsin; Enzyme purification fold; Enzyme kinetics Introduction Fish viscera are a potential source of digestive enzymes, especially digestive proteases. Proteases represent an important class of industrial enzymes, accounting about 50% of the total sale of the enzymes in the world [1] . Various digestive proteases such as aspartic protease pepsin and serine proteases - trypsin, chymotrypsin, and elastase are isolated from the fish viscera. Trypsin (EC 3.4.21.4) plays a pivotal role in digestive physiology. This endopeptidase hydrolyzes peptide bonds at the carboxyl end of lysine and arginine residues. Trypsin plays major roles in biological processes including digestion and activation of zymogens [2] . Proteolytic enzyme (proteases) also has the diverse industrial uses such as in detergent, food, pharmaceutical, leather industries [3] . Fish viscera have wide biotechnological potential as a source of digestive enzymes, especially digestive proteases that have high activity over a wide range of temperature and pH and exhibit high catalytic activity at a relatively low concentration [4, 5] . In most teleosts, trypsin is synthesized in the cells of pyloric caecum as an inactive precursor trypsinogen, which is secreted into the intestinal lumen and activated by enteroproteases [6] . Though trypsin and trypsin-like serine proteases are isolated from several marine species, there's scarcity of data on the digestive proteases of tropical freshwater fishes, especially of carps. Trypsin was earlier purified from the intestine of hybrid tilapia Oreochromis niloticus × O. Aureus [7] and also from the intestinal caeca of Amazonian tambaqui Colossoma macropomum [6] . The grass carp Ctenopharyngodon idella is one of the most important farmed freshwater species in rivers, lakes and ponds in India. The grass carp can utilize both plant and animal matter for food but shows a clear dietary preference for certain aquatic weeds. For a better understanding of the physiology and farming of this species (for example developing new feed protein resources and improving feeding regimes) and utilization of its processing wastes, more precise information about its digestive proteinases is needed. This paper describes the isolation, purification, inhibitory, kinetic, stability and thermodynamic properties of trypsin from the intestines of grass carp (Ctenopharygodon idella), and these properties are compared with those of trypsin from other fishes and aquatic organisms.