Helmholtz-Gemeinschaft

Conclusion & Outlook
the hydrophobic surface of the magnetic stir bar. As a consequence, partial unfolding and
aggregation of the BFD-variant occurs. Besides BAL is less affected by such adsorption
processes. The differences in the stability of both enzymes towards magnetic stirring may be
based on their unequal isoelectric points (pI). Protein adsorption onto interfaces occurs mainly
when pH is near their pI. The pI of BFD is at pH 6.5 (Hasson et al., 1995) and the pI of BAL
is at pH 4.6 (Janzen et al., 2006). Mean that an adsorption onto interfaces is more likely for
BFDH281A then for BAL at the investigated pH (pH 6.5-8).
Interface effects and molecular toxicity of organic solvent:
The organic solvent methylisobutylketone (MIBK) has no negative effect on the stability of
BAL up to its solubility limit (5% v/v), which means that its molecular toxicity can be
neglected. However, above its solubility limit MIBK results in faster enzyme inactivation,
already without mixing the two phases (t 1/2 : 108 ± 17 h), which suggest an interface toxicity
of MIBK. The inactivation was again 75% faster in an emulsion by continuous mixing of the
two phases (t 1/2 : 26 ± 7 h). This effect is supposed to be based on the enlarged interface and
stirring effects.
Inactivation by aromatic aldehydes:
In contrast to the inactivation by mechanical stirring, BAL showed a very high sensitivity
towards the aromatic aldehydes even at low concentrations (≤ 2 mM). Here inactivation
occurs within minutes instead of hours. Before analysing the effects with different substituted
benzaldehydes on the enzyme stability a HPLC-based test system was developed. This test
offers the possibility to determine the residual activity of BAL after incubation with the
substrates at distinct time points, without using a second substrate for activity measurements.
Therefore the potential formation of mixed benzoins and thus inaccurate results was
prevented. The inactivation by the aromatic aldeydes was found to be very complex and
inhomogeneous. Half-lifes of BAL varied between 4-100 minutes under the same conditions,
depending on the substrate used for inactivation (table 10). However, no correlation of the
activity (total turn over) of BAL and its inactivation with different aromatic aldehydes was
observed with respect to the electronegativity, the steric demand of the substitution or the
position of the substituent. For further analyses three aldehydes were chosen as standard
substrates: benzaldehyde (BA), 4-chlorobenzaldehyde (4-ClBA) and 3,5-dimethoxybenzaldehyde
(DMBA). It turned out that the inactivation caused by these aldehydes was partially
reversible. With 2-methoxybenzaldehyde even a full reactivation of BAL was observed, after
156