Purification and biochemical properties of a thermostable xylose-tolerant beta- D-xylosidase from Scytalidium thermophilum.
The thermophilic fungus Scytalidium thermophilum produced large amounts of periplasmic beta- D-xylosidase activity when grown on xylan as carbon source. The presence of glucose in the fresh culture medium drastically reduced the level of beta- D-xylosidase activity, while cycloheximide prevented induction of the enzyme by xylan. The mycelial beta-xylosidase induced by xylan was purified using a procedure that included heating at 50 degrees C, ammonium sulfate fractioning (30-75%), and chromatography on Sephadex G-100 and DEAE-Sephadex A-50. The purified beta- D-xylosidase is a monomer with an estimated molecular mass of 45 kDa (SDS-PAGE) or 38 kDa (gel filtration). The enzyme is a neutral protein (pI 7.1), with a carbohydrate content of 12% and optima of temperature and pH of 60 degrees C and 5.0, respectively. beta- D-Xylosidase activity is strongly stimulated and protected against heat inactivation by calcium ions. In the absence of substrate, the enzyme is stable for 1 h at 60 degrees C and has half-lives of 11 and 30 min at 65 degrees C in the absence or presence of calcium, respectively. The purified beta- D-xylosidase hydrolyzed p-nitrophenol-beta- D-xylopyranoside and p-nitrophenol-beta- D-glucopyranoside, exhibiting apparent K(m) and V(max) values of 1.3 mM, 88 micromol min(-1) protein(-1) and 0.5 mM, 20 micromol min(-1) protein(-1), respectively. The purified enzyme hydrolyzed xylobiose, xylotriose, and xylotetraose, and is therefore a true beta- D-xylosidase. Enzyme activity was completely insensitive to xylose, which inhibits most beta-xylosidases, at concentrations up to 200 mM. Its thermal stability and high xylose tolerance qualify this enzyme for industrial applications. The high tolerance of S. thermophilum beta-xylosidase to xylose inhibition is a positive characteristic that distinguishes this enzyme from all others described in the literature.
Zanoelo
Fabiana Fonseca
FF
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brasil.
Polizeli Md
Maria de Lourdes Teixeira de Moraes
Mde L
Terenzi
Héctor Francisco
HF
Jorge
João Atílio
JA
D001203:Ascomycota / Q000201:enzymology* / Q000254:growth & development; D003470:Culture Media; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007700:Kinetics; D025282:Mycelium / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D003470:Culture Media; D014990:Xylans; D014994:Xylose; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase