Purification and characterization of xylan-degrading enzyme from Tricholoma matsutake

Abstract

Tricholoma matsutake NBRC 30605 shows xylan-degrading activity when cultured in xylan-supplemented medium. In the present study, we purified a xylan-degrading enzyme to homogeneity. The molecular mass of the enzyme was found to be 81 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 88 kDa by size-exclusion chromatography. The purified enzyme showed maximum activity at a temperature of 55℃ and pH 4.0. The enzyme was completely stable at temperatures up to 30℃ and at pH 3.0 － 4.0. Km and Vmax values of the purified enzyme against p-nitrophenyl β-D-xylopyranoside were 1.28 mM and 14.45 U/mg, respectively. The enzyme released xylose from β-1,4-linked xylo-oligosaccharides with chain lengths of 2 to 5 and beechwood xylan. Treatment with 10 mM MnCl2 (29%), ZnCl2 (28%), NiCl2 (29%) and with 5 mM EDTA (15%) strongly inhibited the activity of the purified enzyme. The N-terminal amino acid sequence of the enzyme was identical to that of the deduced amino acid sequence of T. matsutake β-xylosidase. These findings indicate that the purified enzyme is a xylan-degrading β-xylosidase belonging to the glycosyl hydrolase family 3.