Monday, May 2, 2011
Grand Ballroom C-D, 2nd fl (Sheraton Seattle)
Enzymatic hydrolysis of cellulose often involves cellulases produced by Trichoderma reesei (T. reesei). Cellobiohydrolase1 (CBH1) is the most abundant cellulase generated by T. reesei (about 60% of total cellulases) and plays an important role in the hydrolysis of crystalline cellulose. A complete mechanism on how CBH1 interacts with cellulose is not yet clear in detail, so a method for separating sufficient quantities from the bulk cellulase cocktail is desirable for studies that aim to characterize binding and hydrolysis kinetics of CBH1 on substrate. In this project, CBH1 was separated from Spyzyme CP cellulases using ion-exchange columns and a protocol modified from a smaller scale process. The larger scale separation was performed by connecting several columns in parallel to a vacuum manifold system. With five columns running in parallel, a sum of about 68 mg of CBH1 was separated from 200 mg Spzyme CP during one separation. Due to the manifold system, step elution replaced the continuous gradient. After loading of CBH1 onto each 5 ml anion column, an amount of 10 ml of 0.1M, 15 ml of 0.25M, or 8 ml of 0.33M sodium chloride in 20 mM TEA-HCl pH 7.0 buffers were applied to elute the column successively. The purified CBH1 was collected in the third fraction. The separated CBH1 was examined by SDS-PAGE for its purity and showed a single band. It had a pNPC activity of 0.042 U/mg, similar to CBH1 (0.038 U/mg) separated using an FPLC system.