Features of Fibrobacter intestinalis DR7 mutant which is impaired with its ability to adhere to cellulose

A spontaneous adhesion-defective mutant (DR7-M) of Fibrobacter intestinalis DR7 was isolated which was capable of growing on glucose and cellobiose, but impaired in its capacity to degrade cellulose. Levels of enzyme activities were determined in solubilized fractions of DR7 and DR7-M. Total endoglucanases and xylanase activity values of parent DR7 fractions were 2.84 and 1.85 folds higher than those of the mutant, and were distributed mainly in the bacterial envelope fractions, with some activity also found in the extracellular fluid. Ln a separate assay, measurement of the enzymatic activity bound to cellulose showed that a portion of the endoglucanase activity bound to cellulose while most xylanase activity did not bind. Notwithstanding, the wild type DR7 cells had 26-fold higher total activities of cellulose-degrading enzymes than the mutant, and 96% of its activity was exclusively located in outer membrane and periplasm fractions. Ln the mutant, the lower cellulose degrading enzymes activity was located only in the extracellular fluid. Most of the cellulose degrading enzymes activity of DR7 had the capability to bind to cellulose. SDS-page electrophoresis of outer membrane and periplasm cell fractions showed that DR7 and DR7-M possess similar molecular weight (MW) profiles but different quantities of 16 cellulose-binding-proteins (CBPs) in the MW range of 36 up to 225 kDa. Zymogram analysis with soluble substrates, either carboxymethylcellulose or soluble xylan, following SDS-page of DR7 and DR7-M fractions, suggested that CBPs of approximate MW 120, 110, 100, 90, 70 and 40 kDa have endoglucanase activity, and that CBPs of all fractions lack any xylanase activity. (C) 1998 Academic Press.