ABSTRACT
Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.
Subject(s)
Tribolium , Trypsin Inhibitors , Animals , Trypsin Inhibitors/pharmacology , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/isolation & purification , Tribolium/enzymology , Tribolium/drug effects , Insect Proteins/chemistry , Insect Proteins/isolation & purification , Insect Proteins/antagonists & inhibitors , Seeds/chemistry , Insecticides/pharmacology , Insecticides/chemistry , Insecticides/isolation & purification , Plant Proteins/pharmacology , Plant Proteins/isolation & purification , Plant Proteins/chemistryABSTRACT
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.
Subject(s)
Crotalaria , Seeds , Crotalaria/chemistry , Seeds/chemistry , Seeds/enzymology , Hydrogen-Ion Concentration , Trypsin/metabolism , Trypsin/chemistry , Kinetics , Substrate Specificity , Temperature , Molecular WeightABSTRACT
Animal venoms are complex mixtures, including peptides, proteins (i.e., enzymes), and other compounds produced by animals in predation, digestion, and defense. These molecules have been investigated regarding their molecular mechanisms associated with physiological action and possible pharmacological applications. Recently, we have described the presence of a type of angiotensin converting enzyme (ACE) activity in the venom of Thalassophryne nattereri. It is a zinc-dependent peptidase with a wide range of effects. By removing dipeptide His-Leu from terminal C, the ACE converts angiotensinI (AngI) into angiotensin II (AngII) and inactivates bradykinin, there by regulating blood pressure and electrolyte homeostasis. The fractionation of T. nattereri venom in CM-Sepharose indicated a peak (CM2) with angiotensin-converting activity, converting AngI into Ang II. Electrophoresis on polyacrylamide gel (12%) revealed one band with 30kDa for CM2 similar in size to natterins, which are toxins with proteolytic activity found in T. nattereri venom. Mass spectrometry indicated that the protein sequence of the ACE purified from T. nattereri venom corresponds to natterin 1. The isolated protein has also demonstrated inhibition through captopril and EDTA and is characterized as a classic ACE. Thus, the isolated enzyme purified from T. nattereri venom is the first ACE isolated from fish venom.
Subject(s)
Batrachoidiformes/metabolism , Fish Venoms/enzymology , Peptidyl-Dipeptidase A/metabolism , Amino Acid Sequence , Animals , Cations , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/isolation & purification , Sequence AlignmentABSTRACT
Trypsin from L. alexandri was purified using only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose. Trypsin mass was estimated as 24 kDa through SDS-PAGE, which showed only one band in silver staining. The purified enzyme showed an optimum temperature and pH of 50 °C and 9.0, respectively. Stability was well maintained, with high levels of activity at a pH of up to 11.0, including high stability at a temperature of up to 50 °C after 60 min of incubation. The inhibition test demonstrated strong inhibition by PMSF, a serine protease inhibitor, and Kinetic constants km and kcat for BAPNA were 0.517 mM and 5.0 S-1, respectively. The purified enzyme was also as active as casein, as analyzed by zymography. Therefore, we consider trypsin a promising enzyme for industrial processes, owing to its stability in a wide range of pH and temperature and activity even under immobilization.