Helmholtz-Gemeinschaft
Einflussfaktoren auf die Stabilität und Aktivität der ... - JuSER
Einflussfaktoren auf die Stabilität und Aktivität der ... - JuSER
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Conclusion & Outlook<br />
removal of the aldehyde from the enzyme by gel filtration. Furthermore, it was found that the<br />
inactivation depends on the excess of the substrate relative to the enzyme concentration. An<br />
excess of the substrate of around 2,500 to 50,000 per mol of enzyme (tetramer) is sufficient to<br />
reach the maximum inactivation rate. This led to the assumption that BAL exhibits at least<br />
one distinct binding position (covalent or non covalent) with different affinities depending on<br />
the character of the substrate.<br />
pH-depending effects:<br />
The inactivation of BAL by the aromatic aldehydes was found to be pH-dependent. A<br />
stabilisation up to 5-fold could be observed by lowering the pH from pH 8 to pH 7. Again the<br />
extend of pH-dependency of stabilisation was very different for the investigated aldehydes.<br />
The possibility to improve the process stability at neutral pH, where BAL shows higher<br />
stability was investigated in more detail for the standard aldehydes (BA, DMBA and<br />
4-ClBA). Only 4-chlorobenzaldehyde was detected as a potential candidate for this<br />
optimisation process. However, it was found that BAL could not maintain its catalytic activity<br />
long enough to reach almost complete conversion at pH 7. Besides, the catalytic activity at<br />
pH 8 and pH 9 is high enough to reach almost full conversion, before inactivation occurs.<br />
Therefore an optimisation of synthesis processes by lowering the pH is not the matter of<br />
choice.<br />
Molecular reasons for the inactivation by aromatic aldehydes:<br />
All previous studies which were discussed in literature deduced the inactivation of BAL under<br />
process conditions to Schiff base formation of the aldehydes with lysine residues of the<br />
enzyme. Schiff bases can only be formed, if free amino-groups are deprotonated, especially<br />
those which are surface exposed (N-terminus or ε-amino groups of lysine residues). Therefore<br />
the dissociation constant (pK a ) should be lower than the environmental pH. A lysine residue<br />
(Lys127) was identified as the only potential modifiable partner, by PROPKA-calculation of<br />
the pK a and structural comparison to BFDH281A, which is much less sensitive towards<br />
aldehydes. However, amino acid exchanges at this position did not result in stabilisation of<br />
BAL towards the aromatic aldehydes. Based on these results, an inactivation mechanism<br />
based on Schiff base formation can almost be excluded.<br />
Promising results were obtained with BAL-variants which resembles the BFDH281A in the<br />
C-terminal part. These BAL-variants showed a significantly higher stability at least towards<br />
157