Production of xylan-hydrolyzing enzymes by Aureobasidium pullulans

Production of xylan-hydrolyzing enzymes by Aureobasidium pullulans

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 72, No. 2, 135-137. 1991 Production of Xylan-Hydrolyzing Enzymes by Aureobasidium pullulans JACOBUS ...

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JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 72, No. 2, 135-137. 1991

Production of Xylan-Hydrolyzing Enzymes by Aureobasidium pullulans JACOBUS MYBURGH, BERNARD A. PRIOR,* AND STEPHANUS G. KILIAN Department of Microbiology and Biochemistry, University of the Orange Free State, PO Box 339, Bloemfontein 9300, South Africa Received 25 December 1990/Accepted 9 May 1991

Aureobasidium pullulans growing on arabinoxylan secreted ~-xylanase, acetyl esterase (AE), and produced p-nitrophenyl xylosidase (PNPX) and a-L-arabinofuranosidase (AAF) as cell associated forms. The highest respective extracellular volumetric and specific activities of ~-xylanase (82 U/ml; 1370 U/rag) and PNPX (0.44 U/mi; 4.9 U/rag) were obtained on arabinoxyulan. Only ~-xylanase and PNPX were produced when grown on a-xylose.

Xylan is hydrolyzed by fl-xylanases attacking internal xylosidic linkages on the xylan backbone while the fl-xylosidases release xylosyl residues by endwise attack of xylooligosaccharides (Wong et al., 1988). In addition, xylan is frequently substituted with groups such as acetyl and arabinose and the complete hydrolysis requires the action of enzymes that remove these substituents (Johnson et al., 1989). Several xylan-degrading enzymes (principally endofl-l,4-xylanase) have been isolated and characterized from many filamentous fungi and bacteria (Wong et al., 1988). Information on xylan-degrading enzymes produced by yeasts, however, is quite limited (Pou-Llinas and Driguez, 1987) and only Aureobasidium pullulans (Leathers et al., 1984), Cryptococcus albidus (Biely et al. , 1980) and Pichia stipitis (Lee et al., 1987) are known to produce fl-xylanase. The occurrence of other xylan-hydrolyzing enzymes such as p-nitrophenyl-fl-xylosidase (PNPX), a-L-arabinofuranosidase (AAF) and acetyl esterase (AE) in A. pullulans is unknown. A. pullulans NRRL Y 2311-1 was cultivated in yeast nitrogen base (YNB; Difco) in Erlenmeyer flasks (250 ml) containing 45 ml medium with either 10 g/l arabinoxylan (oats spelts xylan; Sigma, St. Louis, USA) or D-xylose (Merck Darmstadt, FRG) as substrates at 28°C on a rotary shaker at 130 rev/min. In some instances, the yeast was cultivated in a Multigen F-2000 fermentor (New Brunswick Scientific Co., Edison, N J, USA) equipped with a 2 l glass vessel containing 980ml medium with either 2 5 g / / arabinoxylan or 25 g/1 o-xylose as substrate. An agitation speed of 400 rev/min, an aeration rate of 1,000ml/min, 28°C and pH 4.5 (by titrating with 5 N NaOH) were maintained during the cultivation. The enzymes in the medium were separated from the yeast cells by centrifugation (15,000 g; 5 min) and both yeast cells and the supernatant were stored at 4°C until analysis, fl-Xylanase activity was determined by measuring the release of reducing sugars from 1.0% arabionxylan as substrate at 30°C (Leathers, 1986). PNPX, AAF and AE were assayed by measuring respectively the release of p-nitrophenol from the substrates p-nitrophenyl-fl-o-xyloside, p-nitrophenyl a-Larabinofuranoside and p-nitrophenyl acetate at 50, 60, or 55°C (MacKenzie et al., 1987). One unit of activity is defined as the release of one pmol ofp-nitrophenol or xylose per min. Cell-associated enzyme activity (with the exception of AE) was determined by permeabilizing washed

whole cells with toluene and sodium deoxycholate (Matlib et al., 1979) to facilitate the diffusion of the chromogenic substrate into the cells (Clarke, 1971). Protein assays of the supernatant were performed by the Coomassie Blue (Bradford, 1976) method with bovine serum albumin as standard. Cultivation of A. pullulans on 1% xylan resulted in the concomitant synthesis of fl-xylanase, PNPX, AAF and AE although the synthesis of AAF was somewhat delayed relative to the other enzymes (Fig. 1). This indicates that the synthesis of AAF may be dependent upon release of an inducer from xylan degradation. When grown on 1% xylose, fl-xylanase and PNPX but not AAF and AE were produced (Fig. 1). On both substrates, fl-xylanase was largely extracellular. On xylan, however, enzyme production continued for a considerably longer period than on xylose (Fig. 1A) and the maximum levels of volumetric and specific activity were significantly higher (Table 1). PNPX was largely cell-associated (Table 1). Irrespective of the substrate, the rate of enzyme production (Fig. 1B) and the maximum volumetric and specific activity levels in the culture fluid were similar (Table 1). Although AAF was mainly cell-accociated, significantly extracellular levels of this enzyme in comparison to PNPX were observed (Table 1). The maximum volumetric and specific activities of flxylanase attained when A. pullulans was cultivated at controlled pH in a fermentor on 2.5% xylan were somewhat lower than the levels observed in shake flasks on 1% xylan (Table 1) whereas the reverse was observed with PNPX. Cultivation on 2.5% xylose, however, led to considerably higher levels of fl-xylanase accumulation than in the case of 1% xylose in shake flasks and compared favourably with levels attained on 1 and 2.5%o xylan under similar conditions (Table 1). This improved production of ~-xylanase on 2.5% xylose may be ascribed to pH control in the fermentor as the low pH values observed in shake flask cultivation reduces the pH stability of p-xylanase (Myburgh et al., 1991). Maximum volumetric and specific activities of PNPX were also higher when cultivated on 2.5%o xylose than on 1% xylose. These results confirm and extend studies that show that A. pullulans secretes a fl-xylanase of high specific activity when cultivated on xylose and xylan (Leathers et al., 1984; Leathers, 1986; Leathers et al., 1986; Leathers, 1988). Only Aspergillus niger (Johnson et al., 1989), Trichoderma reesei (Poutanen et al., 1987; Suh et al. 1988) and a manipulated gene of Streptomyces lividans (Mondou et

* Corresponding author. 135

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FIG. 1. Production of extracellular (squares) and cell-associated (circles) fl-xylanase (A), p-nitrophenyl-xylosidase (B), a-L-arabinofuranosidase (C) and acetyl esterase (D) by A. pullulans NRRL Y 2311-1 on 1% (w/v) arabinoxylan (closed symbols) and on 1% (w/v) o-xylose (open symbols) in shake flasks.

al., 1986) are reported to produce a fl-xylanase with enzyme activities and specific activities of the same order as for A . pullulans. The ability of A . pullulans to produce P N P X , A A F and AE offers a number of advantages since complete degradation of hemicellulose generally requires the synergistic action of substituent-hydrolyzing enzymes in addition to fl-xylanase (Johnson et al., 1989). Although the activities and specific activities of P N P X , A A F and AE are considerably lower than fl.xylanase levels (Table 1), they compare favourably with values reported for other organisms. Only organisms such as T. reesei (Dekker, 1983; Poutanen et al., 1987; Poutanen and Sundberg, 1988),

Schizophyllum c o m m u n e (Biely et al., 1988) and Streptomyces flavogriseus (MacKenzie et al., 1987) have enzyme levels similar to or greater than those observed here. Considerable variation in the levels and inducibility of xylanhydrolyzing enzymes between organisms, between strains (Biely et al., 1988) and as a result of growth conditions (Suh et al., 1988) has been reported. The enzymes A A F and AE are apparently induced only by xylan, as A. pullulans failed to produce these enzymes when grown on xylose. The ability of other substrates to induce these enzymes was not tested. The greater levels of acetyl esterase produced on xylan compared to xylose contrasts with T. reesei and S. c o m m u n e where similar levels were found when these organisms when grown on glucose, xylose or acetyl xylan (Biely et al., 1988). The extracellular location of fl-xylanase and cell-association o f P N P X and A A F is consistent with previous descriptions of the xylan-hydrolyzing enzymes of microorganisms (Biely et al., 1980; Dekker, 1985). The exact cellular location of these enzymes in A. pullulans was not determined here. However, treatment o f the cells with toluene and deoxycholate to permeabilize the cells increased the enzyme activities compared to whole cell assays (unpublished data) thereby indicating activity of enzymes with an intracellular or periplasmic location. Wherther A. pullulans produces a complete range of enzymes necessary to hydrolyze xylan is uncertain. Recently enzymes such as 4-O-methylglucuronidase (Puls et al., 1987; Johnson et al., 1989) and ferulic acid esterase (MacKenzie and Bilous, 1988; Johnson et al., 1989) were also shown to be necessary to remove substituents of xylan. Furthermore the acetyl esterase activity measured here may have only restricted application in the removal of acetyl groups from xylan. Poutanen and Sundberg (1988) found that the purified acetyl esterase o f T. reesei liberated only limited amounts of acetic acid from acetylated xylooligomers consisting of more than ten monomers and they suggested that other esterases with higher substrate specificity to long acetylated xylooligomers may be present. Furthermore it is unclear whether A. pullulans produces optimal ratios of enzymes for synergistic hydrolysis of xylan. Deficiencies in the levels of side-group cleaving activities of xylanolytic enzyme preparations of various microorganisms leads to incomplete hydrolysis of substituted xylan (Poutanen et al., 1987). The production o f

TABLE 1. Maximum levels of hemicellulose-hydrolyzing enzymes in the extracellular fluid of A. pullulans NRRL Y 2311-1 Enzyme fl-Xylanase

p-Nitrophenyl-fl-xylosidase

a-L-Arabinofuranosidase d Acetyl esterase~

Substrates 1% xylana 2.5% xylanb 1% xylose 2.5o//00xylose 1% xylan 2.5% xylan 1% xylose 2.5% xylose 1% xylan I% xylose 1% xylan 1% xylose

a Cultivations on 1% substrate in shake flasks. b Cultivation on 2.5°~o substrate in a fermentor. ND, Not determined. d Enzyme levels were not determined on 2.5°//oosubstrate concentration.

Enzyme activity (U/ml)

Specific activity (U/mg)

% extracellular

82 68 15 46 0.06 0.44 0.04 0.11 0.17 0 1.35 0

1370 760 370 1000 1 4.9 1 2.4 2.8 0 22.5 0

78 NDc 68 ND 2.0 ND 3.6 ND 43 -ND --

VOL. 72, 1991

NOTES

these enzymes by A . p u l l u l a n s s h o u l d be o p t i m i z e d so that the culture filtrate can be used for efficient hydrolysis o f xylan. 12. This project was supported financially by the Foundation for Research Development, the University of the Orange Free State and SAPPI SAICCOR. P. J. du Toit is thanked for advice. REFERENCES 1. Biely, P., MacKenzie, C. R., and Schneider, H.: Production of acetyl esterase by Trichoderma reesei and Schizophyllum commune. Can. J. Microbiol., 34, 767-772 (1988). 2. Biely, P., Vrsanska, M., and Kratky', Z.: Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization. Eur. J. Biochem., 108, 313-321 (1980). 3. Bradford, M.: A rapid and Sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254 (1976). 4. Clarke, P . H . : Method for studying enzyme regulation. Meth. Microbiol., 6A, 269-326 (1971). 5. Dekker, R . F . H . : Bioconversion of hemicellulose: aspect of hemicellulase production by Trichoderma reesei QM 9414 and enzymic saccharification of hemicellulose. Biotechnol. Bioeng.,25, 1127-1146 (1983). 6. Dekker, R. F. H.: Biodegradation of hemiceliuloses, p. 505-533. In Higuchi, T. (ed.), Biosynthesis and biodegradation of wood components. Academic Press, New York (1985). 7. Johnson, K. G., Silva, M. C., MacKenzie, C. R., Schneider, H., and Fontana, J. D.: Microbial degradation of hemicellulosic materials. Appl. Biochem. Biotechnol., 21, 245-257 (1989). 8. Leathers, T.D.: Color variants of Aureobasidium pullulans overproduce xylanase with extremely high specific activity. Appl. Environ. Microbiol., 52, 1026-1030 (1986). 9. Leathers, T. D.: Amino acid composition and partial sequence of xylanase from Aureobasidium. Biotechnol. Lett., 10, 775-780 (1988). 10. Leathers, T.D., Detroy, R.W., and Bothast, R.J.: Induction and glucose repression of xylanase from a color variant strain of Aureobasidium pullulans. Biotechnol. Lett., 8, 867-872 (1986). 11. Leathers, T. D., Kurtzman, C. P., and Detroy, R. M.: Overpro-

13. 14.

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duction and regulation of xylanase in Aureobasidium pullulans and Cryptococcus albidus. Biotechnol. Bioeng. Symp., 14, 225240 (1984). Lee, H., To, R. J. B., Latta, R. K., Biely, P., and Schneider, H.: Some properties of extracellular acetytxylan esterase produced by the yeast Rhodotorula mucilaginosa. Appl. Environ. Micorbiol., 53, 2831-2834 (1987). MacKenzie, C.R. and Bilous, B.: Ferulic acid esterase activity from Schizophyllum commune. Appl. Environ. Microbiol., 54, 1170-1173 (1988). MacKenzie, C.R., Bilons, D., Schneider, H., and Johnson, K. G.: Induction of cellulolytic and xylanolytic enzyme systems in Streptomyces spp. Appl. Environ. Microbiol., 53, 2835-2839 (1987). Matlib, M. A., Shannon, W. A., and Srere, P. A.: Measurement of matrix enzyme activity in situ in isolated mitochondria made permeable with toluene. Meth. Enzym., 56, 544-550 (1979). Mondou, F., Shareck, F., Morosoli, R., and Kiuepfel, D.: Cloning of the xylanase gene of Streptomyces lividans. Gene, 49, 323329 (1986). Myburgh, J., Prior, B.A., and Kilian, S. G.: The temperature and pH properties of the extracellular hemicellulose-degrading enzymes of Aureobasidium pullulans. Process Biochem. Int. (submitted) (1991). Pou-Llinas, J. and Driguez, H.: D-Xylose as inducer of the xylan-degrading enzyme system in the yeast pullularia pullulans. Appl. Microbiol. Biotechnol., 27, 134-138 (1987). Poutanen, K., R~itt6, M., Puls, J., and Vaikari, L.: Evaluation of different microbial xylanolytic systems. J. Biotechnol., 6, 4960 (1987). Poutanen, K. and Sundberg, M.: An acetyl esterase of Trichoderma reesei and its role in hydrolysis of acetyl xylans. Appl. Microbiol. Biotechnol.,28, 419-424 (1988). Puls, J., Schmidt, O., and Granzow, C.: a-Glucuronidase in two microbial xylanolytic systems. Enz. Microbiol. Tchnol., 9, 83-88 (1987). Suh, H. D., Becker, T. C., Sands, J. A., and Montencourt, S. B.: Effects of temperature on xylanase secretion by Trichoderma reesei. Biotechnol. Bioeng., 32, 821-825 (1988). Wong, K. K. Y., Tan, L. U. L., and Saddler, J. N.: Multiplicity of fl-l,4-xylanase in microorganisms: functions and applications. Microbiol. Rev., 52, 305-317 (1988).