A glucose oxidase peroxidase-coupled continuous assay protocol for the determination of cellulase activity in the laboratory: the Abuajah method

Abstract

This present study reports a novel laboratory protocol, the glucose oxidase peroxidase (GOPOD)-coupled continuous assay (GcCA) which is based on the general principle that all cellulases generate β-d-glucose as one of their hydrolytic products. Resultantly, the β-d-glucose generated serves as a substrate to an auxiliary enzyme, GOPOD, coupled to the cellulase hydrolytic process, in a simultaneous secondary reaction which continuously quantifies the concentration of glucose generated as a measure of incremental cellulase activity in the laboratory. The protocol is initial hydrolytic rate-driven. Thus, making it very suitable for kinetic studies of pure cellulase samples in the laboratory. GcCA protocol is simple, rapid, specific, highly sensitive and accurate, non-laborious and saves cost in terms of time and materials. It is robust and dynamic and could be modified or adapted to suit either standard, 1000 μL cuvette or 96-well microtiter plate formats. Its optimized total assay reaction mixture of 500 μL constitutes a substrate, enzyme, buffer, distilled water and coupled GOPOD components. The GcCA protocol was validated for linearity, applicability, sensitivity, precision and compared with a standard and widely used assay method for optimized enzyme activity. It was further compared with the most widely used routine assay procedure for determination of reducing sugars, the DNS method. The GcCA protocol is a high throughput technique which enables the assay of over 120 samples in one day.