Eva Petráková

Induction of cellulose- and xylan-degrading enzyme systems in Aspergillus terreus by homo- and heterodisaccharides composed of glucose and xylose

Synthetic heterodisaccharides composed of glucose and xylose were tested as inducers of cellulose- and xylan-degrading enzymes in Aspergillus terreus, and the inducing abilities were compared with those of sophorose and xylobiose or their positional isomers. Measurement of secreted and cell-associated enzyme activities revealed that the heterodisaccharides induced the synthesis of the cellulolytic and xylanolytic enzymes, 2-O-beta-D-glucopyranosyl D-xylose (Glcbeta 1-2Xyl) being the most powerful inducer. Sophorose and 2-O-beta-D-xylopyranosyl D-Xylose (Xylbeta 1-2Xyl), or their positional isomers, selectively induced the synthesis of cellulases and beta-xylanases, respectively. An analysis of the extracellular enzymes (which were separated by isoelectric focusing followed by detection using chromogenic and fluorogenic substrates) showed that Glcbeta 1-2Xyl initiated the synthesis of specific endo-1,4-beta-glucanases and specific endo-1,4-beta-xylanases identical to those produced separately in response to sophorose or Xylbeta 1-2Xyl. Glcbeta 1-2Xyl also induced specific endo-1,4-beta-glucanases that hydrolysed 4-methylumbelliferyl beta-lactoside at the agluconic bond. The results strengthen the concept of separate regulatory control of the synthesis of cullulases and beta-xylanases. The results also suggest that mixed disaccharides, composed of glucose and xylose moieties, which may occur in nature, could play an important role in regulating the synthesis of wood-degrading enzymes.

Structural features of a water-soluble l-arabino-d-xylan from rye bran

A water-soluble L-arabino-D-xylan, obtained from the chlorite holocellulose of de-lipidated, de-starched, and de-pectinated rye bran by ammoniacal extraction, was composed of L-Ara and D-Xyl in the molar ratio 7.8:10 and had Mw 36,500 and Mn 26,950. The backbone of the polysaccharide was shown to comprise (1----4)-linked beta-D-Xylp residues, with approximately 41% unsubstituted, approximately 33% 2- or 3-substituted, and approximately 26% disubstituted. Single alpha-L-Araf groups were attached to the xylan core; only a small proportion was 2-, 3-, or 5-linked.

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and β-xylanase, intracellular, cell wall bound and extracellular β-glucosidase and β-xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3-β-xylobiose, 1,2-β-xylobiose, 1,4-β-xylosyl-L-arabinose, 1,4-β-xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular β-xylosidase precedes extracellular β-xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular β-glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced β-xylosidase of xylobiose type and a non-specific aryl β-D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.

Structural features of a rye-bran arabinoxylan with a low degree of branching

Abstract Two fractions (AX-1 and AX-II) of an l -arabino- d -xylan with a low degree of branching were isolated from rye bran by different extraction procedures with and without a chlorite-delignification step. The yields and compositions of AX-I and AX-II were similar, but the d.p. of AX-II was ∼100 units higher. AX-I was shown by partial hydrolysis, methylation analysis, and 13 C-n.m.r. spectroscopy to contain a backbone of (1→4)-linked β- d -xylopyranosyl residues substituted with single α- l -arabinofuranosyl groups at position 3 of every sixth or seventh d -xylosyl residue.

An approach to the systematic synthesis of (1→4)-β-d-xylo-oligosaccharides☆

Abstract The first approach to a general synthesis of (1→4)-β- d -xylo-oligosaccharides is described. Thus, 1,2,3-tri- O -acetyl-4- O -benzyl-β- d -xylopyranose was treated with hydrogen bromide in dichloromethane to give the corresponding glycosyl bromide 3 , which was condensed with 1,2,3-tri- O -acetyl-β- d -xylopyranose. The β-linked disaccharide formed, bearing at O-4 of its non-reducing end the selectively removable benzyl group, was submitted to hydrogenolysis to afford a disaccharide nucleophile 21 , having HO-4′ free, which was again treated with 3 . This sequence of reactions was repeated two more times and the isolated products 17–20 were deprotected to give a homologous series of lower xylo-oligosaccharides (from the disaccharide to the pentasaccharide), demonstrating thus the feasibility of stepwise construction of xylo-oligosaccharide molecules. The corresponding α-linked oligosaccharides 5–8 , present in the mixtures from the condensation reactions, were also isolated and characterized. 13 C-N.m.r. spectral characteristics for six homologous series of variously substituted (1→4)- d -xylo-oligosaccharides are presented.

Stereoselective synthesis and 13C-N.m.r. spectra of two isomeric methyl β-glycosides of trisaccharides related to arabinoxylan

Abstract Methyl 2-O-acetyl-4-O-(2,3,4-tri-O-acetyl-β- d -xylopyranosyl)-β- d -xylopyranoside (4) and methyl 2,3-di-O-acetyl-4-O-(2,4-di-O-acetyl-β- d -xylopyranosyl)-β- d -xylopyranoside were separately condensed with 2,3,5-tri-O-benzoyl-α- l -arabinofuranosyl bromide under modified Koenigs-Knorr conditions, to give high yields ( > 90%) of trisaccharide derivatives containing a (1→3)-linked α- l -arabinofuranosyl residue. Removal of the protecting groups gave methyl 3-O- and 3′-O-α- l -arabinofuranosyl-β- d -xylobioside. 13C-N.m.r. data for these compounds are presented and the homo- and hetero-nuclear 2D-n.m.r. spectra of 4 are discussed.

Trimethylsilylation - Aid in NMR Analysis of Oligosaccharides. Assigment of 29Si and 13C NMR Spectra of Trimethylsilylated Methyl β-D-Xylobiosides by 2D NMR

Abstract The 1H, 13C and 29Si NMR spectra of methyl β-D-xylopyranoside and three methyl β-D-xylopyranosyl-β-D-xylopyranosides have been measured and assigned by two-dimensional NMR spectroscopy. According to the determined proton-proton coupling constants, the ring conformer ratio is essential ly the same in the studied compounds. The assigned chemical shifts provide correct substituent chemical shifts for assignments in the spectra of higher trimethylsilylated xylooligosaccharides. Heteronuclear chemical shift correlated 2D NMR spectroscopy is proven to be a usable experimental method for 29Si NMR line assignment in carbohydrates. The assigned silicon shifts identify the site of glycosidation.

Infrared study of intramolecular hydrogen bonds in methyl mono- and di-O-substituted β-d-xylopyranosides

Abstract The strength of the intramolecular hydrogen bonds of the methyl mono- and di- O -methyl-β- d -xylopyranosides, methyl mono- and di- O -benzyl-β- d -xylopyranosides, benzyl di- O -benzyl-β- d -xylopyranosides, and mono- and di- O -acetyl-β- d -xylopyranosides was measured in terms of the frequency of the hydroxyl stretching vibration. The spectral data of the compounds measured in a carbon tetrachloride solution indicated the existence of intramolecular hydrogen bonds and conformational equilibria of the pyranoid ring in 4 C 1 and 1 C 4 chair forms resulting from the hydrogen bond properties, and position, number and steric effects of the substituents.