Sunday, April 29, 2007

A high-throughput assay to measure cellulase binding and synergism in ternary mixtures

Navaneetha Santhanam and Larry P. Walker. Department of Biological and Environmental Engineering, Cornell University, B79 Riley Robb Hall, Ithaca, NY 14850

A rapid high-throughput cellulase binding assay using microwell plates was developed to quantify bound fractions of fluorescently labeled Thermobifida fusca cellulases Cel5A, Cel6B and Cel9A alone or in synergistic mixtures.  These cellulases were labeled with Alexa Fluor 594, Alexa Fluor 350 and Alexa Fluor 488, respectively, without losses in activity on bacterial micro-crystalline cellulose. Controlled experiments were conducted (1) to ascertain whether individual labeled cellulases can be accurately quantified using microwell plates; (2) to investigate whether the fluorescent signal of one labeled cellulase can be reliably filtered from the signals of other labeled cellulases in a ternary mixture to accurately quantify individual cellulases; (3) to verify the thermostability of fluorescent signals of the labeled cellulases; and (4) to assess cooperative or competitive cellulase binding in ternary mixtures.  Experiments clearly demonstrated that the microwell plate reliably yielded accurate measurements of cellulase concentrations in single cellulase reactions or when in the presence of other labeled cellulases.  In addition, the fluorescent signals remained stable at 50°C over the entire 4h 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 than what was observed when these cellulases were reacted with cellulose alone.  A major conclusion drawn from this research is that a strong linear relationship exists between DSE and the molar ratio of Cel6B-AF350 in ternary mixtures, with DSE increasing in proportion to the increase in exocellulase mole fraction.