In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic acid, gallic acid, d,L-dithiothreitol, tropolone, glutathione, sodium azide, benzoic acid, salicylic acid, and 4-aminobenzoic acid using 4-methylcatechol, pyrogallol, and catechol as substrate. The presence of EDTA, 4-aminobenzoic acid, salicylic acid, gallic acid, and benzoic acid did not cause the inhibition of artichoke PPO. A competitive-type inhibition was obtained with sodium azide, L-cysteine, and d,L-dithiothreitol inhibitors using 4-methylcatechol as substrate; with L-cysteine, tropolone, d,L-dithiothreitol, ascorbic acid, and sodium azide inhibitors using pyrogallol as substrate; and with L-cysteine, tropolone, d,L-dithiotreitol, and ascorbic acid inhibitors using catechol as a substrate. A mixed-type inhibition was obtained with glutathione inhibitor using 4-methylcatechol as a substrate. A noncompetitive inhibition was obtained with tropolone and ascorbic acid inhibitors using 4-methylcatechol as substrate, with glutathione inhibitor using pyrogallol as substrate, and with glutathione and sodium azide inhibitors using catechol as substrate. Fr...

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Pichia pastoris beta-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate, gel filtration, and ion-exchange chromatography with Q-Sepharose and CM-Sepharose. The enzyme is a tetramer (275 kD) made up of four identical subunits (70 kD). The pH optimum is 7.3, and it is fairly stable in the pH range 5.5-9.5. The temperature optimum is 40 degrees C. The purified beta-glucosidase is effectively active on p-/o-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with Km values of 0.12, 0.22, and 0.096 mM and Vmax values of 10.0, 11.7, and 6.2 micromol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-beta-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/o-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with Ki value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with Ki value of 7.2 mM for p-NPG. Pichia pastoris has been employed as a host for the functional expression of heterologous beta-glucosidases and the risk of high background beta-glucosidase activity is discussed.

β-Glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) is a catalytic enzyme present in both prokaryotes and eukaryotes that selectively catalyzes either the linkage between two glycone residues or between glycone and aryl or alkyl aglycone residue. Growing edible mushrooms in the soil with increased cellulose content can lead to the production of glucose, which is a process dependent on β-glucosidase. In this study, β-glucosidase was isolated from Agaricus bisporus (white button mushroom) using ammonium sulfate precipitation and hydrophobic interaction chromatography, giving 10.12-fold purification. Biochemical properties of the enzyme were investigated and complete characterization was performed. The enzyme is a dimer with two subunits of approximately 46 and 62 kDa. Optimum pH for the enzyme is 4.0, while the optimum temperature is 55 °C. The enzyme was found to be exceptionally thermostable. The most suitable commercial substrate for this enzyme is p-NPGlu with Km and Vmax values of 1.751 mM and 833 U/mg, respectively. Enzyme was inhibited in a competitive manner by both glucose and δ-gluconolactone with IC50 values of 19.185 and 0.39 mM, respectively and Ki values of 9.402 mM and 7.2 µM, respectively. Heavy metal ions that were found to inhibit β-glucosidase activity are I(-), Zn(2+), Fe(3+), Ag(+), and Cu(2+). This is the first study giving complete biochemical characterization of A. bisporus β-glucosidase.

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