|Maglione, Giuseppe - CORNELL UNIVERSITY|
|Wilson, David - CORNELL UNIVERSITY|
Submitted to: Journal of Applied & Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 3, 1996
Publication Date: N/A
Interpretive Summary: Providing adequate dietary sources of energy is a major cost of meat and milk production. Ruminant farm animals (cattle, sheep, goats) harbor cellulolytic (cellulose-digesting) bacteria in their gastrointestinal tract. This symbiotic relationship allows ruminants to utilize dietary cellulose, a major component of many animal feeds. There have been many attempts to purify cellulases (cellulose-digesting enzymes) to a crystalline state. Access to crystalline cellulase would provide a means of cloning cellulase gene(s) which, in turn, could lead to methods of increasing the efficiency of cellulose digestion. Fibrobacter succinogenes is one of the most active cellulolytic bacteria in the rumen. We found, in our experiments with this bacterium, that cellulases were very sensitive to feedback inhibition, a process whereby end products (sugars) of cellulose digestion bind to the enzyme and either reduce or block subsequent cellulase activity. Since only growing cells can remove sugar from the active site of the enzyme, it is difficult to monitor cellulase activity once the cells have been disrupted. This has added to the difficulty of purifying cellulase to its crystalline state and is a barrier to cloning of the enzyme. This information could contribute to development of methods for improving cellulose digestion by ruminants, thereby increasing the efficiency with which feeds are utilized for meat and milk production.
Technical Abstract: Fibrobacter succinogenes cells grown on either cellobiose or cellulose digested cellulose at a rapid rate, but nitrogen- or 2- methylbutyrate - limited cultures stopped digesting cellulose as soon as growth ceased. Extracts of cells growing on cellulose had carboxymethylcellulase but not crystalline cellulase activity. Cells that had been growing exponentially on cellulose initiated cellulose digestion immediately, and cellulose- dependent succinate production could be used as an index of crystalline cellulase activity. The rate of cellulose digestion was linear if the time was less than 60 min and the cell concentration was less than 150 ug protein/ml. Cells that were pre-incubated for a short time with thiocellobiose lost their ability to digest cellulose, and thiocellobiose was a competitive inhibitor. Since the Ki of thiocellobiose was only 0.2 mg/ml, and extracellular cellobiose did not accumulate, the crystalline cellulase of F. Succinogenes appears to be very sensitive to feedback inhibition. Cellulose sources bound different amounts of congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red (250 ug/ml) did not inhibit the growth of F. Succinogenes S85 grown on cellobiose, but this concentration of congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver Burk plot of ball-milled cellulose digestion rate versus the cellulose concentration indicated that congo red was a competitive inhibitor of cellulose digestion (Ki was 250 ug/ml). Congo red binding capacity was highly correlated with the Vmax values of cellulose digestion and inversely related to Km.