The use of capillary viscometry, reducing end-group analysis, and size exclusion chromatography combined with multi-angle laser light scattering to characterize endo-1,4-β-D-glucanases on carboxymethylcellulose :: A comparative evaluation of the three methods

Three methods were used to characterize seven purified endo-1,4-beta-D-glucanases derived from different microbial sources (Trichoderma reesei, Thermomonospora fusca, and Acidothermus cellulolyticus) on carboxymethylcellulose (CMC). The methods included capillary viscometry, reducing end-group analysis, and high performance size exclusion chromatography combined with multi-angle laser light scattering (HPSEC-MALLS). The investigation,vas performed with the objective of comparative evaluation of the different methods for characterizing endoglucanases, particularly in relation to their endo- and exomode of action. The measurement of the initial rate of reducing end-group formation using disodium 2,2'-bicinchoninate (BCA) was found to be the must accurate method for determination of endoglucanase activity. The BCA method was highly sensitive, simple to perform, and directly gave the number of bonds broken, thus allowing for expression of endoglucanase activity in international units (mu mol of beta-1,4-glucosidic bonds hydrolyzed in I min during the initial period of hydrolysis). The viscometric method was simple to perform and highly sensitive for the internal bonds cleaved bur did not account for the hydrolysis of CMC near the chain end and thus only allowed for expression of endoglucanase activity in arbitrary viscometric units. The HPSEC-MALLS technique provided the number-average molecular weight (M-n) of CMC, thus allowing the quantification of the number of the bonds broken during degradation of CMC; however, reproducibility of the method was low, especially for the high-molecular weight fragments of CMC at the beginning of hydrolysis. As hydrolysis proceeded to the more advanced stages, the HPSEC-MALLS method gave an overestimated (compared to the reducing end-group analysis) values for M-n, probably due to insufficient sensitivity of the light-scattering detector for the low-molecular weight products of CMC degradation. The combined use of the three methods allowed characterization of endoglucanases according to their selectivity for hydrolysis of internal bonds within a CMC molecule which was expressed as the ratio of the initial rate of viscosity decrease to the initial rate of glucosidic bonds broken. This ratio was found to be unique for each endoglucanase, and therefore, no universal equation could be established for all endoglucanases for conversion of arbitrary viscometric units to international units of activity. (C) 1998 Elsevier Science Inc.