Purification and properties of a thermostable inulinase (beta-D-fructan fructohydrolase) from Bacillus stearothermophilus KP 1289

A thermophilic soil isolate, Bacillus stearothermophilus KP1289, that grew from 41¦C to 69¦C, produced extracellular inulinases in the presence of inulin. One (inulinase II) of these enzymes was purified to homogeneity. The molecular weight M(-r) and the isoelectric point of the enzyme were estimated as 54,000 and 5.0, respectively The enzyme was active between 30 and 75¦C and at pH 4.5-8.6 with an optimum at 60¦C and pH 6.1. At 69¦C and pH 7.0 the half-life of the enzyme was 10 min. The enzyme released fructose exo-wise from the non-reducing end of inulin (M-r = 4,5000). The Michaelis constant, catalytic center activity, and specificity constant for inulin at 60¦C and pH 5.0 were 80 mM (360 mg/mL), 460 s(-1), and 5.8 s(-1) mM(-1), respectively. The ratio of specificity constants for inulin, sucrose, and raffinose was 1:0:50.0.16. The enzyme was classified as a thermophilic thermostable beta-D-fructan fructohydrolase.