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Title:Investigation of Ruminococcus flavefaciens FD-1 cellulase
Author(s):Doerner, Kinchel Clay
Doctoral Committee Chair(s):Mackie, Roderick I.
Department / Program:Animal Sciences
Discipline:Animal Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Microbiology
Agriculture, Animal Culture and Nutrition
Abstract:The cellulase system of the anaerobic bacterium Ruminococcus flavefaciens FD-1 was investigated. Through the use of ion-exchange chromatography and non-denaturing polyacrylamide gel electrophoresis, crude cellulase was found to contain no fewer than 18 endo-$\beta$-1,4-glucanase components and present in two cellulase complexes. No synergistic effect on $\sp{14}$C-cellulose degradation was observed when these two cellulase complexes were incubated together or when the cellulase complexes were incubated together or separately in the presence of an exo-$\beta$-1,4-glucanase produced by this organism (Exoglucanase A). Both cellulase complexes degraded xylan and the xylanase components migrated coincidently with all endo-$\beta$-1,4-glucanase components present in both cellulase complexes.
A monoclonal antibody (MAb S1) generated against Exoglucanase A was used to investigate cellulolysis by R. flavefaciens. MAb S1 recognized few components in crude cellulase demonstrating its specificity for Exoglucanase A. MAb S1 was used to investigate the cellular location of the Exoglucanase A. Thin-sectioning of R. flavefaciens followed by immunolabelling, and examination using a transmission electron microscope showed a non-specific interaction of the monoclonal antibody for the embedding resin. Although, various treatments were tried to prevent this interaction, no conclusions about the cellular location of Exoglucanase A could be drawn. MAb S1 also inhibited the action of Exoglucanase A in vitro but failed to inhibit growth of R. flavefaciens when added to culture medium. This suggests the action of Exoglucanase A is not necessary for growth or the antibody concentration in the medium was too low to affect growth.
Nutritional factors affecting cellulase expression in R. flavefaciens were also investigated. Cellobiose-grown cells produced more endo-$\beta$-1,4-glucanase and $\sp{14}$C-cellulase, but an equal amount of exo-$\beta$-1,4-glucanase compared to cellulose-grown cells. Growth on cellobiose or cellulose did not affect cellular location of either endo-$\beta$-1,4-glucanase or exo-$\beta$-1,4-glucanase, however cellulose-grown cells exhibited higher $\sp{14}$C-cellulase levels present in the supernatant as opposed to the cell pellet. Furthermore, addition of cellulose to a growing culture using cellobiose did not increase cellulase production compared to a control with no addition. Cellulase was constitutively produced in cellobiose- and cellulose-grown R. flavefaciens cells.
Issue Date:1992
Rights Information:Copyright 1992 Doerner, Kinchel Clay
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9215803
OCLC Identifier:(UMI)AAI9215803

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