Helmholtz-Gemeinschaft

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