Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
There are a variety of inconclusive comparisons of the effectiveness of pretreatment methods ability to enhance enzymatic hydrolysis. Determining the reducing ends on a cellulose surface accessible to exocellulases, such as Cel7A, provides a direct measure of cellulose reactivity. Especially useful to understanding a substrate’s susceptibility to enzymatic digestion is differentiating between productively bound and non-productively bound enzymes on a cellulose surface. To that end, we use Cel7A enzymes as molecular probes for interrogating cellulose surfaces. How much total enzyme a substrate will adsorb is a reasonable, but imperfect, predictor of hydrolysis rate. The catalytic domain must find a reducing end of the cellulose chain and pull it from the crystal face to form a catalytically active, threaded complex. We developed a novel assay to differentiate between adsorbed and threaded enzyme populations and couple it with well-characterized model cellulose substrates including a variety of polymorphs and range of crystallinities. Competitive inhibition between cellulose substrate and soluble species must be overcome to reliably distinguish between threaded and non-productively bound Cel7A. Once the relative activities are deconvoluted, the relative populations of threaded Cel7A correlate with predicted free energies of decrystallization for different crystalline polymorphs.