Purification of two immunologically distinct endoglucanases without affinity for microcrystalline cellulose from Cellulomonas sp. ATCC 21399.
Two endoglucanases (endoglucanase B and endoglucanase C) without affinity for cellulose were purified from the culture broth of Cellulomonas sp. ATCC 21399 using gelfiltration and ion exchange chromatography. Fused rocket immunoelectrophoresis was used to select the fractions with the highest content of endoglucanase and lowest content of contaminating proteins. The endoglucanases were purified to immunological homogeneity. In addition both endoglucanases were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (molecular weights of endoglucanase B and endoglucanase C were 67000 and 25000, respectively). Endoglucanase B was homogeneous when studied by isoelectric focusing showing one protein band at pl 4.3. Both endoglucanases lacked activity against microcrystalline cellulose (Avicel) and showed similar endo action on carboxymethylcellulose (CMC). Endoglucanase B had a high specific activity against CMC, H(3)PO(4)-swollen Avicel and xylan, but showed no activity against galactomannan. In contrast, endoglucanase C showed activity against both CMC, xylan, and galactomannan all being polysaccharide substrates linked with beta-1-4-D-glucoside bonds. The specific activity of endoglucanase C against H(3)PO(4)-swollen Avicel was low.
Poulsen
O M
OM
Institute of Plant Physiology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353, Copenhagen K, Denmark.
Petersen
L W
LW