Molecular size characterization and kinetics studies on hydrolysis of pullulan by pullulanase in an entangled alginate medium.

The behavior of a hydrolytic enzyme (pullulanase) toward its substrate (pullulan) in the presence of a nonsubstrate (alginate), both below and above the critical entanglement concentration (C*), was studied. The hydrolysis kinetics were studied with the enzyme and alginate concentrations varied using two main methods: a colorimetric assay of the reducing extremities (RE), which allowed the number-average molar masses (Mn) of the oligosaccharides to be determined, and size exclusion chromatography with on-line, multiangle light scattering, viscometer, and differential refractive index detectors, which allowed the average molar masses, Mn and Mw, of the oligosaccharides during hydrolysis to be determined. Free pullulanase acts via an "endo" process. The presence of alginate slows the hydrolysis kinetics, particularly when the alginate concentration is greater than the C*. These results were confirmed by the evolution of the kinetic parameters (KM, Vmax) obtained via isothermal titration calorimetry (ITC). The amount of oligosaccharides produced is not dependent on the alginate concentration, and the endo enzyme behavior is not modified by the entanglement in the medium. These observations were also confirmed by ITC analysis in the presence of degraded alginate (without entanglement). Our results correlated with the substrate diffusion in entangled media. The pullulanase reaction in the presence of alginate is shown to be diffusion-dependent.

Covalent immobilization of pullulanase on alginate and study of its hydrolysis of pullulan.

The immobilization of pullulanase from Klebsiella pneumoniae by grafting was investigated. Pullulanase was linked after activation of alginate via a covalent bond between the amine groups of the enzyme and the carboxylic acid groups of alginate. The immobilization yield was 60%. The activity of free pullulanase and immobilized pullulanase was followed by the quantification of reducing ends by colorimetric assay and the determination of the molar masses of the hydrolyzed pullulan by SEC/MALS/DRI. Compared to free pullulanase, the kinetics is largely slowed. The evolution of the weight average molar mass of pullulan leading to high production of shorter oligosaccharides during hydrolysis is not the same as that obtained with free enzyme. Immobilized pullulanase retained 75% and 30% of its initial activity after 24 h and 14 days of incubation at 60°C, respectively while free pullulanase lost its activity after 5 h of hydrolysis at the same temperature. The kinetic parameters of immobilized pullulanase were also investigated by isothermal titration calorimetry (ITC). The affinity of immobilized enzyme to its substrate was reduced compared to the free pullulanase due to steric hindrance and chemical links.

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.