Whole cell biotransformation processes are of special interest for the synthesis of chiral compounds since microorganisms offer some advantages in comparison to chemical catalysts or even isolated enzymes. Due to their internal production of cofactors biotransformation processes using whole cell catalysts can be operated without addition of external cofactors. In this study recombinant Escherichia coli cells were applied as biocatalysts for the reduction of ketones. By the expression of suitable alcohol dehydrogenases (adh) both enantiomeres of chiral alcohols can be produced. The syntheses of enantiopure (R)-3-hydroxybutyrate and (S )-3-hydroxybutyrate were carried out in continuously operated biotransformation processes with membrane retention of the whole cell biocatalysts. Recombinant E. coli expressing adh from Lactobacillus brevis turned out to be stable during biotransformation processes with substrate-coupled cofactor regeneration even when exceedingly high concentration of substrate and cosubstrate were applied. In contrast to that processes with enzyme-coupled cofacor regeneration affects the biocatalyst stability more intensely. For the substrate-coupled approach further investigations deal with the application of in situ acetone removal techniques in whole cell transformation processes. Stripping, pervaporation and extraction with an ionic liquid were applied as strategies for in situ acetone removal. In all cases higher conversion was achieved by overcoming thermodynamic and kinetic limitations. The pervaporation procedure turned out to be the most gentle method of acetone removal which only causes negligible damage to the whole cell biocatalysts. It was also applied for continuously operated biotransformation processes. For the knowledge of whole cell biocatalysts it is desirable to quantify intracellular concentrations of cofactors during a biotransformation process. A new method for quantification of several intracellular metabolites was developed and now transferred to biotransformation processes with recombinant E. coli. The concentrations of intracellular cofactor concentrations were determined during the reduction of methyl acetoacetate and 2,5-hexanedione with substrate-coupled cofactor regeneration by oxidation of 2-propanol. Die praktischen Arbeiten zur vorliegenden Dissertation wurden in der Zeit von August 2004 bis Oktober 2007 am Institut für Biotechnologie 2 im Forschungszentrum Jülich durchgeführt. Ich möchte mich an dieser Stelle bei einer Reihe von Personen bedanken, die mir bei der Anfertigung dieser Arbeit sehr viel Unterstützung geboten haben.
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