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Biotechnology and Applied Biochemistry (1999) 30, (155–162) (Printed in Great Britain)
A novel efficient enzyme-immobilization reaction on NH2 polymers by means of L-ascorbic acid
Jörg Tiller*,2, Peter Berlin*1 and Dieter Klemm†
*Forschungszentrum Jülich GmbH, ZEL, D-52425 Jülich, Germany, and †Institut für Organische Chemie und Makromolekulare Chemie, Friedrich-Schiller-Universität, Humboldtstraße 10, D-07743 Jena, Germany
Abbreviations used: ABTS, 2,2´-azino bis-(3-ethylbenzothiazoline-6-sulphonic acid); ASA, L-ascorbic acid; DMA, N,N-dimethyl acetamide; DS, degree of substitution; LOD, lactate oxidase; GOD, glucose oxidase; GlOD, glutamate oxidase; PVA, poly(vinyl alcohol); POD, horseradish peroxidase; UOD, urate oxidase; PDA cellulose tosylate, 6-deoxy-6-(4-aminophenyl)amino-2(3)-O-tosylcellulose; AH cellulose carbanilate, 6-deoxy-6-(6-aminohexyl)amino cellulose carbanilate; PDA-PVA tosylate, deoxy-(4-aminophenyl)amino O-tosyl poly(vinyl alcohol). 1 To whom correspondence should be addressed. 2 Present address: Institut für Organische Chemie und Makromolekulare Chemie, Friedrich-Schiller-Universität, Humboldstraße 10, D-07743 Jena, Germany. 
A new enzyme-immobilization reaction by means of L-ascorbic acid (ASA) is described 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 and peroxidase can be covalently fixed with a high surface loading to ultrathin and transparent NH2-polymer films if their surfaces are previously treated with an ASA solution, in, for example, N,N-dimethyl acetamide, DMSO or methanol. ASA then obviously reacts like a diketo compound with amino groups of the NH2-polymer film and enzyme protein, forming dehydroascorbic acid derivatives with neighbouring Schiff's-base structures. In a subsequent fragmentation reaction, the latter presumably form stable oxalic acid diamide derivatives as coupling structures between enzyme protein and NH2-polymer film, as suggested by results from investigations of the ASA reaction with n-butylamine. The immobilized enzymes can be stored at 4 °C in bidistilled water for at least 1 month without becoming detached from the NH2-polymer film and without diminished enzyme activity. The apparent Km values of the immobilized enzymes are in part clearly smaller than those of the dissolved enzymes or those found in other immobilization processes such as the diazo coupling or the bifunctional glutardialdehyde reaction. For example, the Km value of the immobilized GOD with different NH2 polymers as the matrix structure is smaller by a factor of approx. 20 than that of the dissolved enzyme.
Received 6 April 1999/21 June 1999; accepted 30 June 1999
Portland Press Ltd © 1999 |
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