This title appears in the Scientific Report :
2000
Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones
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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Personal Name(s): | Iding, G. M. |
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Dünnwald, T. / Greiner, L. / Liese, A. / Müller, M. / Siegert, P. / Grötzinger, J. / Demir, A. S. / Pohl, M. | |
Contributing Institute: |
Institut für Biotechnologie; IBT |
Published in: | Chemistry, 6 (2000) S. 1483 - 1495 |
Imprint: |
Weinheim
Wiley-VCH
2000
|
Physical Description: |
1483 - 1495 |
Document Type: |
Journal Article |
Research Program: |
Verfahrenstechnik der Gewinnung Verfahrenstechnik zur mikrobiellen Gewinnung von Primärmetaboliten |
Series Title: |
Chemistry-a European Journal
6 |
Subject (ZB): | |
Publikationsportal JuSER |
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. |