Purification and characterization of a β‐glucosidase from Evernia prunastri

Abstract

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)...

On the occurrence of peroxidase and laccase activity in lichens

“…While lignin degradation confers no energetic benefits to lichens, it will expose cellulose that may be utilized as a carbon supply. Yagüe & Estévez (1988 a , b ) suggest that lichens can produce all the enzymes needed for direct cellulose degradation. Furthermore, recent studies on free-living fungi indicate that, as for lignin, cellulose breakdown can also be facilitated by ROS production (Martinez et al 2009).…”

“…Lichens from the order Peltigerales are active producers of the phenol oxidases laccase and tyrosinase (Zavarzina & Zavarzin 2006), and tyrosinases also occur in some non-Peltigeralean lichens (Beckett et al 2012). Furthermore, cellulase activity has been well documented in several species, for example Evernia prunastri and Peltigera canina (Yagüe & Estévez 1988 a , b ; de los Rios et al . 1997), and probably occurs in almost all lichens.…”

β-1,4-Glucanase (Cellulase) Location in the Symbionts of Evernia Prunastri

“…In situ localization of P-l,4-glucanase activity may be possible using the cytochemical method of Nolan & Bal (1974). Alternatively, the recent purification of P-1,4-glucanase activity from E. prunastri (Yague & Estevez 1989) may enable the production of anti-cellulase antibodies and the use of immunocytochemical techniques (Sexton et al 1980;Chapman et al 1983). E. Yague gratefully acknowledges support through a Plan de Formation del Personal Investigador fellowship (Ministerio de Education y Ciencia, Spain).…”

Production and enzymatic hydrolysis of carbohydrates in intertidal sediment

“…Differences in the methods for the enzyme assays will also affect the results. For example, high concentrations of substrate are known to cause substrate inhibition of β-glucosidase (Yagüe & Estévez 1988, Woodward & Clarke 1991. The saturating substrate concentration may be determined by measuring exoenzyme rates at a range of substrate concentrations and fitting a Michaelis-Menten equation to the data.…”