ABSTRACT
This work describes purification of a protease from the visceral mass of the mussel Mytella charruana as well as evaluation of its ability to hydrolyze milk casein to generate antimicrobial peptides. The enzyme showed pI of 4.1 and a single polypeptide band of 83.1â¯kDa after SDS-PAGE. Sequence similarities with tropomyosin and myosin from mollusks were detected. The protease showed a trypsin-like activity with optimal temperature of 40⯰C and stability in a wide pH range (3.0-9.0). Km was 4.28⯱â¯0.34â¯mM of the synthetic substrate N-benzoyl-dl-arginyl-ρ-nitroanilide, whereas Vmax was 0.056⯱â¯0.001â¯nmolâ¯min-1. The enzyme hydrolyzed casein, and the hydrolysate inhibited the growth of Escherichia coli, Micrococcus luteus, Bacillus subtilis, and Klebsiella pneumoniae at a minimal inhibitory concentration of 5.0⯵gâ¯mL-1. In conclusion, the visceral mass of M. charruana contains a trypsin-like protease that can generate peptides from casein that have a bacteriostatic effect.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Bivalvia/enzymology , Peptides/pharmacology , Serine Endopeptidases/metabolism , Animals , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemistry , Caseins/chemistry , Caseins/metabolism , Drug Evaluation, Preclinical/methods , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Hydrolysis , Microbial Sensitivity Tests , Peptides/chemistry , Peptides/metabolism , Serine Endopeptidases/isolation & purification , Temperature , Viscera/enzymologyABSTRACT
Moringa oleifera seeds contain a water-soluble lectin [water-soluble M. oleifera lectin (WSMoL)] that has shown coagulant activity. Magnesium ions are able to interfere with the ability of this lectin to bind carbohydrates. In this study, we performed structural characterization of WSMoL and analyzed its effect on the electrical resistance of a kaolin clay suspension in both presence and absence of monosaccharides (N-acetylglucosamine, glucose, or fructose) and magnesium ions. The coagulant activity of WSMoL was monitored by measuring optical density and electrical resistance over a period of 60 min. Native WSMoL had a molecular mass of 60 kDa and exhibited anionic nature (pI 5.5). In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it appeared as three polypeptide bands of 30, 20, and 10 kDa. WSMoL reduced the optical density and electrical resistance of the kaolin suspension, which suggests that suspended particles are destabilized and that this is followed by formation of complexes. The coagulant activity of lectin decreased in the presence of Mg2+ ions and carbohydrates at concentrations that also inhibited hemagglutinating activity. This was most likely due to conformational changes in lectin structure. Our findings suggest that the coagulant activity of WSMoL is enhanced by lowering of electrical resistance of the medium and is impaired by lectin-carbohydrate and lectin-Mg2+ interactions.
Subject(s)
Electric Impedance , Lectins/pharmacology , Magnesium/pharmacology , Monosaccharides/pharmacology , Moringa oleifera/chemistry , Water/chemistry , Animals , Chromatography, Gel , Coagulants/pharmacology , Flocculation/drug effects , Hemagglutination/drug effects , Ions , Rabbits , SolubilityABSTRACT
The digestive apparatus of termites may have several biotechnological applications, as well as being a target for pest control. This report discusses the detection of cellulases (endoglucanase, exoglucanase, and ß-glucosidase), hemicellulases (ß-xylosidase, α-l-arabinofuranosidase, and ß-d-xylanase), α-amylase, and proteases (trypsin-like, chymotrypsin-like, and keratinase-type) in gut extracts from Nasutitermes corniger workers and soldiers. Additionally, the effects of pH (3.0-11.0) and temperature (30-100°C) on enzyme activities were evaluated. All enzymes investigated were detected in the gut extracts of worker and soldier termites. Endoglucanase and ß-xylanase were the main cellulase and hemicellulase, respectively. Zymography for proteases of worker extracts revealed polypeptides of 22, 30, and 43kDa that hydrolyzed casein, and assays using protease inhibitors showed that serine proteases were the main proteases in worker and soldier guts. The determined enzyme activities and their response to different pH and temperature values revealed that workers and soldiers contained a distinct digestive apparatus. The ability of these termites to efficiently digest the main components of lignocellulosic materials stimulates the purification of gut enzymes. Further investigation into their biotechnological potential as well as whether the enzymes detected are produced by the termites or by their symbionts is needed.
