ABSTRACT
The production of proteases by white rot fungi, such as those of the genus Pleurotus, is related to the degradation of wood proteins, the substrate on which these fungi grow in the environment. From the point of view of production, they are still little explored for this purpose. A selection of agro-industrial residues highlighted corn bagasse as the best substrate for solid-state protease production using the basidiomycete Pleurotus pulmonarius. The enzyme production was maximized through a factorial design, where the enzyme activity increased from 137.8 ± 1.9 to 234.1 ± 2.7 U/mL. Factors such as temperature stability, pH, and chemical reagents were evaluated. The optimum temperature was 45 °C, showing low thermal stability at higher temperatures. The enzyme inhibition occurred by Mn2+ (50.3%) and Ba2+ (76.4%); SDS strongly inhibited the activity (82.4%), while pepstatin A partially inhibited (56%), suggesting an aspartic protease character. Regarding pH, the highest protease activity was obtained at pH 5.5. Partial characterization resulted in apparent values of the KM and Vmax constants of 0.61 mg/mL and 1.79 mM/min, respectively.(AU)
Subject(s)
Humans , Pleurotus , Peptide Hydrolases , Fungi , Enzyme Activation , Temperature , Hydrogen-Ion Concentration , Pepstatins , MicrobiologyABSTRACT
The current study sought to evaluate the acute effects of phloretin (PH) on metabolic pathways involved in the maintenance of glycemia, specifically gluconeogenesis and glycogenolysis, in the perfused rat liver. The acute effects of PH on energy metabolism and toxicity parameters in isolated hepatocytes and mitochondria, as well as its effects on the activity of a few key enzymes, were also evaluated. PH inhibited gluconeogenesis from different substrates, stimulated glycogenolysis and glycolysis, and altered oxygen consumption. The citric acid cycle activity was inhibited by PH under gluconeogenic conditions. Similarly, PH reduced the cellular ATP/ADP and ATP/AMP ratios under gluconeogenic and glycogenolytic conditions. In isolated mitochondria, PH inhibited the electron transport chain and the FoF1-ATP synthase complex as well as acted as an uncoupler of oxidative phosphorylation, inhibiting the synthesis of ATP. PH also decreased the activities of malate dehydrogenase, glutamate dehydrogenase, glucose 6-phosphatase, and glucose 6-phosphate dehydrogenase. Part of the bioenergetic effects observed in isolated mitochondria was shown in isolated hepatocytes, in which PH inhibited mitochondrial respiration and decreased ATP levels. An aggravating aspect might be the finding that PH promotes the net oxidation of NADH, which contradicts the conventional belief that the compound operates as an antioxidant. Although trypan blue hepatocyte viability tests revealed substantial losses in cell viability over 120 min of incubation, PH did not promote extensive enzyme leakage from injured cells. In line with this effect, only after a lengthy period of infusion did PH considerably stimulate the release of enzymes into the effluent perfusate of livers. In conclusion, the increased glucose release caused by enhanced glycogenolysis, along with suppression of gluconeogenesis, is the opposite of what is predicted for antihyperglycemic agents. These effects were caused in part by disruption of mitochondrial bioenergetics, a result that should be considered when using PH for therapeutic purposes, particularly over long periods and in large doses.
