Monday, May 4, 2009
5-48
Determination of Product Inhibition of CBH1, CBH2 and EG1 Using a Novel Cellulase Activity Assay
Faye Du, Erin Wolger, Thijs Kaper, and Brad Kelemen. R&D, Genencor, A Danisco Division, 925 Page Mill Road, Palo Alto, CA 94304
Along with other factors, product inhibition limits lignocellulosic biomass hydrolysis. For this reason, we are interested in engineering our cellulases to resist product inhibition. The direct measurement of product is difficult in the presence of large amounts of added product. Instead, we have developed a method of measuring a decrease in substrate, taking advantage of the fluorescent properties of a calcoflour dye. The selected calcoflour dye has greater fluorescence when in the presence of intact cellulose. The hydrolysis of phosphoric acid swollen cellulose by endoglucanases and cellobiohydrolases results in a decrease in fluorescence of the calcoflour dye. We find that, while cellobiohydrolase 1 is strongly inhibited by cellobiose, cellobiohydrolase 2 and endoglucanase 1 are not significantly inhibited.