Characterization of microbial inulases

Abstract

Three forms of inulase were purified from $\underline{Aspergillus}$ $\underline{niger}$ by ultrafiltration, DEAE-Trisacryl chromatography, Sephadex G-150 gel filtration, and preparative isoelectric focusing. The isoelectric focusing resolved inulase into three peaks with isoelectric points of 4.5, 4.9 and 5.2. Among them, peaks II and III were purified to homogeneity as a single band on an analytical isoelectric focusing gel. These peaks represented glycoproteins with similar molecular weights, about $3×10^5$ and appeared to consist of four identical subunits. On the basis of molecular weight, subunit, amino acid analysis, chemical modification, and fluorescence spectrum, the overall molecular characteristics of the three forms were similar. However, the three forms exhibited distinctly different kinetic constants. The enzyme splits β-2, 1-linkage of sucrose and straight chain oligoand polyfructosides of the inulin type, by endwise removal of fructosyl units. A exo-inulase (β-D-fructofuranoside fructohydrolase, EC 3.2.1. 26) from $\underline{Bacillus}$ $\underline{subtilis}$ was partially purified, and its mode of action and general properties were studied. The enzyme had an apparent molecular weight of 49,000 as estimated by gel filtration. Its isoelectric point was 5.2. The enzyme was acid-labile, and had a optimum pH of 6.6. The optimum temperature was 50℃. Kinetic studies showed an apparent Km of 18 mM for sucrose and 25 mM for raffinose. The enzyme degraded sucrose and chain oligoand polyfructosides of the inulin type by endwise removal of fructosyl units.