Stabilization of Activity of Oxidoreductases by Their Immobilization onto Special Functionalized Glass and Novel Aminocellulose Film Using Different Coupling Reagents

Abstract

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°C or after treatment at 65°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°C and 92% after heat treatment at 65°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.

Preparation of Translucent and Flexible Human Hair Protein Films and Their Properties

“…the immobilization of biopolymers and bioactive substances, [2][3][4] the improvement of enzyme stability, [4][5][6] and drug-delivery systems. 7) Various proteins including human hair and wool keratin, [8][9][10] collagen, 7) fibrin, 11) silk fibroin 12) and their mixtures 13,14) have been utilized as the starting materials of protein films.…”

Covalent Immobilization of Glucose Oxidase on Well-Defined Polymer-Silicon Wafer Hybrids via Surface-Initiated Raft-Mediated Process

“…For example, the stability of the immobilized enzyme via a 1,3-bifunctional aromatic coupling agent was reduced, which may results from the conformation deformation of the active enzyme and the loss in the multipoint binding ability of the enzyme. 37,38 As presented in this work, such a problem can be avoided to a large extent through the direct coupling between nucleophilic -NH 2 groups of GOD with the epoxide groups of PGMA.…”

Immobilization of Hansenula polymorpha Alcohol Oxidase for Alcohol Biosensor Applications

“…Activation of DEAE-cellulose with cyanuric chloride. The DEAE-cellulose was activated by slightly modified method proposed by Tiller et al 18 The pre-swollen DEAE-cellulose was washed and suspended with distilled. The suspended solution was saturated by sodium hydroxide.…”