Patents developed with the collaboration of staff members of GENZ.
Inventors: Marin-Zamora, M.E., Garcia-Canovas, F., Garcia-Ruiz, P.A.
Title: Method for obtaining o-diphenols, involves carrying out hydroxylation of monophenol in ortho position with respect to hydroxyl group, and hydroxylation of monophenol is enzymatically catalyzed by tyrosinase in presence of borate buffer.
Request Number: ES2301453-A1.
Priority Country: Spain.
Priority Date: 18-2-2008.
Entity: University of Murcia.
Extention Countries: Spain.
Exploitation Business: In study.
References:
Production of o-diphenols by inmobilized mushroom tyrosinase.
Journal of Biotechnology, 139, 163-168, 2009.
Abstract: The o-diphenols 4-tert-butyl-catechol, 4-methyl-catechol, 4-methoxy-catechol, 3,4-dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acidwere produced fromthe corresponding monophenols (4-tert-butyl-phenol, 4-methyl-phenol, 4-methoxy-phenol, p-hydroxyphenylpropionic acid and p-hydroxyphenylacetic acid) using immobilized mushroom tyrosinase from Agaricus bisporus. In all cases the yield was Rdiphenol ≥88–96%, which, according to the literature, is the highest yield so far, obtained using tyrosinase. The reaction was carried out in 0.5M borate buffer pH 9.0 which was used to minimize the diphenolase activity of tyrosinase by complexing the o-diphenols generated. Hydroxylamine and ascorbic acidwere also present in the reaction medium, the former being used to reduce mettyrosinase to deoxytyrosinase, closing the catalytic cycle, and the latter to reduce the o-quinone produced to o-diphenol. Inactivation of the tyrosinase by ascorbic acid was also minimized due to the formation of an ascorbic acid–borate complex. Concentrations of the o-diphenolic compounds obtained at several reaction times were determined by gas chromatography–mass spectrometry (GC–MS) and UV–vis spectroscopy. The experimental results are discussed.
Inventors: Rodríguez López, J.N., López Molina, D., Tudela, J. y García Cánovas, F.
Title: Enzyme with peroxidase activity isolated from artichoke (Cynara scolymus L.), procedure for its isolation, purification and applications.
Request Number: P200002553/6.
Priority Country: Spain.
Priority Date: 24-10-2000.
Entity: University of Murcia.
Extention Countries: European Union.
Exploitation Business: BIOPRODIN S.L.
References:
Purification and partial characterization of a new cationic peroxidase from fresh flowers of Cynara scolymus L.
J. Inorg. Biochem. 94, 243-254, 2003.
Abstract: A basic heme peroxidase isoenzyme (AKPC) has been purified to homogeneity from artichoke flowers (Cynara scolymus L.). The enzyme was shown to be a monomeric glycoprotein, Mr = 42000 ± 1000, (mean ± S.D.) with an isoelectric point >9. The native enzyme exhibits a typical peroxidase ultraviolet–visible spectrum with a Soret peak at 404 nm (e = 137000 ± 3000 cm ) and a Reinheitzahl (Rz) value (A404nm / A280nm) of 3.8 ± 0.2. The ultraviolet–visible absorption spectra of compounds I, II and III were typical of class III plant peroxidases but unlike horseradish peroxidase isoenzyme C, compound I was unstable. Resonance Raman and UV–Vis spectra of the ferric form show that between pH 5.0 and 7.0 the protein is mainly 6 coordinate high spin with a water molecule as the sixth ligand. The substrate-specificity of AKPC is characteristic of class III (guaiacol-type) peroxidases with chlorogenic and caffeic acids, that are abundant in artichoke flowers, as particularly good substrates at pH 4.5. Ferric AKPC reacts with hydrogen peroxide to yield compound I with a second-order rate constant (k+1) of 7.4 x 105 M-1 s-1 which is significantly slower than that reported for most other class III peroxidases. The reaction of ferric and ferrous AKPC with nitric oxide showed a potential use of this enzyme for quantitative spectrophotometric determination of NO and as a component of novel NO sensitive electrodes.
Compound I formation in artichoke (Cynara scolymus L.) peroxidase is modulated by the equilibrium between pentacoordinated and 6-aquo hexacoordinated forms of the heme and by calcium ions.
Biochemistry, 42, 8799-8808, 2003.
Abstract: Basic artichoke (Cynara scolymus L.) peroxidase (AKP-C), when purified from the plant, has an unusually intense and sharp Soret absorption peak. The resonance Raman spectrum [López-Molina, D., et al. (2003) J. Inorg. Biochem. 94, 243-254] suggested a mixture of pentacoordinate high-spin (5cHS) and 6-aquo hexacoordinate high-spin (6cHS) ferric heme species. The rate constant (k+1) of compound I formation with hydrogen peroxide (H2O2) was also lower than expected. Further stopped-flow studies have shown this reaction to be biphasic: a nonsaturating fast phase and a slow phase with complex H2O2 concentration dependence. Addition of calcium ions (Ca2+) changed the absorption spectrum, suggesting the formation of a fully 5cHS species with a k+1 more than 5 orders of magnitude greater than that in the absence of Ca2+ using the chelator ethylenediaminetetraacetic acid. Ca2+ titrations gave a dissociation constant for a single Ca2+ of approximately 20 mM. The circular dichroism spectrum of AKP-C was not significantly altered by Ca2+, indicating that any structural changes will be minor, but removal of Ca2+ did suppress the alkaline transition between pH 10 and 11. A kinetic analysis of the reaction of Ca2+-free AKP-C with H2O2 supports an equilibrium between a slow-reacting 6cHS form and a more rapidly reacting 5cHS species, the presence of which was confirmed in nonaqueous solution. AKP-C, as purified, is a mixture of Ca2+-bound 5cHS, 6-aquo 6cHS, and Ca2+-free 5cHS species. The possibility that Ca2+ concentration could control peroxidase activity in the plant is discussed.
Inventors: Rodríguez López, J.N., Tudela, J. y García Cánovas, F.
Title: Artichoke (Cynara scolymus L.) extract and its use in the bioreparation of media polluted with phenols, aromatic amines, organic halides and heavy metals.
Request Number: P200002544/7.
Priority Country: Spain.
Priority Date: 23-10-2000.
Entity: University of Murcia.
Extention Countries: Spain.
Exploitation Business: ARTBIOCHEM S.L.
Reference:
Enzymatic renoval of phenols from aqueous solution by artichoke (Cynara scolymus L.) extracts.
Enzyme and Microbial Technology, 33, 738-742, 2003.
Abstract: The effects of extracts from artichoke (Cynara scolymus L.) flower bracts on model wastewaters containing a range of phenolic contaminants have been studied. The extracts contained various isoenzymes of both peroxidase (POD) and polyphenol oxidase (PPO). HPLC measurements showed that the monophenol, 4-chlorophenol, was most effectively oxidized in the presence of both extract and hydrogen peroxide (H2O2), suggesting that this substrate was mainly being acted upon by POD. In similar experiments with l-dopa, an o-diphenol, the presence of H2O2 was not required for the efficient formation of insoluble melanins as PPO was more active in this case. Agitation of the reaction mixture favored oxidation by PPO by ensuring an adequate supply of oxygen to the enzyme. Conversely, bubbling with nitrogen greatly reduced PPO activity but did not affect POD. Some care was required to avoid the presence of large excesses of H2O2, which could lead to the inactivation of the enzymes. The results indicate that using a mixture of enzymes (and possibly also non-enzymic constituents) such as that found in artichoke extract, makes wastewater treatment with extract more effective and flexible than using either POD or PPO alone. Furthermore, artichoke extract was easy and simple to produce from a low value source making the method highly economical.
Inventors: Bañón Arnao, M., Cano, A., García Cánovas, F y Acosta, M.
Title: Method of evaluation of the antioxidant power in foods.
Request Number: P9801295.
Priority Country: Spain.
Priority Date: 19-06-1998.
Entity: University of Murcia.
Extention Countries: Spain.
Exploitation Business: In study.
Reference:
An end-point method for estimation of the total antioxidant activity in plant material.
Phytochemical Analysis 9, 196-202, 1998.
Abstract: The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) or ABTS radical can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase. This ABTS radical (ABTSR), a chromogen, is stable at room temperature but is unstable above 35ºC and/or at pH values of above 7.5. Nevertheless, the most important factor in its stability is the ABTS/ABTSR concentration ratio in the medium. The radical reacts with the antioxidant L-ascorbic acid, with a high rate constant, the stoichiometry of the reaction being 1 mol of L-ascorbic acid per 2 mol of ABTSR reduced. Based on these considerations, a spectrophotometric end-point method has been developed to evaluate L-ascorbic acid in aqueous media, and this represents an improvement over the lag-method previously reported. Under optimal conditions of temperature, pH and reagent concentration, the end-point method was capable of determining L-ascorbic acid with a limit of quantification of 0.38 nmol. In the assay described here, this ability is used to evaluate the total antioxidant activity of commercial citrus juices, in wich ascorbic acid is a principal component. In our opinion this procedure can quickly provide useful information on the antioxidant content of foods and plant extracts.