| The objective of this research was to evaluate the potential of using the Klebsiella oxytoca secreted pullulanase (PulA) as a determinant for secretion of Cellulomonas fimi cellulases as fusion proteins. Fusion proteins were created between PulA from K. oxytoca and each of the following C. fimi cellulose hydrolyzing enzymes: endo-beta-1,4-glucanase A (CenA), endo-beta-1,4-glucanase B (CenB), and exo-beta-1,4-glucanase (Cex). The fusion proteins PulA:CenA, PulA:CenB, and PulA:Cex were produced and found to retain cellulolytic activity when expressed in Escherichia coli cells. Because cellulose hydrolysis by CenB presented the greatest applied potential for degrading cellulose to cellobiose at the outset of my thesis research, PulA:CenB was studied in more detail. Several plasmid systems were tested for expression of the pulA:cenB fusion gene in K. oxytoca and the expression vector pMMB207, which allows for IPTG-regulated expression from the tac promoter, was determined to be the most appropriate. Expression of pulA:cenB led to the accumulation of 17 mg/L of the chimeric PulA:CenB within cultures of K. oxytoca (pMMBpulA:cenB). A portion (8%) of the total chimeric protein produced was located within the membrane-enriched fraction and shown to be exposed to the extracellular medium by immunolocalization, with ca. 6--16% of cell-associated CMCase activity located at the cell surface. This amount of cell-surface exposed PulA:CenB was not sufficient to allow growth on cellulose. Mutagenesis of K. oxytoca (pMMBpulA:cenB) cells with MNNG, followed by selection for growth on cellulose as sole carbon source was done, but mutants able to grow on cellulose were not isolated. Additionally, expression of PulA:CenB in K. oxytoca (pMMBpulA:cenB) cultures grown in a minimal medium resulted in a triphasic growth pattern in this strain, suggesting an abnormality in PulA:CenB membrane translocation that interfered with cell growth. |
