Eva Stratilová

Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides

Oligosaccharide subunits were prepared from xyloglucan (XG) by partial hydrolysis with cellulase and added back at micro- to millimolar concentrations to XG in the presence of nasturtium seed xyloglucanase (XG-ase). The oligosaccharides (0.2 mM) stimulated the capacity of this XG-ase to reduce the viscosity of XG solutions by 10- to 20-fold. Purification and fractionation of seed XG-ase activity by gel permeation fast protein liquid chromatography produced a single peak that was much more active in the presence than absence of added XG oligosaccharide. [14C]Fucose-labeled XG nonasaccharide was synthesized by pea fucosyltransferase and shown to be incorporated into polymeric XG in the presence of seed XG-ase without the net production of new reducing chain ends, even while the loss of XG viscosity and XG depolymerization were enhanced. It is concluded that in vitro seed XG-ase can transfer cleavage products of XG to XG oligosaccharides via endotransglycosylation reactions, thereby reducing XG M(r) without hydrolysis. Since this is the only XG-cleaving enzyme that develops in nasturtium seeds during germination, it may be that its transglycosylase and hydrolase capacities are both necessary to account for the rapid and complete depolymerization of XG that takes place.

An essential tyrosine residue of Aspergillus polygalacturonase

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine‐reactive agents tested, acetic anhydride, N‐acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3–9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

PectinaseAspergillus sp. polygalacturonase: Multiplicity, divergence, and structural patterns linking fungal, bacterial, and plant polygalacturonases

Nine forms ofAspergillus sp. polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing. Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical. Four forms with freea-amino groups have identical start positions but internal amino acid replacements. Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations. Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from otherAspergillus species (72–75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66–71% residue differences toward theErwinia, tomato, and peach enzymes). Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties

Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees

Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β‐glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β‐glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D‐xylose the most frequently of all the yeasts tested. The majority of the fruit‐inhabiting yeasts grew in the medium with higher osmotic pressure.

The glycoprotein character of multiple forms of Aspergillus polygalacturonase.

Comparisons of known primary structures of polygalacturonases show that extent and localization of potential N-glycosylation sites differ. Some sites are similar in position and adjacent to strictly conserved residues at the potential active site. The presence of N-acetylglucosamine and mannose in the molecules of two homogeneous, major Aspergillus sp. polygalacturonase forms was confirmed by IR spectroscopy. The purification method, based on interaction of the carbohydrate part with concanavalin A immobilized on chlorotriazine bead cellulose, was optimized. Deglycosylation with N-glycosidase F under denaturating and nondenaturating conditions led to molecular mass decreases followed by complete inactivation of the polygalacturonase enzyme activity. These results show the importance of glycosylation in these protein forms, while the comparative patterns establish both variability and some similarities in overall glycosylation architectures.

Effect of cultivation and storage pH on the production of multiple forms of polygalacturonase by Aspergillus niger

SummaryThe production of multiple forms of extracellular polygalacturonase by Aspergillus niger is directly dependent on the pH of maintenance media. The pH of cultivation medium as well as the carbon-source are only secondary factors with limited influence on this production.

Affinity Chromatography of Glycoenzymes and Glycoproteins on Concanavalin A-Bead Cellulose

Abstract Concanavalin A immobilized on chlorotriazine bead cellulose was applied to affinity purification of glycoenzymes and glycoproteins. Enzymes such as invertase from bakers yeast, endopolygalacturonase (Rohament P) and exopolygalacturonase from carrot roots, as well as extracellular mannoproteins from the yeast Cryptococcus laurentii were examined. Chromatography was performed on minicolumns filled with Concanavalin A-triazine bead cellulose gel with the content of immobilized Concanavalin A within the range 1.2 – 8.2 mg per mL of gel. The specifically bound glycoenzymes or glycoproteins were eluted with a solution of the corresponding counter-ligand a-methyl mannopyranoside. Individual degrees of purification, estimated from the measurements of specific activity of crude and purified glycoenzymes, were 14.5-fold for invertase, 93-fold for polygalacturonase and 3.9-fold for exopolygalacturonase. The yeast mannoprotein was isolated from the heteroglycoprotein fraction. The purified mannoprotein cont...

Effect of the Pore Size of a Support on Activity and Action Pattern of Immobilized Endopolygalacturonase

Endopolygalacturonase (E.C. 3.2.1.15) was covalently bound to silica supports of different porosity treated with 3-(2′,3′-epoxypropoxy)propyltrimethoxysilane. The activity and action pattern on sodium pectate and tetra(D-galactosiduronic acid) were investigated in batch and continuous flow-reactors. Pore size of the supports affected the loading of the enzyme as well as its action pattern and kinetics. A decrease in randomness of degradation of D-galacturonan, loss of specificity of (3 + 1) splitting of tetra(D-galactosiduronic acid) and decrease in Km value were found with the supports containing predominantly micropores. The extent of the changes decreased with increasing pore size of the support. The catalytic behaviour of endopolygalacturonase bound on the supports with large pores was quite analogous to that of the free enzymes.

Production and biochemical characterization of polygalacturonases produced byAureobasidium pullulans from forest soil

The production of individual form of extracellular polygalacturonase byAureobasidium pullulans from forest soil was found to depend on the pH of cultivation medium as well as on the nitrogen source in the precultivation or cultivation medium. Polygalacturonases were purified and characterized. The pH optima of polygalacturonases produced in the first phases of cultivation (24 or 48 h) and after 10 days as well as their molecular masses, isoelectric points, action pattern and ability to cleave polymeric and oligomeric substrates were different. Generally, polygalacturonases with random action pattern (EC 3.2.1.15) were produced only in the first phases of cultivation in acidic medium. The function of these enzymes for A.pullulans in the colonization of plant material rather than in the destruction of plant was hypothesized in physiological conditions. Exopolygalacturonases (EC 3.2.1.67) with terminal action pattern were produced in later phases of growth. Oligogalacturonate hydrolase as well as strongly basic polygalacturonase with unusual action pattern on substrates were found.

Endopolygalacturonase immobilized on epoxide-containing supports

SummaryEndopolygalacturonase was immobilized onto 3-(2′,3′-epoxypropoxypropyl)-silica and oxirane-acrylic beads. Optimum conditions of immobilization and catalytic properties of the immobilized enzyme preparations are described.