Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones


Iding H., Dunnwald T., Greiner L., Liese A., Muller M., Siegert P., ...More

CHEMISTRY-A EUROPEAN JOURNAL, vol.6, no.8, pp.1483-1495, 2000 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 6 Issue: 8
  • Publication Date: 2000
  • Title of Journal : CHEMISTRY-A EUROPEAN JOURNAL
  • Page Numbers: pp.1483-1495
  • Keywords: decarboxylation, enzyme catalysis, enzyme membrane reactors, stereoselective synthesis, thiamin diphosphate, ALPHA-HYDROXY KETONES, DIPHOSPHATE-DEPENDENT ENZYMES, SILYL ENOL ETHERS, THIAMIN DIPHOSPHATE, PYRUVATE DECARBOXYLASE, ZYMOMONAS-MOBILIS, ENANTIOSELECTIVE OXIDATION, ACID DECARBOXYLASES, KINETIC RESOLUTION, CRYSTAL-STRUCTURE

Abstract

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.