Technology for Regenerable Biosensor Probes Based on Enzyme‐Cellulose Binding Domain Conjugates

Abstract

The application 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 with the potential to address both of these difficulties is presented here. 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 electrode can be configured with a cellulose matrix onto which the enzyme-CBD conjugate can be repeatedly loaded (bound by the CBD) and subsequently eluted by perfusing the cellulose matrix with the appropriate solution. Glucose oxidase (GOx) conjugated to CBD with glutaraldehyde was used in an experimental glucose biosensor to demonstrate the feasibility of multiple cycles of loading and elution of the conjugate. Michaelis-Menten enzyme kinetics provided an empirical model for the calibration of the experimental biosensor. The development of a computer-controlled prototype glucose biosensor and a fermentation monitoring system based on this approach is discussed.

Carbohydrate Binding Modules: Biochemical Properties and Novel Applications

“…In order to optimize bioprocesses such as fermentation, it is very important to monitor the glucose levels. Phelps et al (156) addressed this problem based on the reversible immobilization of chemically conjugated CBM-glucose oxidase (CBM Cex from C. fimi), which can be repeatedly loaded onto a cellulose probe. Given that the binding is reversible, the sensor can be regenerated by replacing the originally bound enzyme with a fresh one (157,200).…”

DNA-Directed Immobilization: Efficient, Reversible, and Site-Selective Surface Binding of Proteins by Means of Covalent DNA–Streptavidin Conjugates

“…To extend the applicability of biomolecular elements, a general need for reversible and site-specific immobilization methods is apparent to allow fabrication of reusable bio-and immunosensor devices. Strategies for reversible immobilization of proteins (1) include reversible chemical interactions (2)(3)(4), in particular metal chelation (5)(6)(7)(8)(9) or disulfide cleavage (10 -15), protein-ligand interactions (16,17), and nucleic acid hybridization (18,19). Moreover, a number of efforts are currently underway to develop methods for siteselective immobilization of biomolecules on surfaces.…”

Monitoring of Submerged Bioprocesses

“…The development of in situ biosensors, although much desired, has been beset with practical problems related to difficulty in sterilization, recalibration, sample pretreatment, and maintenance of optimal operating conditions of the bio-component. Although attempts have been made to overcome these problems in specific cases (e.g., Phelps et al, 1994;de LumleyWoodyear et al, 1997;Rishpon et al, 1990), little has been reported on in situ applications (Bradley and Schmid, 1991, being the exception). However, most reports deal with applications ex situ in integration with FIA systems.…”