Studies of kinetic property of inulinase using fructo-oligosaccharides as substrate

Abstract

The substrate specificities of exoinulinase (2,1-¥â-D-fructan fructohydrolase, E.C. 3.2.1.80) from Bacillus sp. snu-7 (ExIB) on various fructooligosaccharides( degree of polymerization: n = 2-7) were investigated by steadystate kinetic analysis at 37 ¡É and pH 7.5. We observed that the degree of polymerization affected the Km and kcat values of exoinulinase. The subsite affinities Ai (subsite number: i = 1-7) in the active site of ExIB were as follows: A1, -0.18 kcal/mole; A2, 1.39 kcal/mole; A3, 0.136 kcal/mole; A4, 0.026 kcal/mole; A5, 0.040 kcal/mole; A6, 0.033 kcal/mole; A7, 0.085 kcal/mole, and the intrinsic rate constant, kint, was 671 min-1. Therefore, the subsite number 2 and 3, having large positive Ai values, was the most effective for the binding of small FOS to the active site. With respect to inulin with an average n = 30, its discrepancy between the calculated and observed rate parameters exceeded the experimental error, suggesting the participation of the non-productive complexes between ExIB and inulin.