UBC Theses and Dissertations
Development of a regenerable glucose biosensor probe for bioprocess monitoring Phelps, Michael R.
The implementation and commercialization of enzyme-based biosensors for on-line bioprocess monitoring and control has been slowed by problems relating to the in situ sterilizability of the probe and the stability of the enzyme component. A novel technology is presented here which addresses both of these difficulties. The approach is based on the reversible immobilization of enzymes conjugated with the cellulose binding domain (CBD)of cellulases from Cellulomonas fimi. A regenerable biosensor probe is configured with a cellulose matrix onto which the solubilized enzyme-CBD conjugate can be repeatedly loaded (via the attachment of the CBD) and subsequently eluted by perfusing the cellulose matrix with the appropriate loading or eluting solution. The chemical conjugation of the enzyme glucose oxidase (GOx) with CBD by glutaraldehyde is described. The GOx-CBD conjugate retained the enzymatic activity of the glucose oxidase and the binding affinity of the CBD. The GOx-CBD conjugate was used in an experimental glucose biosensor based on a platinum rotating disk electrode fitted with a cellulose immobilization matrix to demonstrate the feasibility of multiple cycles of loading and elution of the conjugate and to develop suitable protocols and reagents for the loading and elution procedures. A prototype glucose biosensor and reagent flow system were designed and built for use in fermentation monitoring. A custom-designed membrane system consisting of a sterilizable, glucose-permeable outer Nafion membrane for the sensor and a cellulose acetate coating on the indicating electrode was developed for use in a microbial fermentation. The prototype glucose biosensor was used successfully to monitor medium glucose concentration for 16.5 continuous hours during a 20 L fed-batch cultivation of E. coli in minimal medium. Michaelis-Menten enzyme kinetics were used as an empirical model for the calibration of the experimental biosensor. The development of a computer-controlled prototype glucose biosensor and fermentation monitoring system is discussed. These results are the first to demonstrate the concept, feasibility, and utility of are generable biosensor based on reversible immobilization of the enzyme using CBD technology and represent a significant step toward better instrumentation for fermentation monitoring and control.
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