Effect of carboxymethyl groups on degradation of modified pullulan by pullulanase from Klebsiella pneumoniae.

Pullulanase is an enzyme that hydrolyses the α-1,6 linkages of pullulan (Pull) to produce maltotriose units. We studied the capacity of pullulanase to cleave its modified substrate: carboxymethylpullulan (CMPull), synthesized with two different degrees of substitution (DS=0.16 and 0.8). Size exclusion chromatography with on line multi angle light scattering and differential refractive index detection (SEC/MALS/DRI) was used to estimate both number and weight average molar masses, respectively, Mn and Mw, of pullulan and CMPulls together with the percentage of maltotriose formed during hydrolysis. Determination of reduced sugars gave also a Mn that is compared to data obtained by SEC. It revealed that CMPull is partially degraded by pullulanase and the rate of hydrolysis decreased with increased DS. At the end of the hydrolysis, Mn is decreased by a factor of 23 and 1.7 for CMPull with a DS of 0.16 and 0.8 respectively. The percentage of produced maltotriose decreased also when increasing DS (24% and 7% for CMPull DS 0.16 and 0.8 respectively). The kinetic properties of pullulanase were also investigated with Pull and CMPulls by isothermal titration calorimetry (ITC) using simple injection method. Based on Michaelis-Menten kinetics, Vmax (maximal velocity) decreased and KM (Michaelis constant) increased when DS of modified pullulan CMPull increased.