Michael K. Weibel

Immobilized enzymes: A prototype apparatus for oxidase enzymes in chemical analysis utilizing covalently bound glucose oxidase☆

Abstract A prototype analytical device incorporating an immobilized oxidase enzyme reactor is described. The monitor assembly involves the continuous measurement of dissolved oxygen in the sample stream and uses a commercially available Clark electrode mounted into a miniature vortex mixer. Utilizing glucose oxidase covalently coupled to porous glass particles a reagentless analytical procedure is presented for glucose in both simple and complex biological fluids. Operational limitations are described and characterized for both kinetic and endopoint methods of analysis.

Insolubilized coenzymes: the covalent coupling of enzymatically active NAD to glass surfaces.

Abstract NAD was attached to the surface of glass beads by a diazo coupling procedure. The insolubilized NAD was shown to function as a coenzyme in the yeast alcohol dehydrogenase reaction.

Biochemical fuel cells. Demonstration of an obligatory pathway involving an external circuit for the enzymatically catalyzed aerobic oxidation of glucose.

Abstract An in vitro biochemical fuel cell based upon the enzymatically catalyzed aerobic oxidation of glucose is described. The anodic half-reaction employs an electron transfer sequence consisting of the glucose oxidase reductive half-reaction and dichloroindophenol. The cathodic half-reaction involves reduction of molecular oxygen. A high Faradic efficiency for the intact cell approaching 100% has been experimentally demonstrated. The steady state current is exponentially related to the concentration of the terminal electron transfer species in the anodic chamber. The behavior is consistent with application of the Nernst relationship to define the cell potential and a simple resistance circuit. The discharge profile of the cell after complete oxidation of the primary fuel, glucose, can be modeled as a capacitor discharging through a resistor.

A coupled enzyme assay for aldose 1-epimerase

Abstract Aldose 1-epimerase (mutarotase) EC 5.1.3.3. catalyzes the mutarotation of selected pyranose sugars (1). The enzyme has been implicated as a component of the sugar transport system in kidney and intestine (2,3,4). Conventional analytical methods for monitoring catalytic activity involve relatively long, finite-interval polarimetric or spectrophotometric measurements of mutarotation rates employing α- d -glucose as the substrate (5). We have found this method to be somewhat cumbersome and time consuming, as α- d -glucose solutions spontaneously epimerize at rates requiring their individual preparation for each experiment. We report here a kinetic assay method for aldose 1-epimerase based upon fast in situ generation of α- d -glucose employing hydrolysis of sucrose by β-fructofuranosidase and a subsequent reporter reaction involving the aerobic oxidation of β- d -glucose via glucose oxidase. Analytical monitoring of the rate limiting epimerization step in the three-enzyme system is achieved by measurement of oxygen depletion in solution employing a conventional Clark electrode assembly.

Application of Immobilized Enzymes to Chemical Analysis

Considerable interest in the analytical applications of immobilized enzymes is focused upon the development of reagentless methods involving redox enzymes. Our operational definition of a reagentless enzyme based analytical method implies only the conservation of catalytic components such as enzymes and, if needed, coenzymes. Substrate acceptors and coenzyme regeneration substrates, which are readily incorporated into the carrier buffer as stable and inexpensive components, are considered endogenous to the sample carrier solution.