Enzymes Involved in the Utilization of the Galactomannan, Guar Gum, by Bacteroides Ovatus

122 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces an (alpha)-galactosidase (I) which is different from the (alpha)-galactosidase (II) it produces when it is grown on galactose, melibiose, raffinose or stachyose. I have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9-6.4 for I, 6.3-6.5 for II). However, (alpha)-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000 whereas (alpha)-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. Alpha-galactosidase I has a lower affinity for melibiose, raffinose and stachyose (K(,m) values of 20.8 mM, 98.1 mM and 8.5 mM, respectively) than (alpha)-galactosidase II (K(,m) value of 2.3 mM, 5.9 mM and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase.Bacteroides ovatus, when it utilizes guar gum, also produces two cell-associated galactomannanases. Cell-associated galactomannanase activity was inducible by guar gum. Approximately 30% of the total cell-associated galactomannanase activity partitioned with cell membranes. Seventy percent of this membrane-associated activity was associated with the outer membrane fraction. The enzyme activity was released when membrane proteins were treated with 3- 3 cholamido-propyl) dimethylammonio -1-propane sulfonate (CHAPS) or with Triton X-100 at a detergent to protein ratio of 1.0. The CHAPS-solubilized galactomannanase was partially purified by chromatography on a FPLC Mono Q column. The enzyme has a pI of 6.9. 60-70% of the cell-associated galactomannanase activity was soluble. I have purified this enzyme from the soluble fraction. The soluble galactomannanase has a pI of 4.9-5.0 and a monomeric molecular weight of 61,000. This enzyme appears to be different from the outer membrane-associated galactomannanase.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD