A covalent two-step immobilization technique using itaconic anhydride |
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Authors: | L Fischer F Peißker |
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Institution: | (1) Institute of Biochemistry and Biotechnology, Technical University of Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany Tel.: +49 531 3915730 Fax: +49 531 3915763 e-mail: L.Fischer@tu-bs.de, DE |
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Abstract: | β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds
were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser
extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity
and the K
m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically
with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability
at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit
immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation
in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately
50% more chloramphenicol β-glucoside was obtained after 5 h.
Received: 22 September 1997 / Accepted: 28 October 1997 |
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