Chemical and biological characterization of siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 and its antibacterial activity

After analysis using HPLC and electronic ion spray mass spectroscopy, the purified siderophore produced by the marine-derived Aureobasidium pullulans HN6.2 was found to be fusigen. The purified desferric fusigen still had strong inhibition of growth of the pathogenic Vibrio anguillarum while the fusigen chelated by Fe³⁺ lost the ability to inhibit the growth of the pathogenic bacterium. The added iron in the medium repressed expression of the hydroxylase gene encoding ornithine N⁵-oxygenase that catalyzes the N⁵-hydroxylation of ornithine for the first step of siderophore biosynthesis in the yeast cells while expression of the hydroxylase gene in the yeast cells grown in the medium plus ornithine was enhanced.     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2

The l-ornithine-N ⁵-monooxygenase structural gene (SidA gene, accession number: FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RT-PCR. An open reading frame of 1,461 bp encoding a 486 amino acid protein (isoelectric point: 7.79) with calculated molecular weight of 55.4 kDa was characterized. The promoter of the gene (intronless) was located from −1 to −824 and had three HGATAR boxes which were putative binding motifs for the respective DNA-binding motifs and one CATA box. The SidA gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase (HPT) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6 (∆sidA) did not synthesize both intracellular and extracellular fusigen so that it could not inhibit growth of the pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. The disruptant S6 did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with 10 μM Fe³⁺ and Fe²⁺. H₂O₂ in the medium was more toxic to the disruptant S6 than to its wild type HN6.2. Thus, we infer that the fusigen produced by the marine-derived A. pullulans HN6.2 can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells and secretion of siderophore may represent an efficient tool to eliminate competitors to compete for limiting nutritional resources in marine environments.     

Pullulan production from brewery wastes by Aureobasidium pullulans

The production of pullulan from brewery wastes by Aureobasidium pullulans in shake flask culture was investigated. The maximum pullulan concentration (6.0g/l) was obtained after 72h of fermentation. The external addition of nutrients into the spent grain liquor improved significantly the production of pullulan. In this case, the highest values of pullulan concentration (11.0±0.5g/l), pullulan yield (48.2±1.5%), and sugar utilization (99.0±0.5%) were obtained in the medium (pH 6.5–7.5) supplemented with K₂HPO₄ 0.5%, l-glutamic acid 1%, olive oil 2.5%, and Tween 800.5%.     

Polysaccharide production by sponge-immobilized cells of the fungus Aureobasidium pullulans

T.P. WEST AND B.R.-H. STROHFUS. 1996. Cells of the fungus Aureobasidium pullulans ATCC 42023 were immobilized in sponge cubes and examined for their ability to elaborate the polysaccharide pullulan in relation to carbon source. It was found that fungal cells grown on corn syrup, sucrose or glucose as a carbon source could be immobilized in sponge cubes and that comparable cell weights and viable cell concentrations were immobilized. Independent of the carbon source tested, the immobilized fungal cells could be used at least three times for the production of polysaccharide. The immobilized A. pullulans cells elaborated the highest polysaccharide levels in the culture medium after 5–7 d of growth at 30°C.     

Elevated polysaccharide production by mutants of the fungus Aureobasidium pullulans

Abstract Two mutants of the fungus Aureobasidium pullulans ATCC 42023 were isolated that exhibited elevated polysaccharide production. Both mutants were isolated using a combination of chemical mutagenesis and resistance to growth inhibitors. It was found that both mutants elaborated higher polysaccharide levels after 7 days of growth on corn syrup or sucrose, respectively, compared to ATCC 42023. The dry weights of the mutant cells were found not to differ greatly from those of the parent cells whether corn syrup or sucrose served as the carbon source. The pullulan content of the polysaccharide synthesized by the mutants or parent cells on sucrose was consistently lower than polysaccharide synthesized on corn syrup. Using corn syrup as a carbon source, the pullulan content of the polysaccharide elaborated by the parent was higher than either mutant. The inverse was found to occur with respect to pullulan content when the strains were grown on sucrose as a carbon source.     

Lipase production by diverse phylogenetic clades of Aureobasidium pullulans

Thirty-nine strains representing 12 diverse phylogenetic clades of Aureobasidium pullulans were surveyed for lipase production using a quantitative assay. Strains in clades 4 and 10 produced 0.2–0.3 U lipase/ml, while color variant strain NRRL Y-2311-1 in clade 8 produced 0.54 U lipase/ml. Strains in clade 9, which exhibit a dark olivaceous pigment, produced the highest levels of lipase, with strain NRRL 62034 yielding 0.57 U lipase/ml. By comparison, Candida cylindracea strain NRRL Y-17506 produced 0.05 U lipase/ml under identical conditions. A. pullulans strain NRRL 62034 reached maximal lipase levels in 5 days on lipase induction medium, while A. pullulans strain NRRL Y-2311-1 and strains in clades 4 and 10 were highest after 6 days. A. pullulans strain NRRL Y-2311-1 and strains in clade 9 produced two extracellular proteins in common, at >50 and <37 kDa.     

Extracellular cellulase production by tropical isolates of Aureobasidium pullulans

Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and alpha-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 micromol glucose.(mg protein)-1.h-1, while activities on alpha-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 micromol glucose.(mg protein)-1.day-1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.     

Optimization of tannase production by Aureobasidium pullulans DBS66

Tannase production by Aureobasidium pullulans DBS66 was optimized. The organism produced maximum tannase in the presence of 1% tannic acid after 36 h. Maximum gallic acid accumulation was observed within 36 h and tannic acid in the fermented broth was completely degraded after 42 h of growth. Glucose had a stimulatory effect on tannase synthesis at 0.1% (w/v) concentration. The organism showed maximum tannase production with (NH4)2HPO4 as nitrogen source. Shaking speed of 120 rpm and 50-ml broth volume have been found to be suitable for maximum tannase production.     

Effect of two-stage temperature on pullulan production by Aureobasidium pullulans

The effect of a two-stage cultivation temperature on the production of pullulan synthesized by Aureobasidium pullulans CGMCC1234 was investigated. Pullulan production was affected by temperature; although the optimum temperature for pullulan production was 26°C, the optimal temperature for cell growth was 32°C. Maximum pullulan production was achieved by growing A. pullulans in a first stage of 32°C for 2 days, and then in a second stage of 26°C for 2 days. Pullulan production using these two-stage temperatures significantly increased: about 27.80% (w/w) compared to constant-temperature fermentation (26°C for 4 days). The morphology of the A. pullulans (CGMCC 1234) was also affected by temperature; the lower temperature (26°C) supported unicellular biomass growth. Results of this study indicate that fermentation using two temperature stages is a promising method for pullulan production.     

4-氯乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

从实验室保藏的菌株中,筛选到一株立体选择性较高的产4-氯乙酰乙酸乙酯(COBE)羰基还原酶的菌株———出芽短梗霉(Aureobasidiumpullulans)SW0202,菌体产酶条件研究表明,最佳的发酵培养基配方为:麦芽糖30.0g/L,酵母膏20.0g/L,蛋白胨3.0g/L,(NH4)2SO45.0g/L,KH2PO42.0g/L,MgSO4.7H2O0.7g/L,最适发酵温度及初始pH分别为:28°C和pH6.0。该菌在此条件下发酵培养24h,产菌丝体生物量16.78g干菌体/L,COBE羰基还原酶酶活力达到1007U/L。在COBE的转化反应中,产物S-CHBE的浓度达到10.12g/L,光学纯度>97%e.e.。     

Polysaccharide production by Aureobasidium pullulans: factors affecting polysaccharide formation

Aureobasidium pullulans NRRL 6220 synthesized polysaccharide most actively in media containing sucrose, fructose or maltose with (NH₄)₂SO₄ (0.6 g/l) or ammonium acetate giving greatest yields of the polysaccharide. With (NH₄)₂SO₄ at 1.2 g/l, production of polysaccharide was decreased considerably. Polysaccharide production was highest with an initial pH of 6.5 while biomass formation was better below an initial pH of 5.5. Optimum phosphate concentration for polysaccharide production was 0.03 m.S.M. Badr-Eldin, H.G. El-Masry and O.A. Abd El-Rahman are with the Microbial Chemistry Department, National Research Center, Dokki, Cairo, Egypt; F.H.A. Mohamad is with the Chemical Engineering and Pilot Plant Department, National Research Center, Dokki, Cairo, Egypt. O.M. El-Tayeb is with the Microbiology Department, Faculty of Pharmacy, Cairo University, Egypt.     

Polysaccharide production by a reduced pigmentation mutant of the fungus Aureobasidium pullulans

Abstract A reduced pigmentation mutant was isolated from Aureobasidium pullulans ATCC 42023 by chemical mutagenesis and was subsequently characterized. The pigment melanin was present not only in A. pullulans cells but also contaminated the elaborated polysaccharide and thus, was measured in both fractions. Cellular and polysaccharide melanin levels of the mutant strain were at least 11-fold and 18-fold reduced, respectivelu, compared toits parent strain after 7 days of growth at 30°C whether sucrose or glucose served as the carbon source in the culture medium. Polysaccharide and cell dry weight levels of the mutant were very similar to those observed for the parent after growth on sucrose or glucose as the source of carbon over a period of 7 days at 30°C. The pullulan content of the polysaccharide produced by the parent or mutant strain was lower for sucrose-grown cells than for glucose-grown cells. It was also noted that the pullulan content of the polysaccharide elaborated by the mutant strain was slightly higher than that of the polysaccharide produced by the parent strain after growth on either sucrose or glucose.     

Polysaccharide production by immobilized Aureobasidium pullulans cells in batch bioreactors.

Cells of the fungus Aureobasidium pullulans ATCC 201253 were entrapped within 4% agar cubes or 5% calcium alginate beads and were examined for their production of the polysaccharide pullulan in batch bioreactors. The batch bioreactors were utilized twice for 168 hours of polysaccharide production in medium containing corn syrup as a carbon source. The agar-entrapped cells produced nearly equivalent pullulan concentrations during both production cycles. The alginate-entrapped cells produced higher polysaccharide levels during the second cycle compared to the levels observed during the initial cycle. The agar-entrapped cells elaborated a polysaccharide with a higher pullulan content than did the alginate-entrapped cells during both production cycles.     

Inorganic Sulfur Oxidation by Aureobasidium pullulans

Aureobasidium pullulans (de Bary) Arnaud isolated from the phylloplane of sycamore exposed to heavy atmospheric pollution oxidized S⁰ to S₂O₃²⁻, S₄O₆²⁻, and SO₄²⁻ in vitro. The intermediates S₂O₃²⁻ and S₄O₆²⁻ were also oxidized to SO₄²⁻. Cell-free extracts of A. pullulans also oxidized reduced forms of S, the oxidation increasing linearly with increasing protein concentration, showing that the process is enzymatic. The possible role of fungi in S oxidation in soils is discussed.     

出芽短梗霉发酵产生普罗蓝糖的研究

对一株野生型的出芽短梗霉(Aureobasidium pullulans)Ft1和从Ft1出发经原生质体再生筛选出的菌株R45进行了摇瓶发酵产普罗蓝糖的比较研究,结果表明R45无论从形态,菌体生长情况,还是从普罗蓝糖的产量,黑色素的产生等方面都与亲株Ft1有明显的区别,说明R45是一株具有一定生产价值的变异菌株.     

Pullulan Elaboration by Aureobasidium pullulans Protoplasts

Protoplasts of Aureobasidium pullulans are capable of producing pullulan. Biosynthesis of the polymer pullulan required induction with kinetics similar to those of whole cells. The protoplasts also produced a heteropolysaccharide component containing mannose, glucose, and galactose. The relative proportions of the pullulan and heteropolysaccharide fractions were a function of glucose concentration, with the pullulan content of the total polysaccharide rising from 20% at 2.5 mM glucose to 45% at 20 mM glucose. Elaboration of pullulan by both cells and protoplasts was sensitive to 0.6 M KCl, which was present as the osmotic stabilizer in protoplast experiments. The presence of KCl resulted in a shift in the pH optimum to a more acidic value. The molecular weight of the protoplast-derived pullulan was sharply reduced from the molecular weight of the whole-cell-derived product. Exposure of the protoplasts to proteolytic enzymes had no effect on polysaccharide elaboration.