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.
Normandie Université, France.|Laboratoire Polymères Biopolymères Surfaces, Université De Rouen, Mont Saint Aignan, F, 76821, France.|CNRS UMR 6270 & Fr3038, Mont Saint Aignan, F, 76821, France.
Ghina
G
Ali
Normandie Université, France.|Laboratoire Polymères Biopolymères Surfaces, Université De Rouen, Mont Saint Aignan, F, 76821, France.|CNRS UMR 6270 & Fr3038, Mont Saint Aignan, F, 76821, France.
Virginie
V
Dulong
Normandie Université, France.|Laboratoire Polymères Biopolymères Surfaces, Université De Rouen, Mont Saint Aignan, F, 76821, France.|CNRS UMR 6270 & Fr3038, Mont Saint Aignan, F, 76821, France.
Sarah N
SN
Gasmi
Normandie Université, France.|Laboratoire Polymères Biopolymères Surfaces, Université De Rouen, Mont Saint Aignan, F, 76821, France.|CNRS UMR 6270 & Fr3038, Mont Saint Aignan, F, 76821, France.
Christophe
C
Rihouey
Normandie Université, France.|Laboratoire Polymères Biopolymères Surfaces, Université De Rouen, Mont Saint Aignan, F, 76821, France.|CNRS UMR 6270 & Fr3038, Mont Saint Aignan, F, 76821, France.
Luc
L
Picton
Normandie Université, France.|Laboratoire Polymères Biopolymères Surfaces, Université De Rouen, Mont Saint Aignan, F, 76821, France.|CNRS UMR 6270 & Fr3038, Mont Saint Aignan, F, 76821, France.
Didier
D
Le Cerf
D000464:Alginates / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D019149:Bioreactors; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D005936:Glucans / Q000737:chemistry / Q000378:metabolism*; D020723:Glucuronic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D006603:Hexuronic Acids / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007711:Klebsiella pneumoniae / Q000201:enzymology; D013696:Temperature
D000464:Alginates; D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D005936:Glucans; D006603:Hexuronic Acids; D020723:Glucuronic Acid; C009109:pullulan; D006026:Glycoside Hydrolases; C100181:pullulanase
alginate; hydrolysis; immobilized enzyme; pullulanase