A High-Throughput Assay to Measure Cellulase Binding and Synergism in Ternary Mixtures

Abstract

A rapid, high-throughput cellulase binding assay using microwell plates was developed to quantify cellulose-bound fractions of fluorescently labeled Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A alone or in ternary mixtures. These cellulases were labeled with Alexa Fluor 594, Alexa Fluor 350, and Alexa Fluor 488, respectively, without losses in activity on bacterial microcrystalline cellulose. Controlled experiments were conducted (1) to ascertain whether individual labeled cellulase species could be accurately quantified using 96-well microwell plates; (2) to investigate whether the fluorescence emission of one labeled cellulase species could be reliably distinguished from the fluorescence emissions of other labeled cellulases in ternary mixtures to accurately quantify individual cellulases; (3) to verify the thermostability of the fluorescence of labeled cellulases; and (4) to assess cooperative or competitive cellulase binding in ternary mixtures. Experiments demonstrated that microwell plates yielded accurate measurements of cellulase concentrations in single cellulase reactions as well as in multi-cellulase mixtures. In addition, fluorescence remained stable at 50°C over the entire 4 h time course of the experiments. This high-throughput measurement system also revealed 13% greater binding for Cel6B-AF350 and 11% lower binding for Cel9A-AF488 in ternary mixtures than was observed when these cellulases individually reacted with cellulose.