Purification and characterization of a new trypsin-like protease from Crotalaria stipularia.

Proteases are the main enzymes traded worldwide-comprising 60% of the total enzyme market-and are fundamental to the degradation and processing of proteins and peptides. Due to their high commercial demand and biological importance, there is a search for alternative sources of these enzymes. Crotalaria stipularia is highlighted for its agroecological applications, including organic fertilizers, nematode combat, and revegetation of areas contaminated with toxic substances. Considering the pronounced biotechnological functionality of the studied species and the necessity to discover alternative sources of proteases, we investigated the extraction, purification, and characterization of a protease from seeds of the C. stipularia plant. Protease isolation was achieved by three-phase partitioning and single-step molecular exclusion chromatography in Sephacryl S-100, with a final recovery of 47% of tryptic activity. The molecular mass of the isolated enzyme was 40 kDa, demonstrating optimal activities at pH 8.0 and 50 °C. Enzymatic characterization demonstrated that the protease can hydrolyze the specific trypsin substrate, BApNA. This trypsin-like protease had a Km, Vmax, Kcat, and catalytic efficiency constant of 0.01775 mg/mL, 0.1082 mM/min, 3.86 s-1, and 217.46, respectively.

Angiotensin converting enzymes in fish venom.

Animal venoms are multifaceted mixtures, including proteins, peptides and enzymes produced by animals in defense, predation and digestion. These molecules have been investigated concerning their molecular mechanisms associated and possible pharmacological applications. Thalassophryne nattereri is a small venomous fish inhabiting the northern and northeastern coast of Brazil, and represents a relatively frequent cause of injuries. Its venom causes severe inflammatory response followed frequently by the necrosis of the affected area. Scorpaena plumieri is the most venomous fish in the Brazilian fauna and is responsible for relatively frequent accidents involving anglers and bathers. In humans, its venom causes edema, erythema, ecchymoses, nausea, vomiting, and syncope. Recently, the presence of a type of angiotensin converting enzyme (ACE) activity in the venom of Thalassophryne nattereri and Scorpaena plumieri, endemic fishes in northeastern coast of Brazil, has been described. The ACE converts angiotensin I (Ang I) into angiotensin II (Ang II) and inactivates bradykinin, there by regulating blood pressure and electrolyte homeostasis, however, their function in these venoms remains an unknown. This article provides an overview of the current knowledge on ACE in the venoms of Thalassophryne nattereri and Scorpaena plumier.