Stabilization of New Imprint Property of Glucose Oxidase in Pure Aqueous Medium by Cross LinkedImprinting Approach

Alankar Vaidya and Lutz Fischer


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; P-d-glucose:oxygen l-oxidoreductase, E.C. 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 galac-tose-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.

Key Words: Tailor-made enzyme catalysis; glucose oxidase; protein imprinting; bioimprinting; biochemical protein engineering; CLIP technique.

1. Introduction

It is known that catalytic properties of enzymes such as stability and substrate/ enantio-selectivity could be modified by genetic engineering (1). This approach requires intricate multisteps such as identification and isolation of a particular gene, rational or random mutagenesis of the gene, expression of the recombinant gene in appropriate vector, and overexpression and screening of the recombinant enzyme using high-throughput assay. Not only are these steps laborious and time consuming, they also necessitate a lot of instruments and monetary investment. Hence, since 1990, intensive research efforts have been directed toward providing new alternatives for genetic engineering in order to develop better enzyme catalysts. Bioimprinting of enzymes is one of those alternatives that is simple to perform, less time consuming, and one of the cheapest chemical modification methods offering improved enzyme properties (2-4). The basic bioimprinting stratgy is outlined in Fig. 1.

Crude enzymes or proteins can be modified by bioimprinting to obtain tailor made enzymes or artificial antibodies. Bioimprinted enzymes that previously catalyzed a particular reaction is modified so as to demonstrate higher reaction rates in nonaqueous organic medium or to begin accepting the "stereo-counterpart" of the same substrate. This is illustrated in following examples: bioimprinting of a-chymotrypsin enhances selectivity during synthesis of an unusal substrate (i.e., D-form of ethyl ester of N-acetylated amino acids) (2), subtilisin lyophilized from aqueous solution in presence of its inhibitor N-acetyl-L-tyrosine amide demonstrates a 55-fold increase in transesterification reaction between N-acetyl-L-ala-nine methyl ester and propanol in anhydrous octane (5), and interfacial activation-based bioimprinting of lipases results in more than a two-magnitude increase in esterification and alcoholysis carried out in nonaqueous medium (3). Furthermore, the advantage of bioimprinting in modification of noncatalytic globular proteins (such as albumins) is to develop artificial antibodies, as exemplified in following: selective adsorbents of L-malic acid from bioimprinted bovine serum albumin recognizes a substrate with 10 times more selectivity in anhydrous organic solvents (6); and enzyme-like properties were induced in P-lactglobulin using N-isopropyl-4-nitrobenzyl-amine as an imprint molecule resulting in three times the enhanced P-elimination of 4-fluoro-4-[p-nitrophenyl] butane-2-one as compared to nonimprinted protein in dry acetonitrile (7).

Close examination of these examples, however, shows that the induced catalytic reactions performed by bioimprinted enzymes or proteins were carried out exclusively in nonaqueous dry organic solvents. This is the major flaw in the classical bioimprinting approach, within which the new memory (created in biomolecules during imprinting without stabilizing the modified conformation of protein) is erasable and sensitive to the water content of the medium (2,4,8). Furthermore, when a percentage of the water in the reaction medium increases it is concomitantly reflected in a decreased reaction rate. Increasing the water content of the medium results in greater flexibility and decreased rigidity of the modified active site of an enzyme, which apparently "erases" the imprint-induced confor-mational chages. Therefore, this chapter describes how to stabilize the bioimprinted memory in aqueous medium, the most common prevalent milieu for many enzymatic biochemical reactions.

As shown in Fig. 1, the formation of a complex between print molecule and enzyme takes place in aqueous solution. This complex is precipitated so that the transition state of the enzyme or protein bound to the print molecule is frozen in organic solvent. Thus, a modified imprinted active site is generated. Now, if this

Enzyme Protein

Aqueous medium

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