Mária Mastihubová

A spectrophotometric assay for feruloyl esterases.

We have developed a spectrophotometric assay for the quantitative determination of feruloyl esterase activity based on release of 4-nitrophenol from a novel substrate, 4-nitrophenyl ferulate in an emulsion of Triton X-100 in aqueous buffer solution. The release of 4-nitrophenol was linear with reaction time at an early stage of the reaction with various esterase preparations. The method proposed here is accurate, rapid, and easy to perform.

Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters

An extracellular feruloyl esterase (FoFAE-I) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel filtration chromatograp ...

Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidium pullulans

ABSTRACT The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 μmol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1,3)-O-β-d-xylopyranosyl-(1,4)-d-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (kcat/Km) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.

Two efficient ways to 2-O- and 5-O-feruloylated 4-nitrophenyl α-L-arabinofuranosides as substrates for differentiation of feruloyl esterases

Abstract 4-Nitrophenyl 2- O -( E )-feruloyl-α- l -arabinofuranoside 1 and 4-nitrophenyl 5- O -( E )-feruloyl-α- l -arabinofuranoside 2 have been synthesized by two different routes. Monoferuloylation was accomplished by a chemoenzymatic sequence employing a regioselective transesterification catalyzed by lipases. The feruloyl group was introduced to enzymatically prepared 2,3- and 3,5-diacetates of 4-nitrophenyl α- l -arabinofuranoside by reaction with 4- O -acetylferuloyl chloride. Removal of the protecting acetyl groups yielded 1 and 2 . An alternative chemical synthesis suitable for preparation of larger quantities of 1 and 2 also is presented. The new substrates represent convenient tools to differentiate feruloyl esterases on the basis of their substrate specificity.

Differentiation of feruloyl esterases on synthetic substrates in α-arabinofuranosidase-coupled and ultraviolet-spectrophotometric assays.

4-Nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside, synthesized by our group (M. Mastihubová, J. Szemesová, and P. Biely), were found to be suitable substrates for determination of activity of feruloyl esterases (FeEs) exhibiting affinity for 5-O- and 2-O-feruloylated alpha-L-arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl alpha-L-arabinofuranoside with its efficient hydrolysis by alpha-L-arabinofuranosidase to release 4-nitrophenol. An alternative assay explores the difference in the molar absorbances at 340 nm of the substrate (ferulic acid esters) and the reaction products, which are (1) free ferulic acid and 4-nitrophenyl alpha-L-arabinofuranoside in samples free of alpha-L-arabinofuranosidase and (2) ferulic acid, 4-nitrophenyl alpha-L-arabinofuranoside, and/or 4-nitrophenol in samples containing alpha-L-arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity.

A common access to 2- and 3-substituted methyl β-d-xylopyranosides

2-Deoxy-, 3-deoxy-, 2-deoxy-2-fluoro- and 3-deoxy-3-fluoro- derivatives of methyl β-d-xylopyranoside diacetates were prepared by a new common route via 2,3-anhydropentosides. The stereo- and regioselective introduction of fluorine or hydrogen was accomplished by epoxide ring opening of methyl 2,3-anhydro-β-d-ribopyranoside and methyl 2,3-anhydro-4-O-benzyl-β-d-lyxopyranoside. Methyl 2,3-anhydro-4-O-benzyl-β-d-lyxopyranoside was originally obtained in three simple steps from readily available methyl 2,3-anhydro-4-O-benzyl-β-d-ribopyranoside.

Salidroside, a Chemopreventive Glycoside, Diminishes Cytotoxic Effect of Cisplatin in Vitro

Natural products represent the source or the inspiration for the majority of the active ingredients of medicines because of their structural diversity and a wide range of biological effects. Our aims in this study were (i) to synthesize enzymatically salidroside (SAL), the most effective phenylethanoid glycoside in Rhodiola species; (ii) to examine its antioxidant capacity using cell‐free assays (reducing power, DPPH radicals scavenging and Fe2+‐chelating assays); (iii) to assess its DNA‐protective potential on plasmid DNA (DNA topology assay) and in HepG2 cells (comet assay) damaged by Fe2+ ions and hydrogen peroxide, respectively; and (iv) to investigate the effects of SAL, cisplatin (CDDP) and combined treatments of SAL + CDDP on cell viability (MTT test), level of DNA damage (comet assay), proliferation, cell cycle (flow cytometry) and the expression of signalling molecules associated with cell growth and apoptotic pathways (Western immunoblotting). We found out that SAL manifested low antioxidant and DNA‐protective capacity in all assays used. In both parental A2780 and CDDP‐resistant A2780/CP human ovarian carcinoma cells, SAL itself exerted in fact no impact on the viability, while in combination with CDDP it showed antagonistic effect supporting the chemopreventive activity on the CDDP‐induced cell damage. These results were confirmed by the partial reversal of the cell cycle alterations and the DNA damage level, as well as with partial restoration of cell survival/signalling pathways, when the expression of these molecules partially returned to their proper levels.

Efficient chemoenzymatic synthesis of 4-nitrophenyl β-d-apiofuranoside and its use in screening of β-d-apiofuranosidases.

4-Nitrophenyl β-d-apiofuranoside as a chromogenic probe for detection of β-d-apiofuranosidase activity was prepared in 61% yield from 2,3-isopropylidene-α,β-d-apiofuranose through a sequence of five reactions. The synthesis involves one regioselective enzymatic step-benzoylation of primary hydroxyl of 2,3-isopropylidene-α,β-d-apiofuranose catalysed by Lipolase 100T and stereoselective β-d-apiofuranosylation of p-nitrophenol using BF3⋅OEt2/Et3N. The product was used for screening of β-d-apiofuranosidase activity in 61 samples of crude commercial enzymes and plant materials. Fifteen enzyme preparations originating from different strains of genera Aspergillus display β-d-apiofuranosidase activity. The highest activity was found in Rapidase AR 2000 (78.27 U/g) and lyophilized Viscozyme L (64,36 U/g).

Effective enzymatic caffeoylation of natural glucopyranosides.

Reaction system was developed for enzymatic caffeoylation of model saccharidic acceptor methyl β-d-glucopyranoside to obtain exclusively methyl 6-O-caffeoyl-β-D-glucopyranoside. Reaction with starting concentration of acceptor 0.2 M provided 73% yield of purified product within 17 days. Reactions with low acceptor concentrations (0.04 and 0.08 M) run to the completion within 7 days. Such highly effective and regioselective reaction was promoted by Lipozyme TL IM in tert-butanol, using vinyl caffeate as acylation donor. The optimized reaction conditions were used in preparative caffeoylation of natural substances-arbutin and salidroside, giving 75% of 6-O-caffeoylated arbutin (robustaside B) and 74% of 6-O-caffeoylated salidroside as the only products after 12 and 16 days, respectively.

Enzymatic synthesis of tyrosol and hydroxytyrosol β-d-fructofuranosides

Abstract Tyrosol β-d-fructofuranoside and hydroxytyrosol β-d-fructofuranoside have been synthesized as new compounds in 27.6 and 19.5% respective yields through transfructosylation of tyrosol and hydroxytyrosol. Yeast β-galactosidase Lactozym 3000 L comprising invertase activity was used as catalyst. Besides the main monofructosides, an equimolar mixture of tyrosol β-d-fructofuranosyl-((2→1)-β-d-fructofuranoside and tyrosol β-d-fructofuranosyl-(2→6)-β-d-fructofuranoside was isolated as additional product fraction in 14.3% yield.