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.

The purpose of this study was to develop suitable matrix-type transdermal drug delivery systems of Ketotifen fumarate (KF) as antiasthmatic drugs. Chitosan-alginate polyelectrolyte complex (PEC) films were used as drug release regulators for KF. Antihistaminic films with variable PEC compositions were prepared using different ratios of chitosan (CTS) to sodium alginate (ALG). Propylene glycol (PG) was used as plasticizer; Tween 80 (T) and Span 20 (S) were used as permeability enhancers. Nine formulations were obtained by film casting method and characterized in terms of weight uniformity, thickness, folding endurance, moisture lost, and moisture absorption. In addition, drug release and permeation through rat abdominal skin mounted in Franz cell were investigated. All formulations were found to be suitable in terms of physicochemical characteristics, and there was no significant interaction between the used drug and polymers. It was noticed that when T is used as permeation enhancer, a satisfactory drug release pattern was found where 99.88% of drug was released and an amount of 2.121 mg/cm of KF was permeated after 24 h. For the optimal formulation, a permeability coefficient of 14.00 ± 0.001 cm h and a latency time of 0.35 ± 0.02 h were found. The in-vitro analysis showed controlled release profile which was fitted by Korsmeyer-Peppas model (R = 0.998). The obtained results suggested that new controlled release transdermal formulations of asthmatic drugs could be suitably designed as an alternative to the common forms.

Pullulanase from Klebsiella pneumoniae was entrapped into calcium alginate beads. Its activity was estimated by the determination of number-average molar masses using two different methods: a colorimetric assay of reducing ends (REs) and a size-exclusion chromatography/multiangle light scattering/differential refractive index. The second method also provided weight-average molar masses of hydrolyzed pullulan and the quantity of maltotriose (DP3) and its multiples (DP6 and DP9) produced by the enzymatic treatment. The alginate beads showed a good retention of the loaded pullulanase (30%), and the system showed a downturn of hydrolysis kinetics in comparison with free pullulanase due to the limiting access of substrate-enzyme. On the contrary with the results obtained from free enzyme hydrolysis, for which a large distribution of pullulan fragments is observed during the treatment, the immobilized enzyme system has evidenced, during the enzymatic treatment, the coexistence of native or only slightly degraded pullulan chains together with maltotriose units. Complete hydrolysis of pullulan chains was achieved once diffused into the gel.