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.

The Effects of Different Media, Sucrose Concentrations and Natural Additives on Plantlet Growth of Phalaenopsis Hybrid 'Pink'

ABSTRACT The effects of various sucrose concentrations as carbon source and natural additives in different media on plantlet growth of Phalaenopsis hybrid 'Pink' were studied. Plantlets were cultured on two media (Murashige and Skoog [MS] and Vacin and Went [VW]) supplemented with 0, 10, 20, 30 and 40 g L-1 sucrose either with 0, 10 and 20% (v/v) coconut water (CW) or carrot juice (CJ) as natural additives. After four months of culture, the combination of sucrose and CW supplemented with both media affected plantlet growth where most of the plantlets showed slow growth and survival frequency (0-80%) with increasing concentrations of CW in all sucrose concentrations. However, plantlet growth on both media containing only 20 g L-1 sucrose without CW was optimal in terms of root number, root length, leaf number, leaf length, leaf width, fresh weight, dry weight and plant height. The combination of sucrose and CJ supplemented with MS medium resulted in overall good plantlet growth with 100% survival frequency. The combination of sucrose (20 g L-1) and CJ (10%) supplemented with MS medium increased root length, leaf length, leaf width and plant height. Plantlet growth was also optimal in the combination of 20 g L-1 sucrose and 10% CJ supplemented with VW medium. The results of this study indicate that Phalaenopsis hybrid 'Pink' cultured on the combination of sucrose (20 g L-1) and CJ (10%) supplemented with either MS or VW media can be used for plantlet growth of this species.

Comparative study of assay of free radical damage in in vivo, in vitro and ex vivo conditions.

Background: Interception of free radical events and damage in clinical situation remain arbitrary with poor understanding on antioxidant pharmacology. Methods: Experiments on in vitro per-oxidation insult as well as assumed free radical damage situation in ischemia reperfusion are examined for profi le of malondialdehyde rise and effect of Gikgobiloba treatment by different schedule. Results: Effect of Ginkgobiloba was seen on ischemia reperfusion induced, hydrogen peroxide induced and ferric chloride induced methods. Antioxidant effect of Ginkgobiloba was consistent in all techniques. Conclusions: Results reveal the signifi cance of complementary experiment to elaborate issue of antioxidants.

Regulation of wound strength by Ocimum sanctum: in silico and in vivo evidences.

Background: The present work has been an attempt to facilitate the scientific understanding of the wound strength by Ocimum sanctum (OS, holy basil) a traditional knowledge practiced since ancient times in India. Methods: The in vivo Incision (wound strength) and Dead space wound models (biochemical estimation of components of ECM) in rats and In silico method, where one of the target proteins from each class of MMPs involved in wound strength was selected for molecular docking with eugenol (one of the flavonoid present in OS). Results: Molecular docking showed that eugenol was able to inhibit all selected MMPs, i.e. collagenase (-6.37 Kcal/mol), gelatinase (-5.99 Kcal/mol), elastase (-6.31 Kcal/mol) and stromelysin (-5.79 Kcal/mol). Ethanolic extract of Ocimum sanctum (OSE, 200-800 mg/kg) when administered as suspension showed dose-dependent increase in wound breaking strength in in vivo Incision wound rat model. OSE 400 mg/kg produced a significant increase in protein and collagen constituents like hydroxyproline, hexuronic acid and hexosamine in the connective tissue content of extracellular matrix when studied in Dead space wound model in rat. Conclusions: The present study is an attempt to correlate the in vivo findings on wound strength promoting activity by Ocimum sanctum with in silico tools.

Molecular construction of NADH-cytochrome b5 reductase inhibition by flavonoids and chemical basis of difference in inhibition potential: Molecular dynamics simulation study.

NADH-cytochrome b5 reductase, a flavoprotein, plays a central role in many diverse metabolic reactions. NADH-cytochrome b5 reductase has been shown to be responsible for the generation of free radicals from heterocyclic amines. Flavonoids compounds share remarkable similarity in structure but showed differences in their cytochrome b5 reductase inhibition pattern. Our molecular dynamics simulation studies revealed that the difference in substitution at C3 position of ring C may lead to difference in interaction with enzyme. Absence of hydroxyl group substitution at C3 in luteolin facilitates the strong cation-π interaction between Lys185 and ring A, and C and π-π between Phe92 and ring A, and C along with h-bonding between Lys185 and oxo group. Ring B of luteolin showed strong π-π interaction with FAD. These interactions were found absent in quercetin and taxifolin. These results suggest that absence of hydroxyl group substitution at C3 increases the potency of flavonoid inhibitors for cytochrome b5 reductase.

Taxifolin acts as type I inhibitor for VEGFR-2 kinase: Stability evaluation by molecular dynamic simulation.

The VEGFR-2 kinase specific intracellular signalling cascades leading to proliferation, migration, survival of endothelial cells and increased permeability of vessels which contributes to angiogenesis. ATP is essentially required by VEGFR-2 to perform phosphorylation of specific proteins and to maintain cascade downstream. Taxifolin (plant polyphenol) inhibit the VEGFR-2 kinase by binding at ATP-binding pocket revealed by molecular docking study. Further, stability of VEGFR-2 kinase-taxifolin complex is validated by molecular dynamic simulation. RMSD analysis for 3800 ps confirmed the stability of complex. Furthermore, thermodynamic stability was evidenced by stable total energy, potential energy, and, temperature and pressure profile. After MD simulation taxifolin was found to stably interact with pocket residues Cys 917 and Lys 1053 along with water molecules. These results suggest that therapeutic inhibition of VEGFR-2 by taxifolin as a type I inhibitor may be a promising ways to retard signaling cascade of specific proteins which play crucial role in cancer proliferation and also in development of second generation type II inhibitors.