Yeast Aureobasidium Pullulans Research Articles

Expression of fatty-acid-modifying enzymes in the halotolerant black yeast Aureobasidium pullulans (de Bary) G. Arnaud under salt stress.

Multiple tolerance to stressful environmental conditions of the black, yeast-like fungus Aureobasidium pullulans is achieved through different adaptations, among which there is the restructuring of the lipid composition of their membranes. Here, we describe three novel genes encoding fatty-acid-modifying enzymes in A. pullulans, along with the levels of their mRNAs under different salinity conditions. High levels of Δ9-desaturase and Δ12-desaturase mRNAs were seen at high salinities, which were consistent with an increased desaturation of the fatty acids in the cell membranes. Elevated levels of elongase mRNA were also detected. Surprisingly, increases in the levels of these mRNAs were also seen following hypo-osmotic shock, while hyperosmotic shock had exactly the opposite effect, demonstrating that data that are obtained from up-shift and down-shift salinity studies should be interpreted with caution.

Immunological Actions of Sophy β‐Glucan (β‐1,3‐1,6 Glucan), Currently Available Commercially as a Health Food Supplement

We examined the immunological actions of Sophy beta-glucan(Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004-329077), a type of beta-1,3-1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO-202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy beta-glucan significantly (P<0.01) stimulated the (3)H-thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme-linked immunoassays (EIAs) revealed that Sophy beta-glucan stimulated the production of interleukin-8 (IL-8) or soluble Fas (sFas), but not that of IL-1beta, IL-2, IL-6, IL-12 (p70+40), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte-like cell line, U937. The induction by Sophy beta-glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen-class II (HLA-class II), Toll-like receptor-2 (TLR-2), and Toll-like receptor-4 (TLR-4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR-2 and TLR-4 did not block the Sophy beta-glucan-induced production of IL-8 in the U937 cells. Furthermore, a mAb to the beta-glucan receptor, Dectin-1, significantly (P<0.05) blocked the Sophy beta-glucan induced DNA synthesis in the PBMCs, and Sophy beta-glucan-induced production of IL-8 in the U937 cells. The Sophy beta-glucan-induced production of IL-8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Sophy beta-glucan stimulated the expression of IL-8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy beta-glucan also blocked the stimulator cell induction of DNA synthesis and IFN-gamma production in the responder cells in a one-way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy beta-glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy beta-glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries.

Production of cellulolytic and hemicellulolytic enzymes from Aureobasidium pulluans on solid state fermentation

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL beta-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and beta-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75 degrees C, respectively. These enzymes remained stable between a wide range of pH. The beta-glucosidase was the most thermostable enzyme, remaining 100% active when incubated at 75 degrees C for 1 h.

Purification and Characterization of an Alkaline Protease from the Marine Yeast Aureobasidium pullulans for Bioactive Peptide Production from Different Sources

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45 degrees C, respectively. The enzyme was activated by Cu(2+) (at a concentration of 1.0 mM) and Mn(2+) and inhibited by Hg(2+), Fe(2+), Fe(3+), Zn(2+), and Co(2+). The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1-10-phenanthroline, and iodoacetic acid. The K(m) and V(max) values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 micromol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.

Amylase production by the marine yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pacific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identification methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 °C, aeration rate 6 Lmin−1 and agitation speed 250 rmin−1. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.

Glucoamylase production by the marine yeast Aureobasidium pullulans N13d and hydrolysis of potato starch granules by the enzyme

Under the optimal conditions, 10 U/ml of glucoamylase was produced by the marine yeast Aureobasidium pullulans N13d. It was noticed that the crude glucoamylase actively hydrolyzed potato starch granules, but poorly digested raw corn starch and sweet potato starch, resulting in conversion of 68.5, 19 and 22% of them into glucose within 6 h of incubation in the presence of 40 g/l of potato starch granules and 20 U/ml of the crude enzyme. When potato starch granules concentration was increased from 10 to 80 g/l, hydrolysis extent was decreased from 85.6 to 60%, while potato starch granules concentration was increased from 80 to 360 g/l, hydrolysis extent was decreased from 60 to 56%. Ratio of hydrolysis extent of potato starch granules to hydrolysis extent of gelatinized potato starch was 86.0% and the hydrolysis extent of potato starch granules by action of the crude glucoamylase (1.0 U/ml) was 18.5% within 30 min at 60 °C. Only glucose was detected during the hydrolysis, indicating that the crude enzyme could hydrolyze both α-1,4 and α-1,6 linkages of starch molecule in the potato starch.

Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion

The extracellular glucoamylase in the supernatant of the cell culture of the marine yeast Aureobasidium pullulans N13d was purified to homogeneity with a 7.3-fold increase in specific activity as compared to the concentrated supernatant by ammonium sulfate fractionation and gel filtration chromatography (Sephadex™ G-75). According to the data on native polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was 98 kDa. However, the data on SDS polyacrylamide gel electrophoresis show that the purified enzyme consisted of two subunits whose molecular weights were 65 and 33 kDa, respectively. The optimal pH and temperature of the purified enzyme were 4.5 and 60 °C, respectively. The enzyme was activated by Ca 2+, Ba 2+, Na +, Cu 2+, Mg 2+ and Co 2+ and stabilized by CaCl 2. On the other hand, Hg 2+ and Ag + inhibited the enzyme. The enzyme was inhibited by EDTA, EGTA and SDS, but was not inhibited by iodoacetic acid and PMSF. K m and V max values of the purified enzyme for soluble starch were 5.75 ± 0.3 mg/ml and 0.25 ± 0.02 mg/ml/min, respectively. Among raw potato starch, corn starch and sweet potato starch tested, all of them were absorbed by the purified enzyme, but only raw potato starch was digested by the purified enzyme. 15.8% of raw potato starch (1.0%, w/v) and 21.1% of cooked potato starch (1.0%, w/v) were hydrolyzed within 30 min by 0.5 U/mg starch of the purified enzyme and only glucose was detected in the hydrolysate, indicating that the enzyme was glucoamylase with debranching activity.

Influence of starvation, surface attachment and biofilm growth on the biocide susceptibility of the biodeteriogenic yeast Aureobasidium pullulans

To investigate the effect of starvation, surface attachment and growth in a biofilm on the susceptibility of Aureobasidium pullulans to the biocides 2-n-octyl-4-isothiazolin-3-one (OIT) and sodium hypochlorite (NaOCl). Fluorescence loss from a green fluorescent protein (GFP)-transformed strain was used to monitor real-time loss in viability as previously described in situ in 96-well plates. Exponential phase, yeast-like (YL) cells were settled in the bottom of the wells as a low-density monolayer (LDM) and were susceptible to all biocide concentrations (25-100 mug ml(-1)). The exponential phase YL cells were either starved for 48 h in suspension or starved for 48 h as LDMs in the wells. Starvation in both cases led to a small reduction in susceptibility to the biocides. In contrast, 48-h biofilms grown in malt extract broth showed an apparent lack of susceptibility to 25 and 50 mug ml(-1) OIT and to 25-100 mug ml(-1) NaOCl. However, when the OIT concentration was increased to compensate for the higher cell density in the biofilm, the biofilms were found to be equally susceptible to the LDM. Starvation of A. pullulans YL cells either in suspension or as attached LDM resulted in a decrease in susceptibility to low concentrations of both OIT and NaOCl while the apparent reduced susceptibility of mature biofilms was due to the increase in biofilm cell density rather than true biofilm resistance per se. Monitoring fluorescence loss from the GFP-transformed strain of A. pullulans can be used as a fast and reliable method for monitoring cell death in real time as a response to biocide and antimicrobial challenge.

Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans

A yeast strain, Aureobasidium pullulans, which could produce the high yield of protease was isolated from sediment of saltern in Qingdao, China. Maximum production of enzyme (623.1 U/mg protein; 7.2 U/ml) was obtained in a medium containing 2.5 g soluble starch and 2.0 g NaNO 3, 100 ml seawater, initial pH 6.0, after fermentation at 24.5 °C for 30 h. The protease had the highest activity at pH 9.0 and 45 °C.

Analysis of microtubules and F-actin structures in hyphae and conidia development of the opportunistic human pathogenic black yeast Aureobasidium pullulans.

Organization of the cytoskeleton was studied in the ascomycetous black yeast Aureobasidium pullulans, an opportunistic human pathogen, in an effort to present it as a potential target of antifungal therapy. Long cytoplasmic microtubules, extending along the hyphae from the base to the growing apex, were the dominant structures in multinucleate interphase cells. Before mitosis these microtubules disappeared and were replaced by intranuclear spindles. This reorganization of microtubules occurred along the whole length of hypha before synchronous division of the nuclei. Actin cytokinetic rings were rarely seen. Cortical actin in the form of patches accumulated in areas of cell wall growth, i.e. in the hyphal apex and near the occasionally formed septum. Actin cables were not seen. During synchronous conidiogenesis, the cytoplasmic microtubules extended along developing conidia, and actin patches lined their subcortical areas. Actin rings were formed regularly at the base of uninuclear conidia. Microtubule inhibitor methyl benzimidazol-2-ylcarbamate disintegrated the microtubules, and inhibited nuclear division, development of hyphae and conidiogenesis. Actin inhibitor Cytochalasin D induced swelling of hyphal apexes and developing conidia. This inhibitory activity ceased after 5 to 12 h when the occasional septa appeared and conidiogenesis was completed. The lack of unicellular organization in multinucleate hyphae of A. pullulans seems be related to a rarity of F-actin structures: i.e. absence of actin cables, the lack of actin cytokinetic rings in particular, resulting in the uncoupling of the nuclear division from cytokinesis; the association of both processes is, however, retained during conidiogenesis.

Comparison of antagonists of Botrytis cinerea in greenhouse-grown cucumber and tomato under different climatic conditions

The efficacy of Trichoderma harzianum T39 and the yeasts Aureobasidium pullulans and Cryptococcus albidus against Botrytis cinerea in cucumber and tomato was compared with chemical control. Four experiments were conducted in cucumber grown under different climatic conditions in The Netherlands, and two experiments were done in tomato both in the Netherlands and in Israel. T. harzianum and A. pullulans showed the most consistent control of B. cinerea, reducing stem lesions and death of plants by 40–100% in most cases. Control of stem lesions and subsequent wilting was generally better than control of symptoms on fruits. In some cases, the biocontrol agents were more effective than the broad-spectrum fungicide tolylfluanid and the selective fungicide iprodione. The climatic conditions did not strongly influence the efficacy of the biocontrol agents, but regression analysis showed that high temperature during the day and high vapour pressure deficit during the night reduced biocontrol efficacy. From the results, prospects for biocontrol of B. cinerea in greenhouse vegetables appear good under a range of conditions.

Three new metabolites from the marine yeast aureobasidium pullulans

Two new diketopiperazines (1 and 2) with a d-cis-4-hydroxyproline residue, and orcinotriol (3), a new 1,3-dihydroxyphenol derivative, were isolated from cultured broth of the yeast Aureobasidium pullulans, which was isolated from an Okinawan marine sponge. The structures of the compounds, including their absolute stereochemistries, were determined by spectroscopic data and chemical means.

Hydrolysis of extracted and fibre-bound xylan with Aureobasidium pullulans enzymes

The enzymatic hydrolysis of arabinoxylan extracted from oat spelts and fibre-bound xylan present in dissolving pulp was investigated and compared using purified β-xylanase and β-xylosidase as well as the crude enzyme preparation of the yeast Aureobasidium pullulans. By means of size-exclusion and anion exchange chromatography both enzymes were purified to homogeneity with apparent molecular masses of 20 kDa ( β-xylanase) and 216 kDa ( β-xylosidase). Addition of greater activities of β-xylosidase (up to 1.5 IU g −1 of araboinoxylan) to β-xylanase (1.5 IU g −1 of araboinoxylan) in the enzyme-arabinoxylan reaction mixture resulted in the amount and rate of release of xylose and xylo-oligomers being increased. In the absence of β-xylosidase only xylobiose, xylotriose and higher oligomers were detected by high performance liquid chromatography. When β-xylanase or β-xylosidase or a β-xylanase/ β-xylosidase mixture was added to dissolving pulp, release of xylose and xylo-oligomers (xylobiose, xylotriose and higher oligomers) was observed in all instances using thin-layer chromatography. The results showed that the purified and crude enzymes of A. pullulans were only partially effective in the removal of xylan from dissolving pulp and the hydrolysis reaction was both time- and substrate-dependent.

A new variety of Aureobasidium pullulans characterized by exopolysaccharide structure, nutritional physiology and molecular features.

The black yeast Aureobasidium pullulans (de Bary) Arnaud is known to synthesize the exopolysaccharide pullulan, a poly-alpha-1,6-maltotriose. Nine strains were found to produce additional aubasidan-like EPS, i.e. glucans with alpha-1,4-D-, beta-1,6-D- and beta-1,3-D-glycosidic bonds. These strains had previously been found to deviate in genotypic characters. Additional physiological differences were found: the optimal nitrogen source for exopolysaccharide production in liquid medium was NaNO3 for aubasidan-producing strains, and (NH4)2SO4 for the remaining strains. A new variety, A. pullulans var. aubasidani Yurlova, is described for the strains producing aubasidan-like components. The new variety can be distinguished from A. pullulans var. pullulans by the absence of assimilation of methyl-alpha-D-glucoside and lactose.

Repeated treatments with Aureobasidium pullulans hemicellulases and alkali enhance biobleaching of sulphite pulps

The yeast Aureobasidium pullulans when grown on xylose produces hemicellulolytic enzymes with predominant xylanase and β-xylosidase activity and no cellulase activity. At pH 4.5 and a temperature of 50°C, the enzyme preparation produced by the means of ultrafiltration and dialysis retained 96 and 62% of its xylanase activity when incubated for 1 and 24 h, respectively. Aureobasidium pullulans enzymes were able to hydrolyze selectively the hemicellulose portion of unbleached sulphite pulps directed toward the manufacture of dissolving pulp. Hydrolysis conditions for the efficient removal of xylan from pulp by the hemicellulase preparation were optimized in terms of pulp consistency (9%), temperature (55°C), time (1 h), enzyme loading (15 IU xylanase g −1 oven-dry pulp), and number of repetitions (three). The repeated consecutive enzymatic and alkali-oxygen treatments were studied in a totally chlorine-free bleaching sequence for their effects on pulp characteristics related to hemicellulose and lignin content of pulp. The best results were obtained when an enzyme-oxygen treatment repeated three times was applied: Pentosan content of pulp was reduced two-fold, the κ number decreased by 60%, brightness was enhanced by 18 points, and α-cellulose content was enriched by 3 points. Monocarbohydrate analysis indicated that treated pulp contained less xylan (48%) and glucomannan (15%) than the untreated reference of dissolving pulp.

Description and evaluation of reciprocating plate bioreactors

The environment in which live microorganisms has a major impact on their productivity. One important factor is the mechanical mixing that is used to promote good heat and mass transfer in bioreactors. In this paper, the performance of reciprocating plate bioreactors is first evaluated for their ability to produce high oxygen transfer coefficients. Pure water and a glycerol water (50∶50 wt%) solution are used for this evaluation. Then, the performance of reciprocating plate bioreactors for the production of an exocellular polysaccharide (pullulan) by yeast Aureobasidium pullulans is analyzed in terms of quantity and quality of the polysaccharide. Results clearly show that a more efficient substrate utilisation is achieved with reciprocating plate bioreactors.

Yeast communities of the cactus Pilosocereus arrabidae and associated insects in the Sandy coastal plains of southeastern Brazil.

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the moth Sigelgaita sp. in the cactus Pilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactus Pilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent species Pichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp., Myxozyma mucilagina and Sporopachydermia sp. A, representing about 80% of the total isolates. Pichia sp. A and a Candida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genus Kloeckera, basidiomicetous anamorphs of the genus Cryptococcus, the black yeast Aureobasidium pullulans, Pichia sp. A, a Candida domercqii-like species, and some cactophilic yeasts, especially Clavispora opuntiae. The feeding site of Sigelgaita sp. larvae had Clavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.

Yeasts and coliform bacteria of water accumulated in bromeliads of mangrove and sand dune ecosystems of southeast Brazil

Yeasts and coliform bacteria were isolated from water that accumulated in the central cups and adjacent leaf axilae of two bromeliads, Neoregelia cruenta of a coastal sand dune and Quesnelia quesneliana of a mangrove ecosystem near the city of Rio de Janeiro, Brazil. The mean total coliform counts were above 10,000 per 100 mL for waters of both plants, but the mean fecal coliform counts were only 74 per 100 mL for Q. quesneliana and mostly undetected in water from N. cruenta. Of 90 fecal coliform isolates, 51 were typical of Escherichia coli in colony morphology and indol, methyl red, Volges-Proskauer, and citrate (IMViC) tests. Seven representatives of the typical E. coli cultures were identified as this species, but the identifications of nine other coliform bacteria were mostly dubious. The yeast community of N. cruenta was typical of plant surfaces with basidiomycetous yeasts anamorphs, and the black yeast Aureobasidium pullulans was prevalent. Quesnelia quesneliana had a substantial proportion of ascomycetous yeasts and their anamorphs, including a probable new biotype of Saccharomyces unisporus. Our results suggested that the microbial communities in bromeliad waters are typically autochtonous and not contaminants.

Interaction of abscisic acid and indole-3-acetic acid-producing fungi with Salix leaves

The molds Botrytis cinerea, Cladosporium cladosporioides, and the yeast Aureobasidium pullulans, isolated from the leaves of three short-rotation Salix clones, were found to produce indole-3-acetic acid (IAA). Abscisic acid (ABA) production was detected in B. cinerea. The contents of IAA and ABA in the leaves of the Salix clones and the amounts of fungal propagules in these leaves were also measured, in order to evaluate whether the amounts of plant growth regulators produced by the fungi would make a significant contribution to the hormonal quantities of the leaves. The content of ABA, and to a lesser degree that of IAA, showed a positive correlation with the frequency of infection by the hormone-producing organisms. The amounts of hormone-producing fungi on leaves that bore visible colonies were, however, not sufficiently high to support the claim that either the fungal production of ABA or IAA would significantly contribute to the hormonal contents of the leaves of the Salix clones. It is therefore suggested that the effect of fungal IAA production on plants is limited to the rhizosphere and that B. cinerea, which is a known pathogen, induces ABA production by the mother plant as a response to physiological stress.

Interaction between the black yeast Aureobasidium pullulans and the gall midge Lasioptera ephedricola in gall formation on the desert shrub Ephedra trifurca

Galls produced by the cecidomynd Lastoptera ephedncola on Ephedia trifurca always have a black ring associated with them while galls produced by the congener L ephedrae never do Black ring material after microscopic examination and culture proved to be Aureobasidium pullulans In addition to lacking black ring material neither L ephedrae galls nor healthy stems consistently yielded Aureobasidium on culture Gall and larva size measurements indicated that continued larval presence is not necessary for gall development, suggesting fungus initiated gall formation However inoculation of healthy stems with Aureobasidium caused lesions hut not galls The mycelium m galls did not appear grazed and neither larvae nor pupae contained Aureobasidium propagules suggesting that larvae do not feed directly on fungi These data also suggest that there is no trans‐pupal passage of fungus from larvae or pupae to adults Newly emerged females do not carry fungal propagules suggesting that thcy are not inoculated upon exiting the gall Gall position leaf culture and stem culture data suggest that the fungus is picked up from leaves prior to oviposition