Since At2g25630 is an intronless gene with a premature stop codon, its cDNA encoding the predicted mature beta-glucosidase isoenzyme was synthesized from the previously isolated Arabidopsis thaliana genomic DNA. The stop codon was converted to a sense codon by site-directed mutagenesis. The native and mutated cDNA sequences were separately cloned into the vector pPICZalphaB and expressed in Pichia pastoris. Only the cells transformed with mutated cDNA-vector construct produced the active protein. The mutated recombinant beta-glucosidase isoenzyme was chromatographically purified to apparent homogeneity. The molecular mass of the protein is estimated as ca. 60 kD by SDS-PAGE. The pH optimum of activity is 5.6, and it is fairly stable in the pH range of 5.0-8.5. The purified recombinant beta-glucosidase is effectively active on para-/ortho-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with K(m) values of 1.9, 2.1, 0.78 mM and k(cat) values of 114, 106, 327 nkat/mg, respectively. It also exhibits different levels of activity against para-/ortho-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), amygdalin, prunasin, cellobiose, gentiobiose, and salicin. The enzyme is competitively inhibited by gluconolactone and p-nitrophenyl-1-thio-beta-D-glucopyranoside with p-NPG, o-NPG, and 4-MUG as substrates. The enzyme is found to be very tolerant to glucose inhibition. The catalytic role of nucleophilic glutamic acid in the motif YITENG of beta-glucosidases and mutated recombinant enzyme is discussed.

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. From these results, it can be said that the most effective inhibitor for artichoke PPO is tropolone. Furthermore, it was found that the type of inhibition depended on the origin of the PPO studied and also on the substrate used.

Dihydropyrimidinase (DHPase) was purified 74-fold over the initial Albizzia extract using heat treatment, ammonium sulphate precipitation and sephadex G-200 column chromatography. Its molecular mass, determined by SDS-PAGE, was approximately 56 kDa. The optimum temperatures of DHPase were 60 o C and 55 o C for dihydrouracil (DHU) and dihydrothymine (DHT), respectively. Optimum pH value of DHPase for two substrates was found to be 9.5. The stability of DHPase was determined both in crude enzyme extract and in the sample obtained from ammonium sulphate precipitation. The effect of some metal ions on this enzyme was also examined. Km values of the enzyme for DHU and DHT were 0.33 mM and 0.37 mM, respectively. Vmax values were found as 0.15 U/mL min -1 and 0.092 U/mL min -1 for DHU and DHT, respectively.

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.

All known pyrimidine and pyrimidine-derived secondary producs originate from uracil or its precursor, uracil-6-carboxylic acid. The biosynthesis of these products has been suggested to be uracil detoxication mechanisms. The possible toxic effects of uracil on the germination and growh of Pisum sativum L. cultivar Meteor, Lathyrus tingitanus L. in which pyrimidine-derived secondary products occur naturally, and of Phaseolus aureus Roxb. and Glycine max (L.) Merr., in which these compouns do not occur, were examined. The results show that the germination and growth of the P. aureus and G. max seeds under investigation were considerably inhibited by exogenous uracil. The effect of uracil was obvious on the non-producer group of experimental plants, especially on G. max. However, there was not any noticeable effect of uracil either on P. sativum, or on L. tingitanus in the experimental periods of germination and growth. These results show that uracil accumulation is most probably toxic to plants and that the production of these pyrimidine-derived secondary compounds from uracil is therefore a detoxication mechanism.

The pyrimidine and pyrimidine-derived secondary compounds of Phaseolus aureus and Glycine max were investigated. Batches, each consisting of one hundred 12-day-old seedlings of either P. aureus or of G. max were separately extracted in cold dilute (0.6 M) perchloric acid. After preliminary purification of extracts by charcoal adsorption and elution, and by PVP treatment, the extracts were subjected to paper chromatography and high voltage paper electrophoresis. The UV- absorbing bands were detected by viewing chromatograms and electrophoretograms under UV-light and, where possible, identified. The results presented in this work show that these experimental plants do not synthesize and accumulate any significant amount of a pyrimidine-derived secondary product.

The presence of relatively high non-specific phosphatase activities are known in various plant tissues. These enzymes also catalyze the removal of inorganic phosphate from nucleoside monophosphates. According to published reports, so far, the experimental investigations, concerning the 5'-nucleotidase activity, have been mostly carried out without consideration of the non-specific phosphatase activities in crude enzymic extracts. However, few researchers have been included some inhibitors of these phosphatases, such as NaMoO 4 or KF, in assay mixtures. But phosphatases are not completely inhibited by these inhibitors and, therefore, interfere the activity of 5'-nucleotidase. Thus, the non-specific phosphatase activities were determined in crude enzymic extracts by using p-nitro-phenyl phosphate as substrate and the apparent 5'-nucleotidase activity was, first time, corrected accordingly in this present work.