Characterization of benzaldehyde lyase from Pseudomonas fluorescens: A versatile enzyme for asymmetric C-C bond formation.

The thiamin-diphosphate-dependent enzyme benzaldehyde lyase is a very import catalyst for chemoenzymatic synthesis catalyzing the formation and cleavage of (R)-hydroxy ketones. We have studied the stability of the recombinant enzyme and some enzyme variants with respect to pH, temperature, buffer salt, cofactors and organic cosolvents. Stability of BAL in chemoenzymatic synthesis requires the addition of cofactors to the buffer. Reaction temperature should not exceed 37 degrees C. The enzyme is stable between pH 6 and 8, with pH 8 being the pH-optimum of both the lyase and the ligase reaction. Potassium phosphate and Tris were identified as optimal reaction buffers and the addition of 20 vol% DMSO is useful to enhance both the solubility of aromatic substrates and products and the stability of BAL. The initial broad product range of BAL-catalyzed reactions has been enlarged to include highly substituted hydroxybutyrophenones and aliphatic acyloins.

HDLalert - a healthcare DL alerting system: from user needs to implementation.

In the health domain, there are many circumstances where clinicians (i.e. doctors, nurses, allied health professionals) and patients wish to track changes in medical knowledge. However, existing 'news' or 'alert' services provide relatively limited means for selecting which information to receive. The result is that clinicians and patients often receive information that is inappropriate, irrelevant or simply too much. In this paper, we detail alert-relevant findings from several international user studies (e.g. UK, Germany and New Zealand) incorporating both clinical staff (across several hospitals) and patients' perceptions. These findings demonstrate the importance of context, in terms of both the user's task and immediate environment. We introduce a novel alerting architecture that can provide a finely tailored stream of alerts to the user, and provides further support to assist the interpretation of received material.

Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution.

Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.

Development of a donor-acceptor concept for enzymatic cross-coupling reactions of aldehydes: the first asymmetric cross-benzoin condensation.

Highly enantioenriched mixed benzoins are obtained selectively through a biocatalytical cross-coupling reaction of aromatic aldehydes using ThDP-dependent enzymes.