Helmholtz-Gemeinschaft

Conclusion & Outlook
6 Conclusion and Outlook
The thiamine diphosphate-dependent benzaldehyde lyase from Pseudomonas fluorescens is a
highly active biocatalyst, which accepts a broad substrate range of benzaldehyde- and benzoin
derivatives. Furthermore, BAL is highly (R)-selective for the ligation and cleavage of chiral
2-hydroxy ketones. The aromatic aldehydes and benzoin are hardly soluble in aqueous media;
therefore the application of water miscible or immiscible cosolvents is necessary to work
efficiently. Reactions performed in stirred aqueous/organic two-phase systems (emulsions)
result in high conversion rates and yields for aromatic and heteroaromatic 2-hydroxy ketones,
starting from cheap aldehydes. However, a very fast inactivation of BAL was observed under
process conditions, which is assumed to be caused by the aldehydes. In the present study the
inactivating processes of BAL were dissected as far as possible, in order to identify the
relevant factors, which can be various in stirred emulsion systems, like:
Stirring effects
Interface effects
Molecular toxicity of the organic solvent
Molecular toxicity of the substrates and products
Oxidation of the enzyme, e.g. by atmospheric oxygen
Kinetic inhibition by the substrates or products
Oxidation and stirring effects:
BAL was investigated relative to a variant of benzoylformate decarboxylase (BFDH281A),
which is generally more stable then the BAL, even towards aldehydes (Kocot, 2010).
Inactivation by oxidation processes could be excluded for both enzymes, in the present study.
BAL showed a quite good stability towards magnetically stirring (t 1/2 : 91-133 h; t 1/2 of non
stirred control: > 170 h), while BFDH281A was rapidly inactivated, especially at pH 6.5.
Inactivation of the BFD-variant was accompanied by precipitation of the enzyme already after
3 hours of stirring. Further, a partial degradation of the protein was deduced from SDS-PAGE
analysis, which is probably caused by the rubbing of the stir bar on the bottom of the vessel.
However, by increasing the pH to pH 7 and pH 8 stabilisation of BFDH281A could be
observed, furthermore with a steel head stirrer the enzyme showed high stability. Due to the
high stability with the head stirrer and high sensitivity of BFDH281A towards the magnetic
stirrer especially at pH 6.5, deactivation process was deduced to adsorption of BFDH281A on
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