Urease is induced by urea in Evernia prunastri (L.) Ach. thallus, but the enzyme becomes inactive from the fifth hour of culture, this process being more complete when the thallus is incubated in white light than when the thallus is aging in darkness. Dithiothreitol prevents this inactivation to some extent by reduction (or protection) of ‐SH groups in the protein. In vivo inactivation, in darkness as well as in the light, is accompanied by increase in the molecular mass of the enzyme; this pattern is not greatly changed by dithiothreitol.

Evernia prunastri thalli were homogenized and separated into algaland fungal-rich fractions. The former was shown to contain most β-l,4-glucanase (cellulase) activity. The significance of this localization in field material is discussed.

Intracellular P-glucosidase from Evernia prunastri has been purified to homogeneity using anion exchange on DEAE-Sephadex A-50, and gel filtration chromatography on Sephadex G-100 and Sepharose 6B. The purified P-glucosidase showed a single protein band on native electrophoresis and its isoelectric point was at pH 3.12. The molecular mass, calculated from its partition coefficient on the Sepharose 6B column, was 311 kDa, being composed of several subunits of 60 and 70 kDa. The highest activity of this enzyme was attained at pH 4.0 and 60°C. The enzyme showed strong resistance to thermal inactivation. Its activation energy was about 15 kJ/mol. Cellobiose, salicin, and p-nitrophenyl P-D-glucoside, but not carboxymethylcellulose, were hydrolyzed by the enzyme, following substrate inhibition kinetics. The purified P-glucosidase was considered a true cellobiase because of its great affinity towards cellobiose. Cellobiose inhibition does not seem to be a physiological phenomenon. Glucose inhibited enzyme activity in a competitive way (Ki = 1.26 mM). Fe3+ and Co2+ inhibited activity notably. Hg2+, Cu2+ and EDTA were practically ineffective. Even 200 mM gluconolactone did not affect enzyme activity.Cellulolytic organisms possess three types of enzymes which enable complete solubilization of mycrocrystalline cellulose. These enzymes are 1,4-P-D-glucan glucanohydrolase, endoglucanase (EC 3.2.1.4), 1,4-P-~-glucan cellobiohydrolase, exoglucanase (EC 3.2.1.91), and P-glucoside glucohydrolase, P-glucosidase (EC 3.2.1.21). The endoglucanase acts in random fashion on the regions of low crystallinity in the cellulose fibre, the exoglucanase removes cellobiose units from the non-reducing end of the cellulose chain and B-glucosidase produces glucose units from cellobiose [l, 21. Although P-glucosidase is not strictly necessary for cellulose solubilization [3,4], its presence enhances the cellulose hydrolysis because the three enzymes act synergistically [5 -71 and inhibition of endoglucanase and exoglucanase by cellobiose is avoided [8, 91.Evernia prunastri synthesizes P-1,4-glucanase and polygalacturonase when incubated with carboxymethylcellulose and polygalacturonic acid, respectively [lo]. We have recently reported the presence of the whole cellulase system in this lichen [ll] and are currently studying the in vivo and in vitro regulation of the enzymes belonging to the system. In this paper we report the purification and characterization of intracellular P-glucosidase. The enzyme hydrolyzes cellobiose, salicin, and p-nitrophenyl P-D-glucoside (NpGlc) following nonMichaelis-Menten kinetics, showing inhibition by substrate and by product. The significance of these inhibitions is discussed and compared with other fungal P-glucosidases.Correspondence to P. Esttvez, C/Santa Brigida 110.6, MajaAbbreviation. NpGlc, p-nitrophenyl /?-~-gIucoside. Enzymes. 1 ,C/f-~-Glucan glucanohydrolase, endoglucanase (EC 3.2.1.4); 1,4-P-~-glucan cellobiohydrolase, exoglucanase (EC 3.2.1.91); 8-D-glucoside glucohydrolase, P-glucosidase (EC 3.2.1.21)...

The production of bioactive peptides from organic by-waste materials is in line with current trends devoted to guaranteeing environmental protection and a circular economy. The objectives of this study were i) to optimize the conditions for obtaining bioactive hydrolysates from chicken combs and wattles using Alcalase, ii) to identify the resulting peptides using LC-ESI-MS2 and iii) to evaluate their chelating and antioxidant activities. The hydrolysate obtained using a ratio of enzyme to substrate of 5% (w/w) and 240 min of hydrolysis showed excellent Fe2+ chelating and antioxidant capacities, reducing Fe3+ and inhibiting 2, 2′-Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. The mapping of ion distribution showed that a high degree of hydrolysis led to the production of peptides with m/z ≤ 400, suggesting low mass peptides or peptides with multiple charge precursor ions. The peptides derived from the proteins of cartilage like Collagen alpha-2(I), Collagen alpha-1(I), Collagen alpha-1(III) and elastin contributed to generation of bioactive compounds. Hydrolysates from chicken waste materials could be regarded as candidates to be used as ingredients to design processed foods with functional properties.

Evernia prunastri Ach., an epiphytic lichen growing on Quercus rotundifolia Lam., produces a β-1,4-glucanase (EC 3.2.1.4) and a polygalacturonase (EC 3.2.1.15). The activity of these polysaccharidases increases as a response to incubation of the lichen with carboxymethylcellulose or sodium polygalacturonate, respectively. This increase in activity is thought to be the result of enzyme induction because it is inhibited by both cycloheximide and 8-azaguanine. Both polysaccharide-degrading enzymes are partially secreted into the incubation media.