Abstract
Bacteroides ovatus, a human colonic anaerobe, utilizes the galactomannan guar gum as a sole source of carbohydrate. Previously, we found that none of the galactomannan-degrading enzymes were extracellular, and we characterized an outer membrane mannanase which hydrolyzes the backbone of guar gum to produce large fragments. We report here the purification and characterization of a second mannanase from B. ovatus. This enzyme is cell-associated and soluble. Using ion-exchange chromatography, gel filtration, and chromatofocusing steps, we have purified the soluble mannanase to apparent homogeneity. The enzyme has a native molecular weight of 190,000 and a monomeric molecular weight of 61,000. It is distinct from the membrane mannanase not only with respect to cellular location but also with respect to stability and isoelectric point (pI of 6.9 for the membrane mannanase and pI of 4.8 for the soluble mannanase). The soluble mannanase, like the membrane mannanase, hydrolyzed guar gum to produce large fragments rather than monosaccharides. However, if galactosyl side chains were removed from the galactomannan fragments by alpha-galactosidase, both the soluble mannanase and the membrane mannanase could degrade guar gum to monosaccharides. Thus either or both of these two enzymes, working together with alpha-galactosidase, appear to be sufficient for the breakdown of guar gum to the level of monosaccharides.
Full text
PDF![2038](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c318/212083/bbdb884d8034/jbacter00195-0266.png)
![2039](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c318/212083/d8ad0f153473/jbacter00195-0267.png)
![2040](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c318/212083/36679563531c/jbacter00195-0268.png)
![2041](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c318/212083/82767e1e9faf/jbacter00195-0269.png)
![2042](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c318/212083/2ef57ac4e484/jbacter00195-0270.png)
![2043](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c318/212083/491cdb2174e9/jbacter00195-0271.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Dygert S., Li L. H., Florida D., Thoma J. A. Determination of reducing sugar with improved precision. Anal Biochem. 1965 Dec;13(3):367–374. doi: 10.1016/0003-2697(65)90327-1. [DOI] [PubMed] [Google Scholar]
- Gherardini F. C., Salyers A. A. Characterization of an outer membrane mannanase from Bacteroides ovatus. J Bacteriol. 1987 May;169(5):2031–2037. doi: 10.1128/jb.169.5.2031-2037.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gherardini F., Babcock M., Salyers A. A. Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus. J Bacteriol. 1985 Feb;161(2):500–506. doi: 10.1128/jb.161.2.500-506.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kranz R. G., Gennis R. B. Isoelectric focusing and crossed immunoelectrophoresis of heme proteins in the Escherichia coli cytoplasmic membrane. J Bacteriol. 1982 Apr;150(1):36–45. doi: 10.1128/jb.150.1.36-45.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
- Salyers A. A., O'Brien M. Cellular location of enzymes involved in chondroitin sulfate breakdown by Bacteroides thetaiotaomicron. J Bacteriol. 1980 Aug;143(2):772–780. doi: 10.1128/jb.143.2.772-780.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]