Book title: Immobilization of Enzymes and Cells
Editors: Guisan JM
Publisher: Humana Press
Series title: Methods in Biotechnology
Volume number: 22
Abstract: Bioimprinting is a well-known strategy to manipulate the catalytic properties of the enzymes. However, the lack of expression of the newly acquired imprinted property by an enzyme in aqueous surrounding is the chief limitation that restricts the application of this technique. In this chapter we used a combinatorial approach which amalgamates the benefits offered by traditional cross-link-immobilization technique (rigidity and stability) and bioimprinting approach (induced tailor-made catalytic property). This contemporary method is termed cross-linked-imprinting (CLIP). In order to extend the range of its application, glucose oxidase (GO; ß-D-glucose:oxygen L-oxidoreductase, E.C. 22.214.171.124) was selected as a biochemically demanding enzyme (FAD-depending, tetrameric glycoprotein). Galactose, a competitive inhibitor of GO was taken as a template molecule during imprinting. It was demonstrated that the induced imprinted memory created for specific galactose -GO interactions was preserved during precipitation and the resultant modified active site was "frozen" by covalent cross-linking carried out in organic solvent. In separate following experiments, it was established that CLIP-GO not only binds galactose as a conventional ligand, but catalyzes the oxidation of galactose to galactono-1,4-lactone in aqueous medium which was not the case without CLIP method.
Template and target information: Bioimprinting
Author keywords: Tailor-made enzyme catalysis, glucose oxidase, protein imprinting, bioimprinting, biochemical protein engineering, CLIP technique