Geobacillus thermoleovorans GE-7 was isolated from the West-Driefontein goldmine in South
Africa. It is a Gram-positive rod showing optimal growth at 65°C. This isolate was found to be
able to grow on olive oil as the sole carbon source and on a variety of other lipid substrates, a
feature which indicated that the bacterium produces lipases.
In 2004, studies aimed at elucidating factors that would improve lipase production by G.
thermoleovorans revealed that when the bacterium was cultured in medium optimized for lipase
production, a production profile characterized by two enzyme activity peaks was observed. In
2005, a lipase (LipA) open reading frame (1251 bp) was amplified from the bacteriumâs genome.
Furthermore, lipase purification studies from the GE-7 culture grown in media containing olive
oil resulted in the purification of a lipase (~45 kDa), which corresponded to the LipA ORF that
had been amplified from the GE-7 genome. However, lipase activity staining with the
supernatant from GE-7 cultured in media supplemented with olive oil revealed the presence of
two lipolytic protein bands of different sizes. These observations and reports on the purification
of a smaller lipase in addition to LipA from the culture supernatant of related Geobacilli led to
the hypothesis that the two lipase production peaks observed with GE-7 represent two distinct
lipases differentially expressed by the bacterium.
The aim of this study was to characterize the lipolytic activity from this bacterium using
molecular and proteomic techniques. The primary objective was to identify from GE-7 a lipase
different from LipA and to profile its expression relative to that of LipA. Three authors had
already reported on the purification of a smaller lipase (>20 kDa), however only two authors had
published sequence information in this regard (Schmidt-Dannert et al., 1994; Lee et al., 2001).
Since bacterial lipolytic enzyme families are characterized by very high intra-family sequence
similarity, the published N-terminal amino acid sequences were used for similarity searches on
the database to identify homologs of this protein from related Geobacilli. Similarity searches
revealed that the published sequences shared very high similarity with a hypothetically
conserved protein (HCP) from G. kaustophilus and no homology to any other known lipase. The
nucleotide sequence of the HCP was used to design primers to facilitate PCR amplification of
the homolog from GE-7. The âsmall lipaseâ ORF (440 bp) was amplified from the GE-7 genome, however, functional expression using tributyrate/olive oil-Rhodamine plate assays revealed that
the protein was not lipolytic. Furthermore, secondary structure predictions using the âsmall
lipaseâ ORF revealed that the sequence shared significant identity to the type II secretory
pathway pseudopilin protein from related Bacillus species contrary to what was reported by the
two authors.
As a result of the above-mentioned findings a protein purification strategy aimed at isolating a
lipase different from LipA was pursued. Since LipA was purified in a previous study from the
second lipase peak and given that it was hypothesized that the first peak indicated the
production of a lipase different from LipA, GE-7 was cultured, lipase production monitored and
the cells harvested prior to the onset of the second peak. Lipase purification experiments from
the culture supernatant corresponding to the second peak resulted in the isolation of a ~45 kDa
protein. The protein band was analyzed by peptide mass fingerprinting and the amino acid
sequence determined. Sequence analysis revealed that the protein that was purified from the
first production peak is LipA, which served as an indication that LipA was produced at both
peaks.
GE-7 was cultured in media with (induced) and without (uninduced) the lipid substrate (olive oil)
and the lipase production profiles compared. It was observed that under induced conditions the
two production peaks persisted while only one peak was observed in the uninduced. However,
reverse transcription-mediated PCR on RNA isolated from GE-7 cultured as described above
with primers specific for lipA revealed that LipA is produced under both culture conditions.
Moreover, LipA is present at both production peaks under induced conditions which supports
the purification results. Detection of LipA from the uninduced culture indicates the GE-7 is
constitutively expressed, contrary to the previous reports.
Bioreactor studies aimed at relating the effect of media components on lipase production by GE-
7 revealed that, under induced conditions, significant lipase production is only observed after
the depletion of glucose and that the second activity peak is only observed upon the uptake of
the free fatty acids by the cells. This observation suggested the fatty acids could act as a signal
for further lipase production which was supported by the one lipase production peak observed
under uninduced conditions (no fatty acids in the culture medium). The observation that significant lipase production was observed at the stationary phase when
the glucose concentration became limiting served as a possible indication of a mechanism of
gene expression regulation known as catabolite repression. Which suggests that lipA could be
down-regulated in the presence of simple sugars (glucose) and up-regulated upon depletion of
glucose to afford the bacterium the ability to utilize olive oil as a carbon source. A conserved
sequence (CRE-box) has been identified on the promoter regions of a number of genes that are
subject to this mode of regulation. As a result, the lipA promoter region was amplified and
sequenced. Analysis of the sequence revealed the presence of the CRE-box. Experiments were
conducted to investigate whether catabolite repression was a possibility for the GE-7 lipA. GE-7
was cultured under induced conditions, lipase activity monitored and the cells spiked with
additional glucose at the onset of the stationary phase. A significant drop in the lipase
production was observed subsequent to the spike.
The above-mentioned observations and experiments where olive oil (used as a sole carbon
source) or stearic acid was used as the inducer revealed that changing certain components of
the media changed the lipase production profile. All the findings in this study suggest that LipA
is the only lipase produced under the culture conditions investigated and that the changes in the
production profile could be due to the regulation of the gene in response to changes in the
culture medium.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-11112011-123025 |
| Date | 11 November 2011 |
| Creators | Tlou, Matsobane Godfrey |
| Contributors | Prof E van Heerden, Dr LA Piater |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-11112011-123025/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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