Trametes versicolor (L.) Lloyd as a source of thermostable serine protease: production and characterization

Proteases are ubiquitously present and are among the largest groups of commercially important enzymes. Here, we investigated a wood-rot basidiomycete Trametes versicolor (L.) Lloyd [Syn. Coriolus versicolor (L.) Quél.; Polyporus versicolor (L.) Fr.] as a source of the enzyme serine protease, its production, and optimized to obtain a higher yield of the enzyme.. The significant variables with optimized values for maximum production of the enzyme were temperature (30?C), incubation time (120 h) and wheat bran (10 g). The yield increased by 30.76% by statistically optimizing the media. The optimized temperature and pH for the maximum protease activity was 50?C and pH 7.0, respectively. The enzyme was purified through ion exchange (using DEAE cellulose 52 resin) and gel filtration chromatography (using Superdex 200 column). The purified enzyme had a retention time of 7 min in RP-HPLC. The enzyme was stable at a broad range of temperature (30-60?C) and pH (5.0-8.0) with a half-life of 58.72 min, Vmax of 37.17 ?M min/mL and Km of 0.657 mg/mL. Its activity was enhanced by Na+, Ca2+, Mg2+ ions and SDS surfactant. These properties make this enzyme a valuable candidate for industrial applications

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective: To explore the effect of the protease inhibitor from Agaricus bisporus (J.E. Lange) Imbach (AbPI) on glucose uptake and oxidative stress in 3T3-L1 adipocytes. Methods: Adipocytes were differentiated and stained with Oil-Red-O staining to confirm adipogenesis. The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay, intracellular reactive oxygen species generation through flow cytometry, and morphologically through confocal microscopy using propidium iodide, 4,6-diamino-2-phenylindol dihydrochloride, and 2',7'-dichlorofluorescein diacetate dyes. The uptake of fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy. Results: MTT assay showed that the cell survival rate was (28.00±3.00)％, (92.33±2.60)％, and (71.34±2.10)％ in the presence of 2 mM H2O2, AbPI alone, and AbPI and H2O2 both, respectively, in comparison to the control. Oil-Red-O staining indicated that AbPI enhanced adipogenesis. AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone, and showed insulin-sensitizing effect in the presence of insulin, but failed to stimulate the uptake in the absence of insulin. Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon AbPI treatment. Confocal microscopy showed that the damaged cell population rose to 3.50％, 117.84％, and 261.50％ in the presence of AbPI alone, AbPI with H2O2, and H2O2 alone, respectively. Conclusions: The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.