Stabilization of activity of oxidoreductases by their immobilization onto special functionalized glass and novel aminocellulose film using different coupling reagents.
Glucose oxidase (GOD), horseradish peroxidase (HRP), and lactate oxidase (LOD) were covalently immobilized on special NH(2)-functionalized glass and on a novel NH(2)-cellulose film via 13 different coupling reagents. The properties of these immobilized enzymes, such as activity, storage stability, and thermostability, are strongly dependent on the coupling reagent. For example, GOD immobilized by cyanuric chloride on the NH(2)-cellulose film loses approximately half of its immobilized activity after 30 days of storage at 4 degrees C or after treatment at 65 degrees C for 30 min. In contrast, GOD immobilized by L-ascorbic acid onto the same NH(2)-cellulose film retains 90% of its initial activity after 1 year of storage at 4 degrees C and 92% after heat treatment at 65 degrees C for 30 min. Unlike GOD, in the case of LOD only immobilization on special NH(2)-functionalized glass, e.g., via cyanuric chloride, led to a stabilization of the enzyme activity in comparison to the native enzyme. The operational stability of immobilized HRP was up to 40 times higher than that of the native enzyme if coupling to the new NH(2)-cellulose film led to an amide or sulfonamide bond. Regarding the kinetics of the immobilized enzymes, the coupling reagent plays a minor role for the enzyme substrate affinity, which is characterized by the apparent Michaelis constant (K(M,app)). The NH(2)-functionalized support material as well as the immobilized density of the protein and/or immobilized activity has a strong influence on the K(M,app) value. In all cases, K(M,app) decreases with increasing immobilized enzyme protein density and particularly drastically for GOD.
Tiller
Joerg C
JC
Forschungszentrum Jülich GmbH, ZEL, D-52425 Jülich, Germany.
Rieseler
Raino
R
Berlin
Peter
P
Klemm
Dieter
D
D001426:Bacterial Proteins / Q000378:metabolism; D002482:Cellulose; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004800:Enzymes, Immobilized / Q000187:drug effects / Q000378:metabolism; D005898:Glass; D005949:Glucose Oxidase / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010088:Oxidoreductases / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D013995:Time; D014227:Triazines / Q000737:chemistry / Q000494:pharmacology
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D014227:Triazines; C019309:cyanuric chloride; D002482:Cellulose; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; C021497:lactate 2-monooxygenase