Acidic polysaccharide from Aureobasidium pullulans

Acidic polysaccharide from Aureobasidium pullulans

[ 57 ] Trans. Br. mycol, Soc. 75 (1) 57-62 (1980) Printedin Great Britain ACIDIC POLYSACCHARIDE FROM AUREOBASIDIUM PULLULANS BY G. LEAL-SERRANO, P. ...

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[ 57 ] Trans. Br. mycol, Soc. 75 (1) 57-62 (1980)

Printedin Great Britain

ACIDIC POLYSACCHARIDE FROM AUREOBASIDIUM PULLULANS BY G. LEAL-SERRANO, P. RUPEREZ AND J. A. LEAL C.S.I.C. Instituto de Inmunologla y Biologia Microbiana, Velazquez 144, Madrid-6, Spain Aureobasidium pullulans forms an acidic extracellular polysaccharide when grown in liquid, shake or stationary cultures on a range of carbon sources and at different sugar concentrations. The polysaccharide contains glucose (90 %), malic acid (9 %), protein (3 %) and phosphate (0'5 %). The polysaccharide forms an insoluble complex with cetyl-trimethylammonium bromide from which it is recovered in more than 70 % yield with the same composition as the original product. Periodate oxidation and Smith degradation indicate that the polysaccharide has 68 % (1 -+ 3) and 32 % (1 -+ 6) linkages. The infrared spectrum shows an absorption band at 890 crrr-' characteristic of ,8-configuration and a band at 1750 characteristic of the carbonyl (-CO-) group of saturated esters. The yeast-like fungus, Aureobasidium pullulans, synthesizes a mixture of extracellular polysaccharides from a variety of sugar substrates. This mixture is composed of pullulan (an a-glucan with (1 -+ 4) and (1 -+ 6) linkages), a ,8-glucan with (1 -+ 3) and (1 -+ 6) linkage and an acidic polysaccharide or mixture of polysaccharides containing galactose, glucose, mannose and hexuronic acid (Bouveng et al., 1963). Early work on composition and structure of these polysaccharides has been reviewed by Gorin & Spencer (1968). We report here the formation of a single acidic ,8-glucan by A. pullulans and the effect of the carbon source and other factors on its production. MATERIAL AND METHODS

Culture media and microorganism Aureobasidium pullulans (de Bary) Arnaud, strain 105-22 was obtained from the Centraalbureau voor Schimmelcultures, Baam (Holland). The organism was maintained on slants of Bacto potato dextrose agar (Difco) supplemented with 1 gil of Bacto yeast extract (Difco). The basal medium for polysaccharide production and physiological studies has been described by Leal & Ruperez (1978). Unless otherwise stated D-glucose 15 gil was used as carbon source and vitamin-free casamino acids (Difco) 3 gil as the nitrogen source. The media were adjusted to pH 6·5 before autoclaving at 120°C for 15 min. The changes in the carbon source and various other details are reported in connexion with individual experiments. I solation of polysaccharide Cell-free culture fluids were treated with a volume of ethanol or methanol, and the polysaccharide

separated as a stringy precipitate that wound around the stirrer. This material was washed twice with the 50 % aqueous alcohol as used for the precipitation and dried at 60°. An aqueous solution of the polysaccharide containing 1 mg/rnl was treated with cetyl-trimethyl-ammonium bromide (CTAB) to separate neutral and acidic polysaccharide (Scott, 1965). Chemical analysis Total hexosan was determined by the anthrone procedure (Dreywood, 1946). Uronic acids were determined by the method of Dische (1947). Total protein was measured by the method ofLowry et al. (1951). The polysaccharide was hydrolyzed with HCI of different concentrations at 1050 and for different periods of time in sealed evacuated tubes. Phosphate was determined in the hydrolysates (Lowry & Lopez, 1946). The hydrolysates were analyzed by paper chromatography on Whatman no. 1 filter paper; for neutral sugars the paper was buffered with a phosphate buffer 0'066 M pH 5'5. The solvent system was: n-butanol :acetone: water (4:5 :1). This solvent was also used for the separation of polyols. The spots were developed with aniline phthalate or silver nitrate (Dawson et al., 1962). The solvent system for uronic acids was: ethyl-acetate :acetic acid :formic acid :water (36:6: 2 : 8). The spots were developed with silver nitrate. Total O-acyl substituents were determined by the method of Ludowieg & Dorfman (1960). The Hill solution was carefully adjusted between 0'18 and 0'22 pH units. Ethyl acetate was used as standard. The acids were liberated by treatment of the polysaccharide with 0'02 N-KOH for 16 h (Sutherland, 1970). The deacylated polysaccharide was collected by precipitation with ethanol and

0007-1536/80/2828-6350 $01.00 © 1980 The British Mycological Society

Polysaccharide from Aureobasidium the supernatant evaporated to dryness. The dry residue was dissolved in water and a sample passed through a column of Dowex 50 WX 4 (H+), 200400 mesh. Total acidity was determined in the effluent by titration with 0'001 M-NaOH to a phenolphthalein end point. The acidic constituents were resolved on Whatman no. 1 filter paper. The solvent system for volatile acids was: 95 % ethanol: 10% NaOH (100:10) or ethanol:concentrated ammonia (100: 1). The spots were developed with 0'05 % bromophenol blue in 0'2 % aqueous citric acid. For non-volatile acids the solvent systems used were the upper phase of a mixture of: n-butanol :formic acid :water (10 :2: 15); or phenol : formic acid:water (75:1:25). The spots were developed with 0'04 % aqueous chlorophenol red, pH 7 (Dawson et al., 1962). Periodate oxidation was performed according to Aspinall & Ferrier (1957). The formic acid formed was titrated with standard 0'002 M-NaOH to a methyl red end point. Smith degradation was performed as reported by Sietsma& Wessels (1977). In the culture fluids residual glucose was determined with an alkaline copper reagent (Somogyi, Table 1. Composition of the acid polysaccharide from A. pullulans

% by weight

Component Carbohydrates (Anthrone) Protein Acyl groups* Total acidity] Phosphate Uronic acids * Calculated as acetic acid. acid.

4000

Fig.

3500

1.

3000

90-93

2'5-3'0 4'0-4'5 8'5-9'0 0'4--0'5

Negative

t

Calculated as malic

2500

1952) and the arsenomolybdate chromogen of Nelson (1944). The pH was measured with a Crison digit 74 pH meter, Infrared spectra were obtained by the KBr technique on a Perkin-Elmer 457 infrared spectro-photometer. RESULTS

Composition and characterization of the polysaccharide The polysaccharide for these studies was obtained from cultures in 2 I Erlenmeyer flasks containing 1 I of the basal medium, incubated at 25° in an orbital incubator, Gallenkamp IH-465 at 150 rev I min, for 4 days. The composition of the polysaccharide is shown in Table 1. The hexosan fraction was identified by paper chromatography as glucose. The infrared spectrum (Fig. 1A) is characteristic of a glucan having the jJ-configuration, absorption band at 890 crrr" and lack of absorption at 850 crrr ! (Barker, Bourne & Whiffen, 1956). It showed also an absorption band at 1750 cm- 1 characteristic of the carbonyl group of esterified organic acids. This band disappeared when the polysaccharide was treated with 0'02 M-NaOH (Fig. 1B) (Sutherland, 1970). Paper chromatographic analysis of the substances liberated by the NaOH treatment revealed a spot with the same Rf value of malic acid and in the volatile acids system another spot which migrated less (Rf = 0'26) than a standard offormic acid (Rf = 0'31). An aqueous solution of the polysaccharide was precipitated with CTAB and from the precipitate the polysaccharide was recovered in a 70 % yield. This material has the same composition and i.r. spectrum as the original polysaccharide.

2000 1800 1600 1400 1200 1000 800

600

400 200

cm! Infrared spectra of (A) acidic polysaccharide, and (B) deacylated polysaccharide, from A. pullulans,

G. Leal-Serrano, P. Ruperez and]. A. Leal Periodate oxidation and Smith degradat ion The polysaccharide consumed 0'64,umoles of periodate per ,umole of anhydro-glucose unit with formation of 0'32 ,umoles of formic acid corresponding to 32 % (1 ~ 6) and 6& % (1 ~ 3) linkages (Jeanes, 1965). After Smith degradation, the remaining product was subjected to hydrolysis. By paper chromatography, only glucose and glycerol were detected on the hydrolysates, which is in agreement with the above results. Polysaccharide elaboration in submerged cultures The basal medium was distributed in 50 ml portions into 250 ml flasks. The cultures were incubated at 25° and 250 rev/min. Growth, polysaccharide production and changes in the culture medium during incubation are pre sented in Fig. 2. The microorganism grew predominantly in the yeast form. Maximum production of polysaccharide (160 mg /100 ml) was achieved at 60 h and coincided with the disappearance of glucose. No changes in polysaccharide or mycelial dry weight were observed up to 4 days. The pH decreased to 3'5 and remained at this value. Polysaccharide elaboration in static cultures Culture flasks prepared as above were incubated statically. The micro-organism grew predominantly

59

in the filamentous form . Growth, polysaccharide production and changes in the culture medium

during incubation are presented in Fig . 3. The maximum growth (550 mg/100 ml) was achieved after 9 days of incubation and polysaccharide (54 mg /100 ml) after 15 days. The pH decreased to 4'0, remained at this value for 4 days, and then increased to 8'0. Effect of the carbon source In the basal medium, glucose was replaced by 15 gil of the sugar under test. The media were distributed in 50 ml portions into 250 ml flasks. The cultures were incubated statically at 25°. Growth, polysaccharide elaboration and changes in the culture media were determined periodically by harve sting two flasks of each treatment. The results are summarized in Table 2. Polysaccharide was produced in all the carbon sources tested. The best carbon sources for polysaccharide production were maltose (68'S mg / aoo ml) and mannose (66'5 mg /100 ml), The lowest polysaccharide yield was obtained in mannitol (12'4 mg/100 ml), but this medium supported the highest growth (883 mg/f oo ml). Effe ct of glucose concent ration on polysac charide elaboration The effect of the glucose concentration was tested in 250 ml Erlenmeyer flasks containing 50 ml of

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Growth, changes in pH of the medium, residual glucose and polysaccharide production for A. pullulans grown in submerged cultures.

Polysaccharide from Aureobasidium

60 600

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500

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400

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Days after inoculation Fig. 3. Growth, changes in pH of the medium, residual glucose and polysaccharide production for A . pullulans grown statically.

T able 2, Effect of carbon source on poly saccharide production Ma ximum polysaccharide Da ys M ycelium, dry w t pH (rng / aoo ml ) (mg'/ roo ml) incubation 8 4'24 59'2 337' 6 4 '88 66'5 15 334'1 26'0 15 164 '5 7'44 8 48'0 4'24 4 64 '5 15 670 '5 4'50 5° '4 15 883'5 12'4 4'9 1 8 4'68 501'0 25'5 11 68'5 540 '0 4'47 8 4'22 477'5 58 '5 4'28 15 685'° 59'5

Glucose Mannose Galactose Fructose Arabinose Mannitol Glycerol Maltose Sucros e Starch

*

The figures correspond to the day of maximum polysacchar ide production.

Table 3. Effec t of glucose concentration on growth and poly saccharide production in static cultures of A. pullulans Polysaccharide (mg/ roo ml)

M ycelium dry weight (rng'/roo ml) Glucose (g/l)

4*

8

11

5 10 15 20 3° 60 100

193'0 241'0 231'0 234'0 253'0 270'0 343'0

232'7 333 '5 3 89 '0 398'0 520 '0 606'5 818'0

230'8 4° 2 '5 416'0 437'0 571'0 854'0 1227'0

15 222'5 410'7 583'° 626' 5 844'4 1220'7 1474'6

4 34'5 33'5 31'0 30 '0 25'0 21'0 16'0

* Da ys in incubation.

8

11

15

47'6 4 6 '3 45'2 52 '3 55'4 49'0 33'0

53'4 56'3 51'5 60'0 67'0 52'0 46'0

56'7 63'6 67'9 54'0 47'7 38'5 35'0

G. Leal-Serrano, P. Ruperez andJ. A. Leal

61

Table 4. Effect of glucose concentration on growth and polysaccharide production in submerged cultures of A. pullulans Mycelium dry weight (mg/100 ml) Glucose (gIl)

10 20 3° 5°

Polysaccharide (mg/100 ml)

r-

2 1* 62'0 265'6 5 249'5 886'0 258'0 892'0 295'0 1°37'0

3 510'0 949'0 1065'° 1153'0

4 486'0 951'7 1168'0 13°8'5

2

20·6 10·6 9'9 8'1

3 4 44'9 48'8 43'1 77'0 94'6 100'3 68'0 106'2 121'2 7°'0 97'4 115'0

* Days in incubation.

the basal medium. The flasks were divided in two lots, one was incubated statically and the other in an orbital incubator at 150 rev/min. Production of polysaccharide occurred at all concentrations of glucose in both culture conditions. Table 3 shows the results of static culture. Growth increased, while the rate of polysaccharide elaboration decreased, with increasing concentrations of glucose. The maximum production was obtained with 15 gil of glucose (68 mg/100 ml). The results in submerged cultures, Table 4, follow the same pattern as those obtained statically. The maximum production was achieved in 4 days with 30 gil glucose (121 mg/100 ml). DISCUSSION The polysaccharide isolated by us differs from pullulan because the anomeric configuration of its D-glucose units is of the fJ-type, as demonstrated by its infrared spectrum, absorption at 890 cm- I and lack of absorption at 840 cm- I , and because it is esterified with an organic acid, absorption at 1750 cm- I • It also differs from the fJ-glucans because it is formed by malic acid (9 %) and glucose (90 %) and no other sugars have been found by paper chromatography. Polysaccharides linked with organic acids, leaving apart acetic acid, have been reported: formic acid (Sutherland, 1970), pyruvic acid (Wheat, Dorsch & Godoy, 1965), malonic acid (Raistrick & Rintoul, 1931; and Fujimoto et al., 1969) and succinic acid (Harada, 1965), but we have not found in the literature any polysaccharide containing malic acid. As in luteic acid and malonogalactan one carboxyl group of the malic acid is esterified with an hydroxyl group in the polysaccharide and the other is free, conferring acidic properties to the polysaccharide. Treatment of the acidic polysaccharide with 0'02 N-NaOH liberated the acid and it was possible to isolate the neutral glucan as has been reported for luteic acid (Raistrick &

Rintoul, 1931) and malonogalactan (Kohama et al., 1974)· We thank Dr Bellanato of the Instituto de Optica Daza Valdes for her help and advice in the infrared analysis, Laboratorios Abello for the Fellowship granted to G. Leal-Serrano, and the C.S.I.C. for the Fellowship granted to P. Ruperez. REFERENCES ASPINALL, G. O. & FERRIER, R. J. (1957). A spectrophotometric method for the determination of periodate consumed during the oxidation of carbohydrates. Chemistry and Industry 1957, 1216. BARKER, S. A., BOURNE, E. J. & WHIFFEN, D. H. (1956). Use of infrared analysis in the determination of carbohydrate structure. Methods of Biochemical Analysis 3, 213-245· BOUVENG, H. 0., KIESSLING, H., LINDBERG, B. & McKAY, J. (1963). Polysaccharides elaborated by Pullularia pullulans. III. Polysaccharides synthesised from xylose solutions. Acta Chemica Scandinavica 17,135 1-1356. DAWSON, R. M. C., ELLIOTT, D. C., ELLIOT, W. H. & JONES, K. M. (1962). Data for Biochemical Research. Oxford: Oxford University Press. DISCHE, Z. (1947). A new specific color reaction of hexauronic acids. Journal of Biological Chemistry 167, 189- 198. DREYWOOD, R. (1946). Qualitative test for carbohydrate material. Industrial and Engineering Chemistry Analytical Edition 18,499. FUJIMOTO, M., KUNINAKA, A., YONEY, S., KOHAMA, T., & YOSHINO, H. (1969). Occurrence of malonogalactan in the nuclease preparation from Penicillium citrinum. Agricultural Biological Chemistry 33, 1666-1668. GORIN, P. A. J. & SPENCER, J. F. T. (1968). Structural chemistry of fungal polysaccharides. Advances in CarbohydrateChemistry 22, 367-417. HARADA, T. (1965). Succinoglucan 10C3: A new acidic polysaccharide of Alcaligenes faecalis var. myxogenes. Archives of Biochemistry and Biophysics 112,65-69. JEANES, A. (1965). Dextrans. Preparation of dextran

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Polysaccharide from Aureobasidium

from growing Leuconostoc cultures. In Methods in Carbohydrate Chemistry 5, 118-127. KOHAMA, T . FUJIMOTO, M., KUNINAKA, A. & YOSHINO, H. (1974). Structure of malonogalactan, an acidic polysaccharide of Penicillium citrinum, Agricultural Biological Chemistry 38,127-134. LEAL, J. A. & RUPEREZ, P. (1978). Extracellular polysaccharide production by A spergillus nidulans. Transactions of the British Mycological Society 70,115-120. LOWRY, O. H. & LOPEZ, J. A. (1946). The determination of inorganic phosphate in the presence of labile phosphate ester. Journal of Biological Chemistry 162,421-428. LOWRY, O. H., ROSEBROUGH, N . J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193,265-275. LUDOWIEG, J. & DORFMAN, A. (1960). A micromethod for the colorimetric determination of N-acetyl groups in acid mucopolysaccharides. Biochimica et Biophysica Acta 38, 212-218. NELSON, N. (1944). A photometric adaptation of the

Somogyi method to the determination of glucose. Journal of Biological Chemistry 153, 375-380. RAISTRICK, H. & RINTOUL, M . L. (1931). Studies in the biochemistry of micro-organisms. XIII. On a new type of mucilaginous material, luteic acid, produced from glucose by Penicillium lut eum Zukal. Transaction s of the Royal Society 220, 255-367. SCOTT, J. E. (1965). Fractionation by precipitation with quaternary ammonium salts. In Methods in Carbohydrate Chemistry 5, 38-44. SIETSMA, J. H. & WESSELS, J. G. H. (1977). Chemical analysis of the hyphal wall of Schizophyllum commune. Biochimica et Biophysica Acta 496, 225-239. SOMOGYI, M. (1952). Notes on sugar determination. Journal of Biological Chemistry 195, 19-23. SUTHERLAND, 1. W. (1970). Formate, a new component of bacterial exopolysaccharides. Nature, London 228,280. WHEAT, R. W., DORSCH, C. & GODOY, J. (1965). Occurrence of pyruvic acid in the capsular polysaccharide of Klebsiella rhinoscleromatis. Journal of Bacteriology 80, 539.

(Received for publication 16 July 1979)