Purification and characterization of two low-molecular weight endoglucanases of Cellulomonas flavigena

Abstract

Two isoforms of endoglucanases of Cellulomonas flavigena were purified to homogeneity after a series of purification steps including precipitation with 80% acetone, gel filtration, ion-exchange chromatography, and nondenaturing gradient 5–20% preparative polyacrylamide gel electrophoresis. Each of the endoglucanases, CM-Cellulase 1 and CM-Cellulase 2, appeared as a single band on SDS-PAGE and had an apparent molecular weight of 20,400. Both of the endoglucanases were comparable to the substrate-bound endoglucanases and the recombinant DNA-derived endoglucanases of C. flavigena expressed in Escherichia coli. The two isoforms of endoglucanase, CM-Cellulase 1 and CM-Cellulase 2, were different in their mobilities on native nondenaturing 5–20% gradient PAGE. The purified endoglucanases displayed significantly similar characteristics, including pH optima (6.5 and 7) and temperature optima (50°C). The purified CM-Cellulases were unable to hydrolyze Avicel, xylan, and filter paper and were only active against CMC. The purified proteins had a K m value of 0.78 g l −1. Heavy metal ions like Ag 2+ and Fe 2+ inactivated the enzymes. NaCl and CaCl 2 had no effect on enzyme activities. β-Mercaptoethanol also inhibited enzyme activity, possibly due to the involvement of sulfhydryl groups .