| Summary: | 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-β-l,4-glucanase
A (CenA), endo-β-l,4-glucanase B (CenB), and exo-β-l,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
(pMMBpulAxenB) 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 (pMMBpulAxenB) 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.
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