Kinetic behaviour and specificity of β-fructosidases in the hydrolysis of plant and microbial fructans

Abstract

Fructosidases, in particular exo-β-fructosidases, may act on fructans such as inulins and levans of plant and bacterial origin to produce fructose. In this paper, the kinetic properties of a commercial preparation (Fructozyme L) and a recombinant exoinulinase (BfrA) from Thermotoga maritima, were studied using fructan polymer substrates from various sources. Both enzymatic preparations preferentially hydrolyzed β2-1 linkages and low molecular weight fructans. We show that chicory inulin is degraded most efficiently by both preparations, followed by bacterial inulin, in spite of its high molecular weight and branching in β2-6 positions. All bacterial levans were more slowly hydrolyzed. Michaelis–Menten kinetics describe the hydrolysis of sucrose and low molecular weight fructans (≤8.3 kDa) by both enzyme preparations, while first order kinetics were observed with respect to bacterial fructans due to the high molecular weight and, therefore, low molar concentrations. Comparison of second order rate constants indicates that bacterial inulin ( Leuconostoc citreum CW28) is hydrolyzed more slowly with both enzyme preparations than chicory inulin by approximately one order of magnitude. For Leuconostoc mesenteroides NRRL B-512F levan, the second order rate constant for Fructozyme L is 200-fold lower than for chicory inulin. However, the second order rate constant for BfrA is only 22-fold lower than for chicory inulin. Taken together, our studies characterize the kinetics of fructan hydrolysis and also suggest that the kinetic parameters may be used to differentiate between fructan structures.