An extracellular galactoxylomannan of acapsular Cryptococcus laurentii mutant.

An extracellular galactoxylomannan (GalXM) composed of D-Gal (34.0%), D-Xyl (26.6%) and D-Man (31.0%), and a small amount of L-Ara (4.0%) and D-Glc (4.4%) has been isolated from culture medium of acapsulated mutant of Cryptococcus laurentii by ethanol precipitation and gel filtration. Phosphorylated polymer of Mw approximately 75,000 contained 90% carbohydrates, 3.9% phosphorus and 5.3% proteins. Results of chemical and spectroscopic studies showed a highly branched structure of GalXM with a 1,6-linked mannopyranosyl-galactopyranosyl backbone (approximately 44%) branched predominantly at C-2 and C-3 of mannosyl, and C-3 of galactosyl residues by side chains terminated mainly by xylosyl and mannosyl residues, and to a less extent by arabinosyl and glucosyl ones.

Conidiation of Trichoderma atroviride isolate during submerged cultivation in a laboratory stirred-tank fermenter.

Conditions for conidiation of a natural isolate of Trichoderma atroviride during submerged cultivation in Erlenmeyer flasks and in a laboratory stirred-tank fermenter were optimized. From the simple sugars tested, cellobiose was the best substrate for conidia production while cellulose fines from paper mill waste proved to be a suitable cheap complex carbon source. Optimum temperature for conidiation was 24-26 degrees C, and the required dissolved oxygen level was > 40% saturation. After initial slight decrease during the 1st d after inoculation, the pH of the culture medium constantly increased throughout the sporulation period. Attempts to regulate the pH during fermentation did not improve the spore yields. The most intense formation of conidia took place between 2nd and 3rd d of growth and the overall volumetric productivity of conidia was 4.1-8.2 x 10(9) conidia per L/h.

Effect of gluco-monosaccharides and different conditions on digestion of hyaluronan by testicular hyaluronidase.

The changes of molecular size of hyaluronan during enzymatic reaction of bovine testicular hyaluronidase at different conditions are monitored by size exclusion high performance liquid chromatography. The effect of glucuronate, galacturonate, glucosamines and pyridoxin as potential inhibitors of hydrolysis is evaluated. The most effective of all tested inhibitors was the presence of glucuronate which not only inhibited the hydrolysis, but also initiated enzymatic reconstruction by transglycosylation reaction at pH 7.0 and absence of any buffer or salt. That effect was not found in the presence of a salt or with any other of the compounds tested.

Production of polysaccharide hydrolases in the genus Rhizopus.

Polysaccharide hydrolase activity was assayed in a group of 28 selected Rhizopus strains. The production of lichenases, mannanases, cellulases, xylanases, amylases and pullulanases was demonstrated using the gel-testing method during growth of the strains on suitably meshed polysaccharide gels.

Conjugation of yeast mannans with protein employing cyanopyridinium agent (CDAP)--an effective route of antifungal vaccine preparation.

The possibility of using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for activation of saccharide hydroxyl groups (instead of hazardous cyanogen bromide) is examined with cell-surface mannans of the yeasts Candida albicans, Candida tropicalis, Candida lambica and galactoglucoxylomannan of Cryptococcus laurentii. Direct conjugation with human serum albumin yielded soluble products with increased molecular size in comparison with the original polysaccharides. Immunodiffusion experiments revealed that conjugation did not affect the immunospecificity of the antigen epitppe.

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.

Light-activated adenyl cyclase from Trichoderma viride.

The effect of light on adenyl cyclase (E.C. 4.6.1.1) and 3':5'-cyclic-AMP-phosphodiesterase (E.C. 3.1.4.17) activity of Trichoderma viride was investigated. Adenyl cyclase proved to be a membrane-associated enzyme, requiring Mn2+ and was activated by light. In contrast, 3':5'-cyclic-AMP-phosphodiesterase showed no light-stimulated activity. The activity of 3':5'-cyclic-AMP-phosphodiesterase was present mainly in the cytosol and was stimulated by Mg2+.

Hyperpolarization and intracellular acidification in Trichoderma viride as a response to illumination.

Using indirect methods based on uptake of [3H]tetraphenylphosphonium cation and [14C]benzoic acid by cells of the fungus Trichoderma viride we found that the illumination-induced transient hyperpolarization of the plasma membrane is followed immediately by a rapid temporary decrease in intracellular pH. Hyperpolarization and intracellular acidification were completely suppressed by 150 mM-KCl and by the K(+)-ionophore valinomycin. The light-induced acidification of the cytoplasm was not observed in the presence of the cytochrome respiratory chain inhibitors antimycin A and mucidin. Based on these results, we hypothesize that the hyperpolarization of the cells is the consequence of an efflux of K+ through a light-activated K(+)-channel in the plasma membrane. The loss of positive charge in the cytoplasm caused by this efflux of cations is counterbalanced by H+ originating from the light-activated mitochondrial respiratory chain.

A third ADP/ATP translocator gene in yeast.

The op1 mutation in yeast is known to be due to a defect in the mitochondrial ADP/ATP translocator. Sequencing of the gene AAC2 revealed that the mutation resulted from a single base change that caused a replacement of arginine 97 by a histidine. The gene encoding AAC2 was also cloned and sequenced from an op1 revertant capable of growth on glycerol as a sole carbon source. Sequence analysis indicates that the reverted gene underwent rearrangement in which a portion of an unknown gene was used to repair the mutation. An oligonucleotide complementary to this insert was used to clone a previously unrecognized gene encoding ADP/ATP translocator in yeast. The newly discovered gene, AAC3, is homologous with the previously known genes AAC1 and AAC2. Gene disruption experiments suggest that AAC2 encodes the majority of the translocator. Expression of AAC1 and AAC2 required derepressed conditions whereas expression of AAC3 occurred almost exclusively under anaerobic conditions. Both the op1 mutant and the strain that contains an interrupted AAC2 were able to grow under anaerobic conditions, suggesting that AAC3 can replace the gene product of AAC2. Indeed, when cloned into multicopy plasmid, AAC3 was able to replace the disrupted AAC2 in the JLY-73 strain. The concomitant disruption of the AAC2 and AAC3, however, results in arrest of cell growth under conditions of low oxygen tension. The discovery of a third gene encoding ADP/ATP translocator helps to clarify certain characteristics of op1 mutants which could not be resolved in the past.

Light-stimulated phosphorylation of proteins in cell-free extracts from Trichoderma viride.

When illuminated by visible light, cell-free extracts from the fungus Trichoderma viride catalysed the phosphorylation of at least two proteins with molecular masses of 18 and 114 kDa which were practically absent when the phosphorylation was performed in the dark. The effect of light could be substituted by 3mM cyclic AMP, not only in the cell-free extract, but also in the separated cytosol. It is concluded that the process of photoinduced conidiation in Trichoderma involves phosphorylation of conidiation-specific proteins by (a) cyclic AMP-dependent protein kinase(s) present in the cytosol.