Determination of the Number-Average Degree of Polymerization of Cellodextrins and Cellulose with Application to Enzymatic Hydrolysis

Abstract

A rapid and accurate method for determining the number-average degree of polymerization (DP(n)) was established for insoluble cellulose and soluble cellodextrins as the ratio of glucosyl monomer concentration determined by the phenol-sulfuric acid method divided by the reducing-end concentration determined by a modified 2,2'-bicinchoninate (BCA) method. The modified BCA method, featuring incubation at 75 degrees C for 30 min, did not result in beta-glucosidic bond cleavage, whereas substantial cleavage was observed at higher temperature. Solubilization of insoluble cellulose in cold phosphoric acid prior to measurement of the reducing-end concentration by the BCA method was found not to be necessary for several model celluloses such as microcrystalline cellulose, but such solubilization was required for large fibers of cellulose such as Whatman No. 1 filter paper. The phenol-sulfuric acid method can be used for measuring the glucosyl monomer concentration of soluble cellodextrins, and also for insoluble cellulose if preceded by a liquefaction step. Standard deviations of < or =2% were obtained for both reducing and glucosyl monomer determination and of < or =3% for overall determination of DP. By use of the reported method, hydrolysis of phosphoric acid-swollen cellulose (PASC) by the Trichoderma reesei cellulase system was shown to result in a rapid decrease in DP as hydrolysis proceeded. By contrast, the DP of Avicel remained nearly constant during hydrolysis. The specific enzymatic cellulose hydrolysis rate is 100-fold higher for PASC as compared to Avicel.