- Quantification of hesperidin in citrus-based foods using a fungal diglycosidase
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A simple enzymatic-spectrophotometric method for hesperidin quantification was developed by means of a specific fungal enzyme. The method utilises the diglycosidase α-rhamnosyl-β-glucosidase (EC 3.2.1.168) to quantitatively hydrolyse hesperidin to hesperetin, and the last is measured by its intrinsic absorbance in the UV range at 323 nm. The application of this method to quantify hesperidin in orange (Citrus sinensis) juices was shown to be reliable in comparison with the standard method for flavonoid quantification (high performance liquid chromatography, HPLC). The enzymatic method was found to have a limit of quantification of 1.8 μM (1.1 mg/L) hesperidin, similar to the limit usually achieved by HPLC. Moreover, it was feasible to be applied to raw juice, without sample extraction. This feature eliminated the sample pre-treatment, which is mandatory for HPLC, with the consequent reduction of the time required for the quantification.
- Mazzaferro, Laura S.,Breccia, Javier D.
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- Enzymatic deglycosylation of flavonoids in deep eutectic solvents-aqueous mixtures: Paving the way for sustainable flavonoid chemistry
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The low solubility of glycosylated flavonoids represents a hurdle to conduct efficient enzymatic deglycosylations in aqueous media. To overcome this drawback, environmentally-unfriendly dimethylsulfoxide (DMSO) is typically used as co-solvent. Using a specific diglycosidase from Acremonium sp. DSM24697 for the deglycosylation of the rutinosylated flavonoid (hesperidin) as model reaction, this communication explores the use of (non-hazardous and biodegradable) deep eutectic solvents (DESs) as co-solvents in flavonoid biocatalysis. The enzymatic deglycosylation was observed when DES composed of choline chloride and glycerol or ethylene-glycol was used at proportions of up to 40% (DES-Buffer, v/v), displaying a promising framework to combine enhanced flavonoid solubilities and high enzymatic activities. The deglycosylation activity significantly increased when the single DES components - glycerol and ethylene-glycol - were added (e.g. 140% of enzyme activity at glycerol at 40% v/v), whereas deleterious effects were observed when choline chloride was solely added, presumably due to its chaotropic effect. Future research opportunities may be envisaged in the genetic design to evolve more robust biocatalysts, and in tailoring DES to deliver more enzyme-compatible solvents.
- Weiz, Gisela,Braun, Lucas,Lopez, Rosana,De María, Pablo Domínguez,Breccia, Javier D.
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- Access to both anomers of rutinosyl azide using wild-type rutinosidase and its catalytic nucleophile mutant
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Rutinosidases hydrolyze β-rutinosylated flavonoids. As retaining glycosidases they also have a transglycosylation activity. Here we show that two newly identified wild-type rutinosidases, which are members of the glycoside hydrolase family 5–23, are capable of glycosylation of an inorganic azide with rutin as a glycosyl donor, yielding rutinosyl β-azide. On the other hand, rutinosyl α-azide was synthesized by the catalytic nucleophile mutant of the rutinosidase from Aspergillus niger, which also belongs to GH5–23. Thus, we were able to synthesize at a preparatory scale both anomers of rutinosyl azide from rutin using either wild-type or mutant rutinosidases of GH5–23.
- Bojarová, Pavla,Brodsky, Katerina,Halada, Petr,Jav?rková, Hana,K?en, Vladimír,Konvalinková, Dorota,Kotik, Michael,Pelantová, Helena
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- Transrutinosylation of tyrosol by flower buds of Sophora japonica
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Dried flower buds of Japanese sophora (Sophora japonica) comprising rutinosidase activity were tested in rutinosylation of tyrosol via transglycosylation process from rutin. Optimal conditions for transrutinosylation of tyrosol were 49 mM rutin and 290 mM
- Karni?ová Potocká, Elena,Mastihuba, Vladimír,Mastihubová, Mária
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- New assay of α-l-rhamnosidase
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Abstract: Free rutinose was prepared by enzymatic hydrolysis of rutin using defatted seed meal from tartary buckwheat. This disaccharide was used as substrate in spectrophotometric assay of α-l-rhamnosidase. The assay is based on hydrolysis of rutinose and subsequent determination of released glucose by a standard glucose oxidase assay kit. The method is easy to perform and requires no expensive equipment. The assay was applied in α-l-rhamnosidase estimation in ten commercial enzyme preparations and compared with standard assay on chromogenic substrate.
- Karni?ová Potocká, Elena,Mastihubová, Mária,?i?ová, Iveta,Mastihuba, Vladimír
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p. 167 - 174
(2017/12/06)
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- Dracopalmaside, a New Flavonoid from Dracocephalum palmatum
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Phytochemical studies of the aerial part of Dracocephalum palmatum (Lamiaceae) isolated the new flavonoid dracopalmaside that was identified based on UV, MS, and NMR spectral data as luteolin-7,4′-di-O-α -Lrhamnopyranosyl-(1→6)-β-D-glucopyranoside (luteolin-7,4′-di-O-rutinoside) and the two known compounds cynarotriside and luteolin-7,4′-di-O-glucoside.
- Olennikov,Chirikova
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p. 1067 - 1069
(2016/02/18)
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- α-L-Rhamnosyl-β-D-glucosidase (rutinosidase) from Aspergillus niger: Characterization and synthetic potential of a novel diglycosidase
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We report the first heterologous production of a fungal rutinosidase (6-O-α-L-rhamnopyranosyl-β-D-glucopyranosidase) in Pichia pastoris. The recombinant rutinosidase was purified from the culture medium to apparent homogeneity and biochemically characterized. The enzyme reacts with rutin and cleaves the glycosidic linkage between the disaccharide rutinose and the aglycone. Furthermore, it exhibits high transglycosylation activity, transferring rutinose from rutin as a glycosyl donor onto various alcohols and phenols. The utility of the recombinant rutinosidase was demonstrated by its use for the synthesis of a broad spectrum of rutinosides of primary (saturated and unsaturated), secondary, acyclic and phenolic alcohols as well as for the preparation of free rutinose. Moreover, the α-L-rhamnosidase-catalyzed synthesis of a chromogenic substrate for a rutinosidase assay - para-nitrophenyl β-rutinoside - is described.
- imkov, Daniela,Kotik, Michael,Weignerov, Lenka,Halada, Petr,Pelantov, Helena,Adamcov, Kateina,Ken, Vladimr
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p. 107 - 117
(2015/01/30)
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- Transglycosylation specificity of Acremonium sp. α-rhamnosyl-β- glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside
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Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-β-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na]+ ions correlated with the theoretical monoisotopic mass (calcd [M+Na]+: 507.44 m/z; obs. [M+Na]+: 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-β-glucosidases by zymographic analysis.
- Mazzaferro, Laura S.,Pi?uel, Lucrecia,Erra-Balsells, Rosa,Giudicessi, Silvana L.,Breccia, Javier D.
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scheme or table
p. 69 - 75
(2012/02/05)
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- α-Rhamnosyl-β-glucosidase-catalyzed reactions for analysis and biotransformations of plant-based foods
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Most aroma compounds exist in vegetal tissues as disaccharide conjugates, rutinose being an abundant sugar moiety in grapes. The availability of aroma precursors would facilitate analytical analysis of plant-based foods. The diglycosidase α-rhamnosyl-β-glucosidase from Acremonium sp. DSM 24697 efficiently transglycosylated the rutinose moiety from hesperidin to 2-phenylethanol, geraniol, and nerol in an aqueous-organic biphasic system. 2-Phenethyl rutinoside was synthesized up to millimolar level with an 80% conversion regarding the donor hesperidin. The hydrolysis of the synthesized aroma precursors was not detected in an aqueous medium. However, in the presence of ethanol as a sugar acceptor, the enzyme was able to transfer the disaccharide residue forming the alkyl-rutinoside. The aroma precursors were significantly hydrolyzed (up to 3-4% in 2 h at 30 °C), which indicated the potential use of the enzyme for biotechnological applications, for example, in aroma modulation of fermented foods.
- Minig, Marisol,Mazzaferro, Laura S.,Erra-Balsells, Rosa,Petroselli, Gabriela,Breccia, Javier D.
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experimental part
p. 11238 - 11243
(2012/03/08)
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- Chemical composition and biological activity of Citrus jambhiri Lush
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The fresh peel of Citrus jambhiri was extracted with aqueous methanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. The chemical structure of the isolated compounds was then identified by MS and NMR. Column chromatography of the petroleum fraction resulted in the isolation of nobiletin, 5-O-demethylnobiletin, tangeretin, 5-hydroxy-3,6,7,8,3′,4′- hexamethoxyflavone, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and a mixture of β-sitosterol and stigmasterol. The chloroform fraction afforded 6-demethoxynobiletin, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, limonin and nomilin. The flavonoid glycosides naringin, hesperidin and neohesperidin were isolated from the ethyl acetate fraction. The chemical structure of the isolated compounds was established by MS and NMR (APT, COSY, HSQC, HMBC, and NOESY). LC-ESI-MS analysis of the ethyl acetate fraction afforded eight flavonoid glycosides, while the dichloromethane fraction of the defatted seeds contained seven limonoid aglycones. The chloroform fraction exerted the strongest DPPH* free radical scavenging activity in comparison to other fractions. The petroleum fraction showed a significant inhibition of lipoxygenase indicating an anti-inflammatory action (IC50 29 ± 1 μg/mL). Some of the isolated polymethoxyflavones exhibited strong cytotoxicity against COS7, HeLa and Caco-2 cell lines.
- Hamdan, Dalia,El-Readi, Mahmoud Zaki,Tahrani, Ahmad,Herrmann, Florian,Kaufmann, Dorothea,Farrag, Nawal,El-Shazly, Assem,Wink, Michael
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p. 394 - 403
(2013/01/09)
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- Synthesis of fluorescently labelled rhamnosides: Probes for the evaluation of rhamnogalacturonan II biosynthetic enzymes
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Three fluorescently labelled saccharides 10-12, representing structures found in pectic glycan rhamnogalacturonan II (RG-II), were synthesised by chemical glycosylation of O-6 of diacetone-d-galactose followed by deprotection and reductive amination with amino-substituted fluorophore APTS. This convenient method installs a common aminogalactitol-based tether in order to preserve the integrity of the reducing end of specific carbohydrates of interest. APTS-labelled glycans prepared in this manner were purified by carbohydrate gel electrophoresis and subjected to capillary electrophoresis analysis, as a basis for the subsequent development of high sensitivity assays for RG-II-active enzymes.
- Prifti, Efthymia,Goetz, Stephan,Nepogodiev, Sergey A.,Field, Robert A.
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experimental part
p. 1617 - 1621
(2011/09/20)
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- Operational stabilization of fungal α-rhamnosyl-β-glucosidase by immobilization on chitosan composites
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The diglycosidase α-rhamnosyl-β-glucosidase from Acremonium sp. DSM24697 was immobilized by adsorption and cross-linking. The supports screened included beads of chitosan composites (gelatin, arabic gum, silica gel), epoxy-activated agarose and chitosan, and macroporous polyvinyl-alcohol cryogel. The chitosan-silica gel beads were selected because of the highest immobilization efficiency obtained and their morphological properties (diameter 1.67 ± 0.99 mm, circularity 0.81 ± 0.05). The optimization of the immobilization - coating with polyethyleneimine, changes in the enzyme load - improved the immobilization efficiency up to 18%. The practical use of the enzyme deals with low water solubility substrates. The higher KM for the immobilized enzyme - 8 mM vs. 1.8 mM hesperidin for the free enzyme - indicated that substrate diffusion limits the enzymatic reaction. The solvent dimethylsulfoxide (50%, v/v) was added in order to increase the substrate solubility, and 80% activity was retained (1 h, 60 °C) in contrast with the complete inactivation of the free form. The stability of the immobilized catalyst was extended to metal catalyzed oxidation where the enzyme was fully preserved in harsh conditions such as 1 mM CuSO4 at 60 °C during 1 h.
- Pi?uel, Lucrecia,Mazzaferro, Laura S.,Breccia, Javier D.
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experimental part
p. 2330 - 2335
(2012/05/20)
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- Mild alkaline hydrolysis of some 7-O-flavone glycosides. Application to a novel access to rutinose heptaacetate
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Alkaline hydrolysis of some 7-O-flavone glycosides was performed through the 6,8-dibromo derivative. When the sugar linked to the aglycon has a 2-hydroxy group trans to the sugar-aglycon bond as in β-D-glucosides or rutinosides, hydrolysis occurred at room temperature under very mild conditions. Application to a novel preparation of rutinose heptaacetate by hydrolysis of a diosmin derivative is described.
- Quintin, Jér?me,Lewin, Guy
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p. 4341 - 4343
(2007/10/03)
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- Purification and characterization of a flavonol 3-O-β heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench
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A flavonol-3-O-β-heterodisaccharide glycosidase (FHG I) was isolated from dried aerial tissues of Fagopyrum esculentum Moench (Fagopyri herba). It has a specific enzyme activity of ca. 3.5 nkat mg-1 protein in buffered extracts when rutin (quercetin-3-O-rutinoside) was used as substrate and an optimal enzyme activity was seen at around pH 4.8 and 30 °C. FHG I was purified about 156-fold to apparent homogeneity by hydrophobic interaction, anion exchange and size exclusion chromatographic steps. The apparent molecular mass of FHG I was 74.5 ± 2 kDa as determined by SDS-PAGE and it is a monomeric glycoprotein with a carbohydrate content of 23%. The isoelectric point as determined by isoelectric focusing was 5.7 and the energy of activation was 32 kJ mol-1. FHG I exhibits a high substrate specificity, preferring flavonol 3-O-glycosides comprising the disaccharide rutinose. The Km and Vmax values for the natural substrate rutin were calculated to be 0.561 μM and 745 nkat mg -1 protein, respectively. Two oligopeptide fragments obtained after enzymatic digestion of FHG I were sequenced and showed similarities to sequences of β-glucohydrolases from other plant species. Polyclonal antibodies were raised and their specificities determined. Another flavonol 3-O-β -heterodisaccharide glycosidase (FHG II) could also be detected in buckwheat herb, having a molecular mass of 85.3 ± 2 kDa and an isoelectric point between pH 6.0 and 6.5.
- Baumgertel, Andreas,Grimm, Rudi,Eisenbeiss, Wilhelm,Kreis, Wolfgang
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p. 411 - 418
(2007/10/03)
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- Anthocyanidin glycosides from the flowers of Alstroemeria
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Three anthocyanins were isolated from the red flowers of four cultivars of Alstroemeria. Two compounds were novel anthocyanidin glycosides; the 3-rutinoside and 3-monoglucoside of 6-hydroxycyanidin. Cyanidin 3-rutinoside was also present in the petals.
- Saito, Norio,Yokoi, Masato,Yamaji, Minako,Honda, Toshio
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p. 2125 - 2126
(2007/10/02)
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- Studies on the Constituents of Apocynacae Plants. Gas Chromatography-Mass Spectrometric Determination of New Flavanoid Triglycosides from the Leaves of Cerbera manghas L.
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Two new flavonol triglycosides, named manghaslin(I) and clitorin(II), were isolated from the leaves of Cerbera manghas L. (Apocynaceae).The structures of I and II were elucidated as quertecin-3-O-L-rhamnosyl-(12)-O6)> D-glucoside(I) and kaempferol-3-O-L-rhamnosyl-(12)-O-6)> D-glucoside(II), respectively, by chemical and gas chromatography-mass spectrometric studies.Keywords-Cerbera manghas L.; Apocynaceae; flavonol triglycerides; manghaslin; clitorin; gas chromatography-mass spectrometry; photohydrolysis; mass spectrum; methanolysis.
- Sakushima, Akiyo,Hisada, Sueo,Ogihara, Yukio,Nishibe, Sansei
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p. 1219 - 1223
(2007/10/02)
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- SYNTHESIS OF 1,2-trans-DISACCHARIDES via SUGAR THIO-ORTHOESTERS
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The reaction of sugar 1,2-thio-orthoesters in the D-gluco, D-galacto, D-manno, and L-rhamno series with primary and secondary trityl ethers of monosaccharides, in the presence of triphenylmethylium perchlorate as catalyst, affords, stereospecifically, derivatives of 1,2-trans-disaccharides in good yields. 4-Trityl ethers of benzyl 2-acetamido-3,6-di-O-acetyl-2-deoxy-α-D-glucopyranoside and methyl 2,3,6-tri-O-benzoyl-α-D-galactopyranoside exhibit low reactivity in glycosylation by thio-ortho-esters.A reaction scheme for the glycosylation is discussed.
- Backinowsky, Leon V.,Tsvetkov, Yury E.,Balan, Nikolay F.,Byramova, Narguiz E.,Kochetkov, Nikolay K.
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p. 209 - 222
(2007/10/02)
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