| In this study, the biochemical characteristics of several hyperthermophilic, exo-acting, glycosyl hydrolases were investigated. The first component of this research focused on the galactomannan-degrading enzymes from Thermotoga neapolitana. In addition to determining that this organism readily grows on galactomannan-based media, the cellular localization of three related enzymes was investigated as a function of growth phase. This pattern of cellular localization was consistent with amino acid sequence information from the corresponding genes in T. neapolitana and for similar enzymes-in a related organism of the same genus, Thermotoga maritima .;The next component of this work focused on the biochemical characterization of recombinant versions of the T. neapolitana alpha-galactosidase and beta-mannosidase, expressed in Escherichia coli. The recombinant alpha-galactosidases from the closely-related T. neapolitana and T. maritima were characterized biochemically to determine how the biochemical features of this enzyme type compared for closely related hyperthermophiles. Results showed that, although the proteins are greater than 84% identical (92% similar) at the amino acid sequence level, the two alpha-galactosidases had different pH optima and kinetic characteristics.;The final phase of the research focused on identifying or creating alpha-galactosidases with higher levels of thermostability and thermoactivity than the Thermotoga versions of this enzyme. However, no putative versions of this enzyme were apparent from genome database searches of hyperthermophiles with higher growth temperature optima than Thermotoga spp. Alternatively, enzymes of related specificity were identified as potential candidates for molecular evolution. The most promising candidate was found to be an alpha-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus, an organism which has a growth Topt of 98°C as compared to 80°C for the Thermotoga spp.;The alpha-glucosidase gene from P. furiosus was subjected to one round of DNA shuffling. Of the 12 colonies, 3 colonies had increased alpha-galactosidase activity concomitant with decreased alpha-glucosidase activity. Although SDS-PAGE showed the shuffled gene expressing proteins with molecular masses different from the control, most colonies still maintained at least partial activity. This preliminary study, therefore, showed DNA shuffling to be a viable option for creating enzymes with novel enzyme activities. (Abstract shortened by UMI.)... |
