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.     

Siderophore production by the marine-derived Aureobasidium pullulans and its antimicrobial activity

Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore. Among them, only the yeast strain HN6.2 which was identified to be Aureobasidium pullulans was found to produce high level of the siderophore. Under the optimal conditions, this yeast strain could produce 1.1 mg/ml of the siderophore. The crude siderophore produced by the yeast strain HN6.2 was able to inhibit cell growth of Vibrio anguillarum and Vibrio parahaemolyticus, isolated from the diseased marine animals.     

β-Mannanolytic system of Aureobasidium pullulans

A xylanolytic yeast strain Aureobasidium pullulans NRRL Y 2311-1, was found to produce all enzymes required for complete degradation of galactomannan and galactoglucomannan. The enzymes differed in function and cellular localization: endo-β-1,4-mannanase was secreted into the culture fluid, β-mannosidase was strictly intracellular, and α-galactosidase and β-glucosidase were found both extracellularly and intracellularly. Among these enzyme components, only extracellular β-mannanase and intracellular β-mannosidase were inducible. The production of β-mannanase and β-mannosidase was 10- to 100-fold higher in galactomannan medium than in medium with one of the other carbon sources. β-mannanase and β-mannosidase were coinduced in glucose-grown cells by galactomannan, galactoglucomannan, and β-1,4-manno-oligosaccharides. The natural inducer of extracellular β-mannanase and intracellular β-mannosidase appeared to be β-1,4-mannobiose. Synthesis of both enzymes was completely repressed by glucose, mannose, or galactose. The synthetic glycoside methyl β-d-mannopyranoside served as a nonmetabolizable inducer of both β-mannosidase and β-mannanase. Received: 24 June 1996 / Accepted: 26 September 1996     

Production of extracellular polyols in Aureobasidium pullulans

Aureobasidium pullulans produced extracellularly considerable amounts of polyols in the media with sucrose, glucose, fructose and mannose as sole carbon source during the late exponential and stationary phase of growth. The maximum yield of polyol was about 23% in the 20%(w/v) sucrose medium, of which mannitol was the main polyol associated with minute quantities of glycerol. Stress solutes such as NaCl and KCl did not promote polyol production.     

Biosynthesis of novel exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 x 10(6) to 2.12 x 10(6) to 0.85 x 10(6) to 0.77 x 10(6) with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 +/- 3 to 29 +/- 2 mol%, and the molecular weight increased from 2.73 x 10(6) to 4.86 x 10(6). There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 +/- 3:13 +/- 3 to 28 +/- 2:72 +/- 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.     

Biosynthesis of Novel Exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 × 10⁶ to 2.12 × 10⁶ to 0.85 × 10⁶ to 0.77 × 10⁶ with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 ± 3 to 29 ± 2 mol%, and the molecular weight increased from 2.73 × 10⁶ to 4.86 × 10⁶. There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 ± 3:13 ± 3 to 28 ± 2:72 ± 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.     

Lipids of Aureobasidium (Pullularia) pullulans]

Fractional composition of free and bound lipids was studied in Aureobasidium (Pullularia) pullulans 8 by preparative TLC on Silufol. Bound lipids contained a fraction (27.76 +/- 0.5%) of dark brown colour, similar to melanin. The composition of fatty acids was studied by GLC. The following fatty acids were identified and determined quantitatively: C12:0, C14:0, C15:0, C16:0, C18:0, C18:1+C15:2. The following fatty acids predominated in free and bound lipids: C16:0, C18:1+C18:2. The ratio between unsaturated and saturated fatty acids in all fractions of free and bound lipids was more than unity. The following parameters were determined for lipids; ester number (173.89 and 178.53); iodine number (44.1 and 33.10), and saponification number (181.17 and 206.03) (the values are given for free and bound lipids, respectively).     

Growth of Aureobasidium pullulans on straw hydrolysate.

Growth characteristics and cell properties of Aureobasidium (Pullularia) pullulans were studied. The organism grew well on an acid hydrolysate of ryegrass straw over a wide range of pH and temperature. The optimum temperature and pH for the growth of the organism were 32 degrees C and 5.5, respectively. A cell yield of 1.5 g/liter of straw hydrolysate was obtained. The dried cell mass contained 42.6% crude protein, 0.4% crude fat, and 6.4% nucleic acids. The essential amino acid profile of the microbial protein was comparable to that of Candida utilis. A rat feeding study indicated that the A. pullulans cells were not toxic and that the feed intake, weight gain, and protein efficiency ratio values were superior to those obtained with C. utilis. Once the question of mathogenicity is resolved, A. pullulans could be useful for production of single-cell protein from cellulosic wastes.     

Growth of Aureobasidium pullulans on straw hydrolysate.

Growth characteristics and cell properties of Aureobasidium (Pullularia) pullulans were studied. The organism grew well on an acid hydrolysate of ryegrass straw over a wide range of pH and temperature. The optimum temperature and pH for the growth of the organism were 32 degrees C and 5.5, respectively. A cell yield of 1.5 g/liter of straw hydrolysate was obtained. The dried cell mass contained 42.6% crude protein, 0.4% crude fat, and 6.4% nucleic acids. The essential amino acid profile of the microbial protein was comparable to that of Candida utilis. A rat feeding study indicated that the A. pullulans cells were not toxic and that the feed intake, weight gain, and protein efficiency ratio values were superior to those obtained with C. utilis. Once the question of mathogenicity is resolved, A. pullulans could be useful for production of single-cell protein from cellulosic wastes.     

The production of exopolysaccharides by Aureobasidium pullulans in fermenters with low-shear configurations

No increases in exopolysaccharide (EPS) yields in Aureobasidium pullulans were observed when grown with reduced-shear impellers instead of standard Rushton turbines in the same vessel. However, yields were dramatically reduced when the organism was grown in an airlift reactor. This fall in production could be counteracted by improving fluid circulation through the placement of impellers within the draught tube, a strategy that resulted in the highest EPS concentration (approx. 13 g l⁻¹) of all the fermenter configurations tested. Received: 24 June 1997 / Revised revision: 25 September 1997 / Accepted: 29 September 1997     

New Method of Producing Protoplasts of Aureobasidium pullulans

A rapid and relatively inexpensive method for producing protoplasts of the black yeast Aureobasidium pullulans is described. The procedure involves anaerobic incubation with the lytic preparation Driselase.     

Biosynthesis of pullulan using immobilized Aureobasidium pullulans cells

Immobilization of Aureobasidium pullulans by adsorption on solid supports and entrapment in open pore polyurethane foam were attempted. By adsorption, the highest cell loading of 0.012-0.018 g dry wt/cm(2) support was obtained in pH 2.0 medium. Under this acidic condition, the net surface charges (zeta potentials) of both the cells and supports were close to zero and no pullulan was synthesized. Cationic coatings of Cytodex and polyethylenimine were not efficient in enhancing the binding strength between the cells and the supports. Surface immobilized cells and polyurethane foam entrapped cells exhibited a similar fermentation characteristics resulting in ca. 18 g/L pullulan and ca. 5 g/L leaked cells. However, cells entrapped in the polyurethane foam were more shear resistant. The immobilized cells thus could be repeatedly used for pullulan biosynthesis.     

Aureobasidium pullulans metabolism of prostaglandins of the A-type.

Aureobasidium pullulans, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA2 (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9 alpha or 9 beta-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9 alpha, 15 alpha-dihydroxy-2, 3, 4, 5-tetranor-13-trans-prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9 beta-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9, 15-oxo-2, 3, 4, 5-tetranorprostanoic acid and 9, 15-oxo-2, 3, 4, 5-tetranor-13-trans-prostenoic acid. The water soluble metabolites have not been characterized further. The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that A. pullulans can perform the following transformation: beta-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both delta 10,11 and delta 13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (delta 10, 11 leads to delta 11, 12). A. pullulans metabolizes 15-epimeric PGA2 equally readily with the production of similar products. PGA1 affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. A. pullulans displays some enantioselectivity, PGA2 and 15-epi-PGA2 are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.