A novel efficient enzyme-immobilization reaction on NH2 polymers by means of L-ascorbic acid.
A new enzyme-immobilization reaction by means of L-ascorbic acid (ASA) is described using NH(2) 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 NH(2)-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 NH(2)-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 NH(2)-polymer film, as suggested by results from investigations of the ASA reaction with n-butylamine. The immobilized enzymes can be stored at 4 degrees C in bidistilled water for at least 1 month without becoming detached from the NH(2)-polymer film and without diminished enzyme activity. The apparent K(m) 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 K(m) value of the immobilized GOD with different NH(2) polymers as the matrix structure is smaller by a factor of approx. 20 than that of the dissolved enzyme.
Tiller
J
J
Forschungszentrum Jülich GmbH, ZEL, D-52425 Jülich, Germany.
Berlin
P
P
Klemm
D
D
D000081:Acetamides / Q000737:chemistry; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D001205:Ascorbic Acid / Q000737:chemistry*; D002082:Butylamines / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D005949:Glucose Oxidase / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry*
D000081:Acetamides; D002082:Butylamines; D004800:Enzymes, Immobilized; D011108:Polymers; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D000594:Amino Acid Oxidoreductases; C023061:L-glutamate oxidase; C013959:dimethylacetamide; C032462:n-butylamine; D001205:Ascorbic Acid