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

Abstract

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.

Preparation of Streptavidin-Coated Magnetic Nanoparticles for Specific Immobilization of Enzymes with High Activity and Enhanced Stability

“…In addition, pullulanase immobilized on magnetic chitosan beads retained 53.6% of its initial activity after a 6 h incubation period at 70 °C, while the free enzyme retained only 12.3% of its initial activity . Finally, pullulanase immobilized on alginate retained 75% of its activity after a 24 h incubation period at 60 °C, while free pullulanase was completely inactivated after a 5 h incubation period …”

Enzyme Kinetics by Isothermal Titration Calorimetry: Allostery, Inhibition, and Dynamics

“…When one considers the number of multi-substrate enzymes (Wang et al, 2019) and multiple enzyme systems with shared substrates or products (Murphy et al, 2010a), as well as the effects of allostery and substrate or product inhibition, the complexity of the experiment-space and versatility of ITC starts to become apparent. Finally, the ability of ITC to extract meaningful kinetic data from systems as complicated as biopolymer suspensions (Henzler et al, 2008;Ali et al, 2015) or even living cells (Wang et al, 2017(Wang et al, , 2018Zhang et al, 2018;Lv et al, 2019) holds great promise for understanding enzyme behavior in situ and in vivo. One could imagine expanding this approach to a multitude of other complex and heterogeneous media, such as purified cellular components, homogenized tissue or soil samples, and nanostructured materials, to name a few.…”

“…Other examples of opaque reaction mixtures are those involving insoluble substrates (Lonhienne et al, 2001;Murphy et al, 2010aMurphy et al, ,b, 2012Murphy et al, , 2013 or enzymes immobilized on insoluble matrices (Henzler et al, 2008;Henao-Escobar et al, 2014;Ali et al, 2015;Mason et al, 2018) where the components are combined as a suspension or slurry. These mixtures are of great industrial importance, in part because the insoluble carbohydrate polymers cellulose and chitin are the two most abundant organic compounds on earth, present in large quantities in vascular plants and arthropod exoskeletons, respectively (Berlemont et al, 2016).…”

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

“…Several compounds have been used for modification of epoxy groups in commercial supports aiming to evaluate different methods of immobilization. Each compound provides its own characteristics to the material, such as the crosslinking of glutaraldehyde; oxidation through acid solution action to produce carboxyl groups; activation of carboxyl groups with 1,1‐(3‐dimethylamino‐propyl)‐ethyl‐carbodiimide hydrochloride by the reaction of nucleophilic agents on protonated carbodiimide, under slightly acid conditions, with amino groups of proteins, and addition of amine groups using ethylenediamine . However, few manuscripts reported the modification of epoxy groups on Immobead 150, and to our knowledge, the use of acid solution has not yet been described.…”

“…Each compound provides its own characteristics to the material, such as the crosslinking of glutaraldehyde; oxidation through acid solution action to produce carboxyl groups; activation of carboxyl groups with 1,1-(3dimethylamino-propyl)-ethyl-carbodiimide hydrochloride by the reaction of nucleophilic agents on protonated carbodiimide, under slightly acid conditions, with amino groups of proteins, and addition of amine groups using ethylenediamine. [25][26][27][28][29][30] However, few manuscripts reported the modification of epoxy groups on Immobead 150, and to our knowledge, the use of acid solution has not yet been described. Moreover, we report for the first time the use of modified or unmodified Immobead 150 in b-galactosidase immobilization.…”