Kinetic parameters of β-fructofuranosidase from Scopulariopsis brevicaulis

Abstract

β-Fructofuranosidase was purified to homogeneity by a series of column chromatographies on Butyl Toyopearl 650M, DEAE-Sephadex A-50, Toyopearl HW-55F, and hydroxyapatite with a yield of 4.6% and 1,100-fold purification from the cell extract of Scopulariopsis brevicaulis N-01. The activity was optimal at 40°C and pH 6–9. The enzyme was a homologous dimer protein and the molecular weight of a subunit was 110,000. Kinetic studies were carried out using the purified β-fructofuranosidase. The K m and V max values for the formation of 1-kestose, nystose, fructosylxyloside, and difructosylxyloside from sucrose, 1-kestose, sucrose and xylose, and fructosylxyloside were respectively determined. Sucrose inhibited nystose formation from 1-kestose, and glucose inhibited the formation of 1-kestose, fructosylxyloside, and difructosylxyloside competitively. The kinetic constants for the hydrolyses of all the above oligosaccharides were determined at lower concentrations, because at higher concentrations the hydrolyses were inhibited. Taking into the consideration the kinetic studies, the preferential production of 1-kestose over nystose and of fructosylxyloside over 1-kestose and difructosylxyloside in fermentations by S. brevicaulis are discussed.