Věra Přikrylová

Only C-2 specific glucose oxidase activity is expressed in ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium

Abstract Two d-glucose-oxidizing enzymes, glucose 1-oxidase (G1O) and pyranose 2-oxidase (P2O, glucose 2-oxidase), have been proposed to play an important role in the ligninolytic system of the white rot fungus Phanerochaete chrysosporium by producing hydrogen peroxide. The possible simultaneous expression and metabolic cooperation of the two oxidases was studied in strains ME-446 (reported as G1O positive) and K-3 (P2O positive) grown in liquid media and under near natural conditions on birch wood blocks. The presence of G1O and P2O in extracts from mycelia and decayed wood was determined by chromatographic, electrophoretic, and immunological methods. Attempts to separate these enzymes and to detect G1O and its reaction product, d-glucono-1,5-lactone, failed. Evidence was obtained only for P2O expression in both strains. Accordingly, P2O, rather than G1O, represents a major source of sugar-derived H2O2 under the culture conditions used.

Glycosylation of silybin

Silybin glycosides—23-O-β-glucoside 2b, β-galactoside 3b, β-lactoside 4b and β-maltoside 5b—have been synthesized by different methods (Helferich glycosylation, Lewis acid catalysis). Separation of two silybin diastereoisomers in the form of acetylated monoglycosides has been achieved for the first time. These new silybin glycosides are 4–30 times more water-soluble, and their hepatoprotectivity is increased compared with that of the parent compound silybin 1.

C-2 and C-3 oxidation of d-Glc, and C-2 oxidation of d-Gal by pyranose dehydrogenase from Agaricus bisporus

Abstract The quinone-dependent sugar oxidoreductase, pyranose dehydrogenase purified from the basidiomycete Agaricus bisporus catalyzed C-2 and C-3 oxidation of d -Glc to d - arabino -hexos-2-ulose (2-keto- d -Glc) and, preferentially, d - ribo -hexos-3-ulose (3-keto- d -Glc). The two aldoketoses accumulated only transiently in the reaction mixture being continually converted to the same end-product, d - erythro -hexos-2,3-diulose (2,3-diketo- d -Glc). d -Gal was oxidized by pyranose dehydrogenase and 1,4-benzoquinone exclusively at C-2 to produce d - lyxo -hexos-2-ulose (2-keto- d -Gal). Structures of the enzyme reaction products were deduced from their N , N -diphenylhydrazone derivatives.

Conversion of d-glucose to d-erythro-hexos-2,3-diulose (2,3-diketo-d-glucose) by enzyme preparations from the basidiomycete Oudemansiella mucida

Abstract Aerobic incubation of crude enzyme extracts from the white rot fungus Oudemansiella mucida with d -glucose resulted in transient accumulation of d - arabino -hexos-2-ulose ( 1 , 2-keto- d -glucose) which was subsequently quantitatively converted into a new tricarbonyl sugar metabolite. d - erythro -hexos-2,3-diulose ( 2 , 2,3-diketo- d -glucose). The latter end-product was derivatized with N , N -diphenylhydrazine; its structure was deduced from formulae of the four major hydrazones isolated from the reaction mixture using thin layer chromatography, and identified by nuclear magnetic resonance and mass spectroscopy, 2 was also obtained on incubations with chemically synthesized 1 , and with partially purified preparations of pyranose 2-oxidase. A hypothetical reaction scheme was proposed for two step oxidation of d -glucose to 2 by a single enzyme, pyranose 2-oxidase (EC 1.1.3.10).

Enzymatic Synthesis of β-N-Acetylhexosaminides of Ergot Alkaloids

2-Acetamido-2-deoxy-O-β-D-galactopyranosides and 2-acetamido-2-deoxy-O-β-D-glucopyranosides of the representatives of the three classes of the ergot alkaloids, i.e., elymoclavine (1a) (clavines), chanoclavine (2a) (secoclavines) and ergometrine (3a)/ergometrinine (4a) (lysergic/iso-lysergic acid derivatives) were prepared via transglycosylation catalysed by β-N-acetylhexosaminidase from Aspergillus oryzae.

Clustered ergot alkaloids modulate cell-mediated cytotoxicity

Dimers of agroclavine (1) and terguride (2), as well as a series of terguride oligomers, for example trimers (5, 6), tetramer (7), hexamer (8) and functionalized tergurides for further complex clustering were synthesized. Terguride oligomers were screened for their direct cellular toxicity on lymphoma cell lines in vitro and for their immunomodulating activities, represented by the natural killer (NK) cell-mediated cytotoxicity, as the most sensitive screening marker during immune responses. Dimers linked via aromatic spacer showed a high toxicity (1 microM) to lymphoma cells, which was not detected in other derivatives. In vitro and ex vivo experiments performed on mouse spleen lymphocytes in the presence of terguride oligomers demonstrated an immunosuppressive effect of dimers with aromatic spacer (4c-d) and NK cell stimulatory effect of terguride hexamer (8) and trimer with aliphatic spacer (5c). There is a considerable evidence that indolic part of molecule contributes to immunosuppressive action of terguride, which is potentiated in dimers carrying aromatic linker. This effect can be reversed by higher oligomerization of the respective alkaloids.

Preparation of Mannac Containing Chitooligomers By Isomerisation and their Binding to Nkr-P1 Protein

Abstract 4-O-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-2-acetamido-2-deoxy-D-mannopyranose (2) was prepared from diacetylchitobiose (1) by Lobry de Bruyn-Alberda van Ekenstein rearrangement under catalysis of Ca(OH)2. The disaccharide 2 shows about ten times higher affinity than 1 towards NKR-P1 protein acting as a crucial activating receptor of rat natural killer cells (leukocytes). Chitotriose (3) can be epimerized analogously to give GlcNAcβ1-4GlcNAcβ1-4ManNAc (4) and its binding properties to NKR-P1 are also about ten times higher than those of 3.

Isolation of nonactic acids fromStreptomyces griseus fermentation broth by thin-layer and high-performance liquid chromatography

Nonactic acid, homononactic acid and their 2-diastereoisomers were isolated by thin-layer chromatography on silica gel and by high-performance liquid chromatography on a reversed phase from the fermentation broth ofStreptomyces griseus, and identified by1H and13C NMR spectroscopy.

Insecticidal properties of nonactic acid and homononactic acid, the precursors of macrotetrolide antibiotics

Abstract(±)-Nonactic acid (1) and (±)-homononactic acid (2), the non-antibiotic precursors of macrotetrolide antibiotics, showed significant insecticidal effects (comparable with those of the commercial synthetic pesticide Metathion) onLeptinotarsa decemlineata, Epilachna varivestis andEuproctis chrysorrhoea. Anisoplia austriaca, Aphis fabae andCalandra granaria were less sensitive in this respect. A low acaricidal activity againstTetranychus urticae (not reaching that of the commercial miticide Acarition) was also found. Both1 and2 exhibited growth-stimulating properties for plants.

Biotransformation of ergot alkaloids by plant cell cultures with high peroxidase activity

Ergot alkaloids agroclavine and elymoclavine have been modified using plant cell cultures exhibiting high peroxidase activity. Setoclavine and isosetoclavine have been isolated from media after transformation of agroclavine on a semipreparative scale. 10-Hydroxyelymoclavine resulted from similar treatment of elymoclavine.