A novel efficient enzyme-immobilization reaction on NH2 polymers by means of L-ascorbic acid

Citation
J. Tiller et al., A novel efficient enzyme-immobilization reaction on NH2 polymers by means of L-ascorbic acid, BIOT APP B, 30, 1999, pp. 155-162
Citations number
22
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology","Biochemistry & Biophysics
Journal title
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
ISSN journal
0885-4513 → ACNP
Volume
30
Year of publication
1999
Part
2
Pages
155 - 162
Database
ISI
SICI code
0885-4513(199910)30:<155:ANEERO>2.0.ZU;2-N
Abstract
A new enzyme-immobilization reaction by means of L-ascorbic acid (ASA) is d escribed using NH2 polymers based on cellulose or poly(vinyl alcohol) with the example of oxidoreductase enzymes. In this way, enzyme proteins such as glucose oxidase (GOD), glutamate oxidase, lactate oxidase, urate oxidase a nd peroxidase can be covalently fixed with a high surface loading to ultrat hin and transparent NH2-polymer films if their surfaces are previously trea ted with an ASA solution, in, for example, N,N-dimethyl acetamide, DMSO or methanol, ASA then obviously reacts like a diketo compound with amino group s of the NH2-polymer film and enzyme protein, forming dehydroascorbic acid derivatives with neighbouring Schiff's-base structures. In a subsequent fra gmentation reaction, the latter presumably form stable oxalic acid diamide derivatives as coupling structures between enzyme protein and NH2-polymer f ilm, as suggested by results from investigations of the ASA reaction with n -butylamine. The immobilized enzymes can be stored at 4 degrees C in bidist illed water for at least 1 month without becoming detached from the NH2-pol ymer film and without diminished enzyme activity. The apparent K-m values o f the immobilized enzymes are in part clearly smaller than those of the dis solved enzymes or those found in other immobilization processes such as the diazo coupling or the bifunctional glutardialdehyde reaction. For example, the K-m value of the immobilized GOD with different NH2 polymers as the ma trix structure is smaller by a factor of approx. 20 than that of the dissol ved enzyme.