School of Engineering and Science - Jacobs University

Results
After changing the mobile phase to methyl t-buthyl ether (MTBE), methanol,
acetic acid and water (3:1:1:1, v/v) the retention factor of the CAS-active
substance was increased to 0.45 (Fig.19B). Interestingly, the retention factor for
authentic achromobactin differs from that (0.35). The smearing of the CASactive
band of Pss22d∆Pvd (Fig.19B) was likely due to a relatively long
incubation of the overlay in order to visualize a second signal above the major
CAS-active band. This additional signal could be observed in both siderophorepositive
samples and may correspond to a second conformation of the
siderophore.
The original CAS-active band of Pss22d∆Pvd corresponds to the more
intensive spot indicated by the arrow (Fig.19B). Approximately the same
amount of XAD4-purification was applied for both strains and the intensity of
the different fluorescent bands did not differ significantly between both lanes. In
contrast, the siderophore activity detected for D. chrysanthemi cbsE-1 tonB60
was much lower as compared to Pss22d∆Pvd.
The modified separation conditions are currently applied on the next large-scale
purification. After this step, another TLC-separation using a different stationary
phase will be used for further purification. Macherey-Nagel offers a range of
differently modified silica plates with varying hydrophobicities of the matrices.
The affinity of the CAS-active substance to silica modified by addition of a diolor
an amino group was tested (Fig.20). The affinity of the CAS-active substance
towards the stationary phase was lower for the diol modification and higher for
the amino modified plate. Thus the diol modified plate could be optimized for
better focusing of the CAS-active band, while the amino-modified phase could
be used to separate other disturbing substances.
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