beta-Xylosidases in the yeast Cryptococcus albidus var. aerius.

beta-xylosidase activity has been detected in cell-free extracts and in culture fluids when Cryptococcus albidus var. aerius was grown on glucose as the sole carbon source. The enzyme appears to be constitutive. Mild acid treatment of whole cells suggested that the total activity is located in the periplasmic space and some experiments indicated that it is partially associated with the cell walls. DEAE-Sephadex A50 chromatography has shown that there are two different forms of beta-xylosidase in the cell-free extracts, but only one form is present in the supernatants of culture.     

Effect of tunicamycin on xylanase secretion in the yeast Cryptococcus albidus

The yeast Cryptococcus albidus secretes a glycosylated xylanase (48 kDa) in the culture medium in response to beta-methylxyloside as inducer. Addition of tunicamycin to the medium results in the formation of a modified xylanase (40 kDa) which is depleted in carbohydrate content and whose enzymatic activity is 2.5 times less than that of the glycosylated xylanase. The secretion of xylanase was followed under both conditions by pulse-chase experiments. The half-time of secretion of the glycosylated and nonglycosylated forms was 5 and 2 h, respectively. Cell-associated xylanase activity was not detected when the cells were treated with the antibiotic. The absence of cell wall-associated xylanase, after tunicamycin treatment, was confirmed by immunolocalization with anti-xylanase antibodies at the electron microscopic level. The results suggest that the interactions of carbohydrate moiety within the cell wall retarded the secretion of the enzyme to the medium.     

Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization

During growth on wood beta-1,4-xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo-1,4-beta-xylanase. The function of the extracellular beta-xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell-bound beta-xylosidae (xylobiase). Aryl beta-xylosidase activity detected in the culture fluid was snown to be due to low affinity of beta-xylanase for p-nitrophenyl beta-D-xylopyranoside. This property of beta-xylanase was preserved after purification of the enzyme by chromatography on DEAE-cellulose, CM-Sephadex and Biogel A 1.5 m or Biogel P 100. Purified beta-xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular beta-xylanase and intracellular beta-xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified beta-xylanase were unable to hydrolyze cellulose or its soluble derivatives.     

β-xylosidases and a nonspecific wall-bound β-glucosidase of the yeast cryptococcus albidus

Cryptococcus albidus grown on wood xylans possesses a soluble intracellular β-xylosidase (EC 3.2.1.37) as an additional constituent of the xylan-degrading enzyme system of this yeast. The enzyme attacks linear 1,4-β-xylooligosaccharides in an exo-fashion, liberating xylose from the non-reducing ends. The activity of the enzyme increases in the cells during growth on xylan and incubation with xylobiose or methyl β-D-xylopyranoside which are the best inducers of extracellular β-xylanase (EC 3.2.1.8). Various alkyl-, alkyl-1-thio- and aryl β-D-xylopyranosides were excellent of a different β-xylosidase of Cryptococcus albidus. This enzyme is localized outside the plasma membrane and is principally associated with cell walls. Unlike the soluble intracellular β-xylosidase, the wall-bound enzyme does not hydrolyze xylooligosaccharides. Evidence has been obtained that β-xylosidase activity in the cell walls is not due to the presence of a specific aryl β-xylosidase, but is exhibited by a nonspecific β-glucosidase (EC 3.2.1.21) inducible by β-D-xylopyranosides. The ratio of β-glucosidase and β-xylosidase activity in the cells and isolated cell walls from yeast induced by various β-xylopyranosides and β-glucopyranosides was very similar. Both wall-bound activities were inhibited in a similar pattern by inhibitors of β-glucosidases, 1,5-gluconolactone and nojirimycin. This bifunctional enzyme does not bear any relationship to the utilization of xylans in Cryptococcus albidus.     

Cloning and expression in Escherichia coli of a xylanase-encoding gene from the yeast Cryptococcus albidus.

In the yeast, Cryptococcus albidus, a comparison between the sequence of the xylanase (XLN)-encoding chromosomal gene (XLN) and the cDNA sequence reveals the presence of seven introns, ranging in length from 51 to 69 bp. One of their 5' splice site sequences is similar to the consensus sequence for yeast, while the other six resemble the consensus sequence for higher eukaryotes. Their 3' end splice site sequences are representative of the conserved sequence found in eukaryotes. Their putative branching point sequences are different from the well-known conserved sequence, 5'-TACTAAC, observed in yeast, but again resemble the mammalian one. The cDNA encoding XLN is expressed by Escherichia coli, under the control of the lacZ promoter. The gene product remains inside the cell and has a molecular size of 40 kDa, which matches the size of the nonglycosylated protein. When compared to the glycosylated enzyme, the nonglycosylated XLN from E. coli shows twofold less affinity for substrate and its Vmax is 100-fold lower. Moreover, the nonglycosylated XLN only acts on large xylan polymers and very slightly on xylohexaose.     

Simulated in situ competitive ability and survival of a representative soil yeast,Cryptococcus albidus

Microcosms containing an air-dried autoclaved loamy sand (Eufala A) with low salt and organic content were inoculated with a representative (obligately aerobic, encapsulated) soil yeast, Cryptococcus albidus var. albidus ^T ATCC 10666, singly (for growth rate and survival determinations) and together with the bacterial biota native to Eufala A. The yeast competed successfully with the more rapidly growing bacteria in the presence of added water from 1% (5.7% of field capacity) to 14% (80% of field capacity) but grew for shorter times than when grown alone; times correlated with the lag phase of the bacterial biota. When well-watered (10 and 14%) competition cultures were allowed to dry and used as inoculum for subcultures, the yeast made significant growth only at 1% added water but survived at the higher moisture concentrations. The competitive ability of Cr. albidus confirms the previously reported advantages of the cryptococcal capsule in hydration and desiccation and, together with lengthy survival, suggests that the importance of such yeasts in the biogeochemistry of arid soils has been seriously underestimated.     

Ability of the antagonistic yeast Cryptococcus albidus to control Botrytis cinerea in strawberry

The yeast Cryptococcus albidus, originally isolated from mature strawberry fruits, was tested for antagonistic activity against Botrytis cinerea, the causal agent of grey mould in strawberries. Conidial germination and germ tube growth of conidia of B. cinerea were inhibited by a cell suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) after 6 and 24 hours of incubation. Application of a cell suspension (1 × 10⁶ cells/ml) on detached strawberry leaf disks incubated at 10°C reduced incidence and conidiophore density of B. cinerea by 86 and 99%, respectively, but effectiveness was reduced at higher temperatures. Treatments with C. albidus during bloom of strawberries reduced incidence of grey mould on ripe strawberry fruits after harvest by 33, 28 and 21% in three years of field trials. The effectiveness of the yeast was increased when formulation substances (alginate, xanthan and cellulose) were added to the cell suspension.     

Purification of a soluble and a wall-bound form of beta-glucosidase from Mucor racemosus.

beta-Glucosidase activity in crude extracts of Mucor racemosus exists in a soluble form and in a wall-bound form which sediments at 3,500 x g. The soluble form and a wall-bound form were purified to homogeneity by ammonium sulfate fractionation. DEAE-Sephadex chromatography, and SP-Sephadex chromatography. Both forms were identical in all parameters measured. Each enzyme is a glycoprotein of 91,000 daltons, with an identical amino acid composition and N-terminal amino acid of lysine; both contain about 10% carbohydrate. Both forms catalyze the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside with identical kinetic constants.     

Diversity in the yeast Cryptococcus albidus and related species as revealed by ribosomal DNA sequence analysis

Evidence accumulated from studies based on physiological, biochemical, and molecular characteristics has pointed to the heterogeneity of the ubiquitous anamorphic basidiomycetous yeast species Cryptococcus albidus (Saito) Skinner, with its current varieties and synonyms. The taxonomic status of this species has not been reappraised because different studies, mostly involving limited numbers of strains, have not been integrated. To assess species diversity within the clade containing Cryptococcus albidus and other phylogenetically related Cryptococcus and Filobasidium species, we determined ribosomal DNA (rDNA) sequences of 69 strains from the 5' end of the 26S gene, D1/D2 region, and in some cases, the non-coding ITS2 region. Analysis of the sequence data together with available physiological, biochemical, and molecular characteristics, showed the segregation of C. albidus into at least 12 species, leading to the elevation of former varieties to the rank of species (C. aerius, C. diffluens), the reinstatement of synonyms (C. liquefaciens, C. terricola), and the proposal of new species (C. arrabidensis, C. chernovii, C. cylindricus, C. oeirensis, C. phenolicus, C. saitoi, C. uzbekistanensis, C. wieringae). The overall analyses of the results argue in favour of the use of rDNA sequence data to improve species delineation when integrated with other available physiological and molecular characteristics.     

Uptake and utilization of glutamic acid by Cryptococcus albidus

Cryptococcus albidus utilizes glutamate as a sole carbon source. The kinetics of uptake of this amino acid were studied. l-Glutamic acid was taken up by two saturable systems: a high affinity system with a Michaelis constant (K(m)) of 1.15 x 10(-5) M and a V(max) of 0.049 mumol per mg per h and a low affinity system with a K(m) of 2.5 x 10(-3) M and a V(max) of 3.61 mumol per mg per h. Both systems possessed characteristics of active transport which were dependent on temperature and pH and which required metabolic energy. Uptake was inhibited at 37 C but the temperature-sensitive step was reversible. Chemical fractionation of cells with 5% trichloroacetic acid showed that glutamic acid initially entered a soluble pool which decreased after 1 h as the amino acid was incorporated into the protein and nucleic acid fractions of the yeast. Some of the glutamate was completely oxidized and could be recovered as (14)CO(2). Therefore, the amino acid was also used as an energy source.     

Molecular expression of xylanase gene in Cryptococcus albidus

In the yeast Cryptococcus albidus, the utilization of xylan as compared to xylose requires at least an inducible endoxylanase enzyme, secreted in the culture medium. The endoxylanase induction was monitored by immunoprecipitation of in vivo and in vitro synthesized products. The mature endoxylanase is a highly glycosylated enzyme with an apparent molecular weight of 48 000. Upon chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight was reduced to 40 000. Addition of tunicamycin to the culture medium resulted in the synthesis of a modified polypeptide having a molecular weight of 40 000. Poly(A)-containing RNA isolated from the yeast was translated in the rabbit reticulocyte protein-synthesizing system. The appearance of a translatable xylanase mRNA was observed in xylan-grown cells but not in xylose-grown cells. The polypeptide identified as xylanase had a molecular weight of 44 000. This suggests that the xylanase is synthesized as a precursor, containing a peptide signal sequence of 35 residues.     

Comparison of serological and chemical characteristics of capsular polysaccharides of Cryptococcus neoformans var. neoformans serotype A and Cryptococcus albidus var. albidus

The antigenic formula and chemical structure of capsular polysaccharide (CPS) of Cryptococcus albidus var. albidus (C. albidus) were studied in relation to those of C. neoformans var. neoformans serotype A (C. neoformans A). The results of slide agglutination tests with factor sera and reciprocal adsorption experiments showed that antigenic formula of C. albidus was the same as that of C. neoformans A. The soluble CPSs from the two species were obtained from culture supernatants by precipitation with ethanol followed by purification by chromatography on DEAE-cellulose column. The structural analyses of such CPSs from the two species showed that the antigenic CPS fractions consisted of a backbone of alpha(1-3)-linked D-mannopyranosyl residues with a single branch of beta(1-2)-xylose or glucuronic acid, and mostly with O-acetyl groups, in which side chains and O-acetyl groups were responsible for antigenic specificity. It was found that there was a minor difference between the CPS of C. neoformans A and that of C. albidus; in the former, unsubstituted mannose residues existed in a low frequency, but in the latter none. Moreover, the 1H-nuclear magnetic resonance spectra of partially hydrolyzed acidic fragments of the two CPSs indicated that two xylose side chains were present between glucuronic acid side chains. Taken together, it was suggested that these two species of C. neoformans A and C. albidus are closely related to each other in their CPSs.     

Characterization of laccase activity produced by Cryptococcus albidus

This study deals with the characterization of laccase enzyme activity produced by Cryptococcus albidus. Industrial wastes like effluent and sludge are complex mixtures of a number of chemicals. These chemicals can interfere with the proper functioning of the enzymes used for bioremediation. Thus, it is important to study the effect of such interfering solvents, detergents, metal chelators, and other chemicals on enzyme activity before industrial applications. Laccase showed maximum activity at pH 2.5 and temperature 20-30°C when ABTS was used as a substrate. The enzyme followed Michaelis-Menten kinetics: K(m) was 0.8158 mM and V(max) was 1527.74 U/mg. Laccase showed good thermostability with a half-life of 81 min at 25°C, 77 min at 35°C, 64 min at 45°C, 36 min at 55°C, and 21 min at 65°C. There was no effect of sodium dodceyl sulfate (SDS) (0.1-1.0%) and EDTA (0.1-0.5%) on laccase activity. Sodium azide and 2-mercaptoethanol showed complete inhibition of laccase activity at 0.1% concentration. At lower concentrations of acetone and acetonitrile, laccase was able to maintain its activity. However, the activity was completely inhibited at a concentration of 50% or above of acetone, methanol, 1,4-dioxan, and acetonitrile.     

Beta-glucanases in the yeast Cryptococcus albidus var. aerius. Production and separation of beta-glucanases in asynchronous cultures.

beta-Glucanases were detected in cell-free extracts of the yeast Cryptococcus albidus var. aerius when grown on glucose as the sole carbon source. The production of beta-glucanases was followed in log-phase cells and stationary-phase cells; the maximal production of beta-(1 leads to 3) and beta-(1 leads to 6) glucanases takes place respectively in log-phase and stationary-phase cells. The results show that there are marked differences in the elution profiles on Sephadex G-50 of fractions containing beta-glucanase from cells grown for 12, 24, 48, 72, and 96 h. The possibility either of replacement changes in fractions containing beta-glucanase activity or of a different synthesis of each beta-glucanase during the growth of the yeast is discussed. The results suggest that all fractions containing beta-glucanases hydrolyze both beta-(1 leads to 3) and beta-(1 leads to 6) linkages. Evidence in support of the conclusion that a low molecular form of beta-glucanase has a molecular weight of 2100 +/- 100 is also shown.     

Positional isomers of thioxylobiose, their synthesis and inducing ability for D-xylan-degrading enzymes in the yeast Cryptococcus albidus.

Isomeric S-linked 2-thioxylobiose 10, 3-thioxylobiose 17, and 4-thioxylobiose 19 were conveniently prepared by SN2 displacement of suitable triflylglycoses with the sodium salt of 2,3,4-tri-O-acetyl-1-thio-beta-D-glucopyranose, either in N,N-dimethylformamide, or in oxolan in the presence of a sodium complexing agent. Allyl 3,5-O-isopropylidene-2-O-trifluoromethanesulfonyl-beta-D-lyxofu ranoside was a convenient electrophilic precursor for 10, which was smoothly obtained after a short sequence of deprotection involving conversion to the 1-propenyl glycoside. 1,2:5,6-Di-O-isopropylidene-3-O-trifluoromethylsulfonyl-alpha-D-++ +allofuranose and 1,2,3-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-L-arabinop yranose were the respective precursors for 17 and 19. 4-Thioxylobiose has a highly stimulatory effect on the synthesis of enzymes of the xylanolytic system in the yeast Cryptococcus albidus when applied to the cells in the presence of the natural disaccharide inducer (1----4)-beta-D-xylobiose.     

Novel inducers of the xylan-degrading enzyme system of Cryptococcus albidus

A series of compounds structurally related to xylan and 1,4-beta-xylobiose were tested as inducers of the xylan-degrading enzyme system of Cryptococcus albidus. Washed, glucose-grown cells were incubated with alpha- and beta-linked xylobioses, 4-O-beta-D-xylopyranosyl-L-arabinopyranose, 3-O-beta-D-xylopyranosyl-xylobiose, 6-O-beta-D-xylopyranosyl-cellobiose, cellobiose, and methyl beta-D-xylopyranoside. All alpha-xylobioses and cellobiose were inactive as inducers of the xylan-degrading enzyme system. Other compounds served as inducers of varying efficiency, depending on their concentration in the induction medium and the time of incubation of cells. The most rapid response of the cells, i.e., the shortest induction period of beta-xyloside permease, beta-xylosidase (EC 3.2.1.37), and beta-xylanase (EC 3.2.1.8), was observed with 1,4-beta-xylobiose, which was the most efficient inducer at low concentrations (0.1 to 0.2 mM). At higher concentrations (2 to 10 mM) and after long incubations, the highest enzyme yields were obtained with 1,2-beta-xylobiose. The results represent a new example of efficient induction of polysaccharide-degrading enzyme systems by positional isomers of dimers derived from the polysaccharide.     

Stimulation of xylanase synthesis in Cryptococcus albidus by cyclic AMP

Cryptococcus albidus secretes a xylanase when induced by xylan or beta-methylxyloside, a non-metabolizable inducer, and production of the enzyme is repressed by xylose. The effect of exogenous cAMP on xylanase production was tested under different growth conditions. The cAMP elicited a 1.5 to 2 fold increase in xylanase production during the induction by xylan and B-methylxyloside but did not relieve the repression observed during growth on xylose. Cyclic AMP also affected the growth rate of the cells and did not modulate the activity of pure xylanase in vitro. A 15-nucleotide sequence located upstream from the xylanase gene could be part of a cAMP regulatory sequence.     

Influence of cultivation conditions on lipid production by Cryptococcus albidus

Summary Cryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.