Purification and partial characterization of three turnip (Brassicanapus L. var. esculenta D.C.) peroxidases

Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 degrees C. The ABTS K(m) values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K(m) value than commercial cationic HRP. The molecular weights determined by SDS-PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2.     

Broccoli processing wastes as a source of peroxidase

A peroxidase isozyme (BP) was purified to homogeneity from broccoli stems ( Brassica oleraceae var. maraton) discarded from industrial processing wastes. BP specific activity was 1216 ABTS [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)] units/mg, representing 466-fold that of crude extract. BP is a monomeric glycoprotein containing 16% carbohydrates, with a molecular mass of 49 kDa and an isoelectric point close to 4.2. From kinetic data it showed a two-substrate ping-pong mechanism, and the catalytic efficiency measured as the rate-limiting step of free BP regeneration was 3.4 x 10(6) M(-1) s(-1). The ABTS K m value was 0.2 mM, which was about 20 times lower than that reported for acidic commercial horseradish peroxidase (HRP). Assessment of BP secondary structure showed 30% helical character, similar to HRP and cytochrome c peroxidase. BP lost only 25% activity after 10 min of heating at 55 degrees C and pH 6; it was stable in the pH range from 4 to 9 and showed an optimum pH of 4.6 using ABTS as substrate. BP was active on substrates normally involved in lignin biosynthesis, such as caffeic and ferulic acids, and also displayed good catechol oxidation activity in the presence of hydrogen peroxide. Reverse micellar extraction was successfully used as potential large-scale prepurification of broccoli peroxidase, achieving a purification factor of 7, with 60% activity yield. Stems from the broccoli processing industry have a high potential as an alternative for peroxidase purification.     

Isolation and thermal characterization of an acidic isoperoxidase from turnip roots

An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.     

Pseudoperoxidase activity of myoglobin: kinetics and mechanism of the peroxidase cycle of myoglobin with H2O2 and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) as substrates

Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) approximately 5.2 at 25.0 degrees C and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M(-1) s(-1), DeltaH# = 66 +/- 4 kJ mol(-1), and DeltaS# = 30 +/- 15 J mol(-1) K(-1) at 25.0 degrees C and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M(-1) s(-1), DeltaH# = 31 +/- 6 kJ mol(-1), and DeltaS# = -53 +/- 19 J mol(-1) K(-1), indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degrees C to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M(-1) s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M(-1) s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.     

Characterization of a heterodimeric GH2 β-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.     

Characterization and role of polyphenol oxidase and peroxidase in browning of fresh-cut melon

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from two different varieties of melon ( Cucumis melo L. cantalupensis cv. Charentais and C. melo L. inodorus cv. Amarillo) and characterized using reliable spectrophotometric methods. In both cases the enzymes followed Michaelis-Menten kinetics, showing different values of kinetics parameters between the two cultivars: K m = 7.18 +/- 0.70 mM ('Charentais') and 6.66 +/- 0.20 mM ('Amarillo') mM; V max = 7.93 +/- 0.35 units/min ('Charentais') and 13.82 +/- 0.37 units/min ('Amarillo'), relative to PPO; K m = 24.0 +/- 2.10 mM ('Charentais') and 5.05 +/- 0.19 mM ('Amarillo') mM; V max = 344.83 +/- 10.32 units/min ('Charentais') and 80.64 +/- 2.01 units/min ('Amarillo'), relative to POD. Optimum pH for PPO was 7.0 for 'Charentais' and 7.5 for 'Amarillo, whereas it was 4.5 for both cultivars relative to POD. Melon PPO had maximum activity at 60 degrees C in both 'Charentais' and 'Amarillo' cultivars, whereas POD maximum activity was found at 45 degrees C in 'Charentais' and at 25 degrees C in 'Amarillo'. POD from both cultivars showed higher thermolability compared with PPO, losing >90% of relative activity after only 5 min of incubation at 70 degrees C. POD's activation energy was much higher than that of PPO (Delta E (#) = 86.3 and 160.6 kJ mol (-1) for 'Charentais' and 'Amarillo', respectively). PPO and POD activities from both cultivars showed a decreasing pattern as sugar concentration in the assay medium increased, except in POD extract from 'Charentais', which maintained its activity in the presence of high d-glucose concentration (up to 5 M). Changes in L*, a*, b*, chroma, and hue angle values were chosen to describe the browning development in the samples during storage at 5 degrees C. A slight decrease in L* value and a more marked reduction of a* value were noted in both cultivars above all at the end of storage period. POD activity during storage at 5 degrees C was highly correlated with changes of parameters a*, b*, chroma, and hue angle ( r (2) from 0.82 to 0.97) for cultivar 'Charentais'. According to these results, only POD activity seemed to be involved in browning of minimally processed melon.     

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.     

Aroma biosynthesis in strawberry: s-adenosylmethionine:furaneol o-methyltransferase activity in ripening fruits

Among the most important volatile compounds in the aroma of strawberries are 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) and its methoxy derivative (methoxyfuraneol, mesifuran). Three strawberry varieties, Malach, Tamar, and Yael, were assessed for total volatiles, Furaneol, and methoxyfuraneol. The content of these compounds sharply increased during fruit ripening, with maximum values at the ripe stage. An enzymatic activity that transfers a methyl group from S-adenosylmethionine (SAM) to Furaneol sharply increases during ripening of strawberry fruits. The in vitro generated methoxyfuraneol was identified by radio-TLC and GC-MS. The partially purified enzyme had a native molecular mass of approximately 80 kDa, with optimum activity at pH 8.5 and 37 degrees C. A high apparent K(m) of 5 mM was calculated for Furaneol, whereas this enzyme preparation apparently accepted as substrates other o-dihydroxyphenol derivatives (such as catechol, caffeic acid, and protocatechuic aldehyde) with much higher affinities (K(m) approximately 105, 130, and 20 microM, respectively). A K(m) for SAM was found to be approximately 5 microM, regardless of the acceptor used. Substrates that contained a phenolic group with only one OH group, such as p-coumaric and trans-ferulic acid, as well as trans-anol and coniferyl alcohol, were apparently not accepted by this activity. It is suggested that Furaneol methylation is mediated by an O-methyltransferase activity and that this activity increases during fruit ripening.     

Characterization of the dimer-monomer equilibrium of the papaya Copper/Zinc superoxide dismutase and its equilibrium shift by a single amino acid mutation.

The coding region of the copper/zinc superoxide dismutase (Cu/Zn SOD) cDNA from papaya fruit, Carica papaya L. cv. Tainong 2, was cloned into an expression vector, pET-20b(+). The Cu/Zn SOD was expressed in Escherichia coli and purified by His-tag technique. Two active forms of the enzyme (30% dimer and 70% monomer) in equilibrium were observed. The activity of the dimeric enzyme was higher than that of the monomeric form. The thermal inactivation rate constant K(d) values calculated for the dimer and monomer at 90 degrees C were -0.0203 and -0.0216 min(-1), and the half-lives for inactivation were 41.9 and 31.8 min, respectively. This indicated that the dimeric enzyme was more stable than its monomeric form. The dimerization of the enzyme was inhibited under acidic pH (below 3.0) or imidazole buffer (above 0.5 M), whereas it was not affected under alkaline pH (above 9.0). Both activity and forms of the enzyme were not affected by 1-4% SDS. Furthermore, the dimeric enzyme was much more resistant to proteolytic attack after 3 h of incubation at 37 degrees C with trypsin or chymotrypsin. In addition, mutation of the papaya Cu/Zn SOD at position 48 from Leu to Phe (L48F) affected the association of monomer, whereas a mutant with Lys substitution (L48K) at the same position tended to dissociate into monomeric form.     

Cloning, heterologous expression and properties of a recombinant active turnip peroxidase

Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% β-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications.     

Screening of postharvest agricultural wastes as alternative sources of peroxidases: characterization and kinetics of a novel peroxidase from lentil ( Lens culinaris L.) stubble

Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL(-1)) did not vary for at least 15 months when stored at 4 °C (k(inact) = 0.146 year(-1), t(1/2 inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k(inact) = 2.2 × 10(-3) day(-1), t(1/2 inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurified by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H(2)O(2)-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K(m)(appH(2)O(2)) = 1.87 mM; V(max)(appH(2)O(2)) = 6.4 mM min(-1); K(m)(app guaicol) = 32 mM; V(max)(app guaicol) = 9.1 mM min(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases. Finally, after the effectiveness of the crude extracts of LSP in oxidizing and removing from solution a series of last-generation dyes present in effluents from textile industries (1) had been checked, a steady-state kinetic study of the H(2)O(2)-mediated oxidation and decolorization of Green Domalan BL by the catalytic action of the lentil stubble extract was carried out, with the observation of the same apparent Michaelian kinetic behavior (K(m)(appGD) = 471 μM; V(max)(appGD)= 23 μM min(-1)). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.     

Dehydroascorbate reductase cDNA from sweet potato (Ipomoea batatas [L.] Lam): expression, enzyme properties, and kinetic studies

A cDNA encoding a putative dehydroascorbate reductase (DHAR) was cloned from sweet potato. The deduced protein showed a high level of sequence homology with DHARs from other plants (67 to approximately 81%). Functional sweet potato DHAR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 12% native PAGE. The protein's half-life of deactivation at 50 degrees C was 10.1 min, and its thermal inactivation rate constant K(d) was 6.4 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 6.0-11.0 and in the presence of 0.8 M imidazole. The K(m) values for DHA and GSH were 0.19 and 2.38 mM, respectively.     

Phenolic compounds, carotenoids, anthocyanins, and antioxidant capacity of colored maize (Zea mays L.) kernels

In this study, the contents of total phenolics, flavonoids, anthocyanins, β-carotene, and lutein as well as free, conjugated, and insoluble bound phenolic acids were determined in whole kernels of 10 different colored maize genotypes. In addition, the antioxidant activity was evaluated as radical scavenging activity with ABTS (2,2-azino-bis/3-ethil-benothiazoline-6-sulfonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) reagents. Generally, considerable differences in phytochemical contents and antioxidant capacity were observed between the genotypes. The β-carotene and lutein contents ranged from 0 to 2.42 mg/kg d.m. and from 0 to 13.89 mg/kg d.m., respectively, whereas the total anthocyanin contents of anthocyanin-rich colored maize genotypes ranged from 2.50 to 696.07 mg CGE/kg d.m. (cyanidin 3-glucoside equivalent) with cyanidin 3-glucoside (Cy-3-Glu) as the most dominant form. The light blue ZPP-2 selfed maize genotype has a higher content of total phenolics, flavonoids, and ferulic acid as compared to other tested maize and the highest ABTS radical scavenging activity.     

Application of the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation assay to a flow injection system for the evaluation of antioxidant activity of some pure compounds and beverages

The 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(*)(+)) assay was adapted to a flow injection (FI) system to obtain a sensitive and rapid technique for the monitoring of antioxidant activity of pure compounds and complex matrixes, such as beverages and food extracts. The FI system includes a HPLC pump that flows the mobile phase (a solution of ABTS(*)(+) in ethanol) through a 20 microL loop injector, a single bead string reactor filled with acid-washed silanized beads, a delay coil and a photodiode array UV-visible detector. The technique was very sensitive, with limits of detection and of quantification of 4.14 and 9.29 micromol of Trolox/L, respectively, and demonstrated high repeatability and reproducibility. The proposed technique was then applied to the evaluation of the antioxidant activity of some pure compounds, demonstrating good agreement with published data obtained by the original spectrophotometric ABTS(*)(+) assay. Finally, the total antioxidant activity of 10 beverages was determined by both the proposed and the original method. The values ranged from 0.09 mmol L(-)(1) for cola to 49.24 mmol L(-)(1) for espresso coffee and did not result significantly different from those obtained by the original spectrophotometric ABTS(*)(+) assay (Student's paired t-test: t = 1.4074, p = 0.1929). In conclusion, the proposed FI technique seems suitable for the direct, rapid and reliable monitoring of total antioxidant activity of pure compounds and beverages and, due to the ability to operate in continuous, it allows the analysis of about 30 samples h(-)(1) making the assay particularly suitable for large screening of total antioxidant activity in food samples.     

Thermal degradation of anthocyanins from purple potato (cv. Purple Majesty) and impact on antioxidant capacity

Degradation parameters of purified anthocyanins from purple-fleshed potato (cv. Purple Majesty) heated at high temperatures (100-150 °C) were determined. Purified anthocyanins, prepared by removing salts, sugars, and colorless nonanthocyanin phenolics from the crude extract, were monitored and quantified using HPLC and spectrophotometry for heat-induced degradation products. Separation of colorless phenolics from the anthocyanins was confirmed using HPLC at two wavelengths, 280 and 520 nm. The degradation kinetics of purified anthocyanins followed a first-order reaction with reaction rate constants (k values) of 0.0262-0.2855 min(-1), an activation energy of 72.89 kJ/mol, thermal death times (D values) of 8.06-8789 min, and a z value of 47.84 °C over the temperature range of 100-150 °C. The enthalpy and entropy of activation were 59.97 kJ/mol and -116.46 J/mol·K, respectively. The antioxidant capacity in the purified anthocyanins, measured by DPPH and ABTS assays, was increased after the thermal treatment, indicating antioxidant activities of degradation products in the samples.     

Influence of ethylenediaminetetraacetic acid (EDTA) on the structural stability of endoglucanase from Aspergillus aculeatus

The effect of the chelating agent ethylenediaminetetraacetic acid (EDTA) on the structure and function of endoglucanase is studied. In the presence of 2 mM EDTA, endoglucanase showed an enhanced enzymatic activity of 1.5-fold compared to control. No further change in activity was observed with increase in the concentration of EDTA to 5 mM. The K(m) values for control and in the presence of EDTA are 0.060 and 0.044%, respectively, and K(cat) was 1.9 min(-1) in the presence of EDTA. The kinetic parameters indicated a decrease in the K(m) with an increase in the K(cat). Far-ultraviolet circular dichroism (far-UV-CD) results showed a 20% decrease in ellipticity values at 217 nm in the presence of EDTA compared to native enzyme. The apparent T(m) shifted from a control value of 57 ± 1 to 76 ± 1 °C in the presence of EDTA (5 mM). The above results suggested that the enhanced activity in the presence of EDTA is due to an increase in the K(cat) and flexible conformation of the enzyme. The stability of endoglucanase increased in the presence of EDTA.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

Lettuce and chicory byproducts as a source of antioxidant phenolic extracts

A process to obtain enriched antioxidant phenolic extracts from lettuce (baby, romaine, and iceberg cultivars) and chichory byproducts as a way to valorize these byproducts was developed. Two extraction protocols using water and methanol as solvent were used. Amberlite XAD-2 nonionic polymeric resin was used to purify the extracts. The extraction yield, phenolic content, and phenolic yield were evaluated as well as the antioxidant capacity of the extracts (DPPH, ABTS, and FRAP assays). Baby and romaine lettuce byproducts showed the highest water extract yields [27 and 26 g of freeze-dried extracts/kg of byproduct fresh weight (fw), respectively], whereas baby and iceberg lettuce showed highest methanol extract yields (31 and 23 g of freeze-dried extracts/kg of byproduct fw, respectively). Methanol extraction yielded a raw extract with a high phenolic content, the baby and chicory extracts being the richest with approximately 50 mg of phenolics/g of freeze-dried extract. Regarding the purified extracts, water extraction yielded a higher phenolic content, baby and chicory being also the highest with mean values of approximately 190 and 300 mg of phenolics/g of freeze-dried extract, respectively. Both raw and purified extracts from baby and chicory showed the higher antioxidant contents (DPPH, ABTS, and FRAP assays). The antioxidant capacity was linearly correlated with the phenolic content. The results obtained indicate that lettuce byproducts could be, from the industrial point of view, an interesting and cheap source of antioxidant phenolic extracts to funcionalize foodstuffs.     

Antiviral activity of esterified alpha-lactalbumin and beta-lactoglobulin against herpes simplex virus type 1. Comparison with the effect of acyclovir and L-polylysines

The antiviral activity of methylated alpha-lactalbumin (Met-ALA), methylated and ethylated beta-lactoglobulins (Met- and Et-BLG) was evaluated against acyclovir (ACV)-sensitive and -resistant strains of herpes simplex virus type 1 (HSV-1) and compared to that of ACV and L-polylysines (4-15 kDa) using fixed or suspended Vero cell lines. Esterified whey proteins and their peptic hydrolyzates displayed protective action against HSV-1, which was relatively lower than that induced by ACV or L-polylysines. The higher activity of L-polylysines was maintained against an ACV-resistant strain of HSV-1, whereas ACV lost much of its activity. The mean 50% inhibitory concentration (IC50) was about 0.8-0.9 microg/mL for L-polylysines against ACV-sensitive and -resistant strains of HSV-1 when using two concentrations of virus (50% and 100% cytopathic effect, CPE). The IC50 values of ACV against the sensitive strain of HSV-1 were 3 and 15 microg/mL when using the low and high concentrations of virus, respectively. When using 50% CPE, IC50 values for esterified whey proteins ranged from 20 to 95 microg/mL, depending on the nature of the ester group, the degree of esterification, and the nature of the protein. Using the real-time PCR technique, it was shown that Met-ALA inhibited HSV-1 replication.     

Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity

Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.