Summary
β-Xylosidase was obtained from Aureobasidium pullulans CBS 58475 with an activity of 0.35 units/ml culture filtrate. The production of the enzyme was strongly inducible. β-Xylosidase was purified in two steps by anion exchange and gel-permeation chromatography to high purity. The enzyme is a glycoprotein with an apparent molecular mass of 224 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and separates into two subunits of equal molecular mass. After SDS-PAGE β-xylosidase could be renatured and stained with methylumbelliferyl-β-xylopyranoside. The enzyme was able to split substrates of other glycosidases. The maximum activity was reached at pH 4.5 and 80° C. β-Xylosidase showed high stability over a broad pH range from pH 2.0 to 9.5 and up to 70° C. Analysis of cleavage patterns revealed that the enzyme was a typical glycosidase. Larger oligosaccharides consisting of xylose were degraded by an exomechanism together with a transxylosylation reaction.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aspinall GO (1980) Chemistry of cell wall polysaccharides. In: Preiss J (ed), The biochemistry of plants, vol 3. Carbohydrates: structure and function. Academic Press, New York, pp 473–500
Bachmann SL, McCarthy AJ (1989) Purification and characterization of a thermostable β-xylosidase from Thermomonospora fusca. J Gen Microbiol 135:293–299
Beldman G, Voragen AGJ, Rombouts FM, Searle L van, Pilnik W (1988) Specific and nonspecific glucanases from Trichoderma viride. Biotechnol Bioeng 31:160–167
Biely P (1985) Microbial xylanolytic systems. Trends Biotechnol 3:286–290
Biely P, MacKenzie CR, Puls J, Schneider H (1986) Cooperativity of esterases and xylanases in the enzymatic degradation of acetyl xylan. Bio/Technology 4:731–733
Biswas SR, Mishra AK, Nanda G (1987) Xylanases and β-xylosidases by Aspergillus ochraceus during growth on lignocelluloses. Biotechnol Bioeng 31:613–616
Buchert J, Puls J, Poutanen K (1989) The use of steamed hemicellulose as substrate in microbial conversions. Appl Biochem Biotechnol 20/21:309–318
Burnette WN (1981) Western blotting: electrophoretical transfer of proteins from sodium dodecyl sulphite-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203
Dekker RFH, Richards GN (1976) Hemicellulases: their occurrence, purification, properties and mode of action. Adv Carbohydr Chem Biochem 32:277–352
Dobberstein J, Emeis CC (1989) β-Xylanase of Aureobasidium pullulans CBS 58475. Appl Biotechnol Microbiol 32:262–268
Federici F, Miller MW, Petroccioli M (1989) Semicontinuous production of glucoamylase using immobilized growing cells of Aureobasidium pullulans. In: Martini A, Vaughan Martini A (eds) Proceedings of the 7th International Symposium on Yeasts, Perugia, Italy, 1.-5. 08. 1988. Yeast, Vol 5, Special Issue April 1989. Wiley; Chichester, New York, Brisbane, Toronto, Singapore, pp 175–179
Glass WF, Briggs RC, Hnilica LS (1981) Identification of tissue specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels. Science 211:70–72
Khan AW, Tremblay D, Le Duy A (1986) Assay of xylanase and xylosidase activities in bacterial and fungal cultures. Enzyme Microb Technol 8:373–377
Kitpreechavanich V, Hayashi M, Nagai S (1986) Purification and characterization of extracellular β-xylosidase and β-glucosidase from Aspergillus fumigatus. Agric Biol Chem 50:1703–1711
Laemmli U (1970) Cleavage of structural protein during assembly of the head of bacteriophage T4. Nature 227:680–685
Leathers TD (1986) Color variants of Aureobasidium pullulans overproduce xylanase with extremely high specific activity. Appl Environ Microbiol 52:1026–1030
Leathers TD, Kurtzman CP, Detroy RW (1984) Overproduction and regulation of xylanase in Aureobasidium pullulans and Cryptococcus albidus. Biotechnol Bioeng Symp 14:225–240
Lee SF, Forsberg CW, Rarray JB (1987a) Purification and characterization of two endoxylanases from Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 53:644–650
Lee SF, Forsberg CW (1987b) Isolation and some properties of a β-d-xylosidase from Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 53:651–654
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Magee RJ, Kosaric N (1985) Bioconversion of hemicelluloses. In: Fiechter A (ed) Advances in biochemical engineering/biotechnology 32: Agricultural feedstock and waste treatment and engineering. Springer, Berlin Heidelberg New York, pp 61–97
Matsuo M, Yasui T (1984a) Purification and some properties of β-xylosidase from Trichoderma viride. Agric Biol Chem 48:1845–1852
Matsuo M, Yasui T (1984b) Purification and some properties of β-xylosidase from Emericella nidulans. Agric Biol Chem 48:1853–1860
Matsuo M, Fujie A, Wim M, Yasui T (1987) Four types of β-xylosidases Penicillium wortmanni IFO 7237. Agric Biol Chem 51:2367–2379
Mc Neil B, Kristiansen B, Seviour RJ (1989) Polysaccharide production and morphology of Aureobasidium pullulans in continuous culture. Biotechnol Bioeng 33:1210–1212
Morrissey JH (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310
Mulchandani H, Luong JHT, Le Duy A (1989) Biosynthesis of pullulan using immobilized Aureobasidium pullulans cells. Biotechnol Bioeng 33:306–312
Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153:375–380
O'Brien M, Colwell RR (1987) A rapid test for chitinase activity that uses 4-methylumbelliferyl-N-acetyl-β-d-glucosamine. Appl Environ Microbiol 53:1718–1720
Ogata S, Muramatsu T, Kolbata A (1975) Fractionation of glycopeptides by affinity column chromatography on Concanavalin A-Sepharose. J Biochem 78:687–696
Pou-Llinas J, Driguez H (1987) d-Xylose as inducer of the xylan degrading enzyme system in the yeast Pullularia pullulans. Appl Microbiol Biotechnol 27:134–138
Poutanen K, Puls J (1988) Characteristics of Trichoderma reesei β-xylosidase and its role in the hydrolysis of solubilized xylans. Appl Microbiol Biotechnol 28:425–432
Poutanen K, Raettoe M, Puls J, Viikari L (1987) Evaluation of different microbial xylanolytic systems. J Biotechnol 6:49–60
Prasad GS, Prabakaran K, Dube HC (1987) Xylanase production by Fusarium F Sp udum. Curr Sci 56:830–831
Rodionova N, Tavobilov IM, Bezborodov AM (1983) β-Xylosidase from Aspergillus niger 15: Purification and properties. J Appl Biochem 5:300–312
Shinoyama H, Yasui T (1988) Superiority of Aspergillus niger β-xylosidase for the enzymatic synthesis of alkyl-β-xylosides in the presence of a variety of alcohols. Agric Biol Chem 52:2375–2377
Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23
Spiro RG (1966) Analysis of sugars found in glycoproteins. In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 8. Academic Press, New York, pp 3–26
Thomson NS (1983) Hemicellulose as biomass resource. In: Soltes EJ (ed) Wood and agricultural residues; research on use for feed, fuels and chemicals. Academic Press, New York, pp 101–119
Win M, Kaniyama Y, Matsuo M, Yasui T (1988) Transxylosylation products of xylobiose by β-xylosidase-1 from Penicillium wortmanni IFO 7237. Agric Biol Chem 52:1151–1158
Wong KKY, Tan LUL, Saddler JN (1988) Multiplicity of β-1.4-xylanase in microorganisms: functions and applications. Microbiol Rev 52:305–317
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dobberstein, J., Emeis, C.C. Purification and characterization of β-xylosidase from Aureobasidium pullulans . Appl Microbiol Biotechnol 35, 210–215 (1991). https://doi.org/10.1007/BF00184688
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00184688