Benzoylformate Decarboxylase from Pseudomonas putida as Stable Catalyst for the Synthesis of Chiral 2-Hydroxy Ketones

The thiamin diphosphate‐ and Mg2+‐dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2‐hydroxy ketones in a benzoin‐condensation type reaction. Carboligation constitutes a side reaction of...

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Bibliographic Details
Published in:Chemistry : a European journal 2000-04, Vol.6 (8), p.1483-1495
Main Authors: Iding, Hans, Dünnwald, Thomas, Greiner, Lasse, Liese, Andreas, Müller, Michael, Siegert, Petra, Grötzinger, Joachim, Demir, Ayhan S., Pohl, Martina
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Carboxy-Lyases - genetics
Carboxy-Lyases - isolation & purification
Carboxy-Lyases - metabolism
Cloning, Molecular
decarboxylation
DNA, Recombinant
enzyme catalysis
enzyme membrane reactors
Enzyme Stability
Ketones - chemical synthesis
Kinetics
Molecular Sequence Data
Pseudomonas putida
Pseudomonas putida - enzymology
Recombinant Proteins - metabolism
Stereoisomerism
stereoselective synthesis
Substrate Specificity
thiamin diphosphate
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Summary:The thiamin diphosphate‐ and Mg2+‐dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2‐hydroxy ketones in a benzoin‐condensation type reaction. Carboligation constitutes a side reaction of BFD, whereas the predominant physiological task of the enzyme is the non‐oxidative decarboxylation of benzoylformate. For this purpose the enzyme was obtained in sufficient purity from Pseudomonas putida cells in a one‐step purification using anion‐exchange chromatography. To facilitate the access to pure BFD for kinetical studies, stability investigations, and synthetical applications, the coding gene was cloned into a vector allowing the expression of a hexahistidine fusion protein. The recombinant enzyme shows distinct activity maxima for the decarboxylation and the carboligation beside a pronounced stability in a broad pH and temperature range. The enzyme accepts a wide range of donor aldehyde substrates which are ligated to acetaldehyde as an acceptor in mostly high optical purities. The enantioselectivity of the carboligation was found to be a function of the reaction temperature, the substitution pattern of the donor aldehyde and, most significantly, of the concentration of the donor aldehyde substrate. Our data are consistent with a mechanistical model based on the X‐ray crystallographic data of BFD. Furthermore we present a simple way to increase the enantiomeric excess of (S)‐2‐hydroxy‐1‐phenyl‐propanone from 90 % to 95 % by skillful choice of the reaction parameters. Enzymatic synthesis with BFD are performed best in a continuously operated enzyme membrane reactor. Thus, we have established a new enzyme tool comprising a vast applicability for stereoselective synthesis.
ISSN:0947-6539
1521-3765
DOI:10.1002/(SICI)1521-3765(20000417)6:8<1483::AID-CHEM1483>3.0.CO;2-S