Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Cellulosic substrates are hydrolysed by the concerted action of three main enzyme components: endoglucanases (EnG), exoglucanases (ExG) and β-glucosidases (βG). For decades, the activity of these enzymes has been measured by IUPAC methods, applying various substrates such as Whatman # 1 filter paper (WFP), carboxymethylcellulose and p-nitrophenyl glucoside. Unfortunately, these substrates are not able to express the potential of a given cellulase preparation nor the actual synergy between enzyme components. However, WFP may be used as a single substrate for the determination of these enzyme activities if the total amount of reducing sugar release (RStot) is split into insoluble (RSinsol) and soluble (RSsol) reducing sugars, both produced after 60min of incubation. Hence, calculation of EnG activity would be based on RSinsol, ExG on RSsol in the presence of gluconolactone (a potent βG inhibitor) and βG on the difference between these two RS measurements. In this work, the activity of commercial cellulases was characterized based on this method, using 70mg of WFP in 2.0mL of the enzyme in 100mmol/L acetate buffer, pH 4.8. Quantification of RS was always based on the DNS method and critical results were also checked by HPLC. The values obtained for these three main activities were complementary to each other, providing a better view of the enzyme performance as well as the synergy among different enzyme components. Kinetic models were also developed based on these data, showing different levels of competitive, uncompetitive, linear mixed and noncompetitive inhibitions for each class of these hydrolytic enzymes.