Family clustering of Baeyer-Villiger monooxygenases based on protein sequence and stereopreference.
Institute of Applied Synthetic Chemistry, Marie Curie Training Site GEMCAT, Vienna University of Technology, Wien, Austria.Angewandte Chemie International Edition (Impact Factor: 13.73). 07/2005; 44(23):3609-13. DOI:10.1002/anie.200462964
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ABSTRACT: Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large-scale applications. We have studied the enzymatic Baeyer-Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non-growing Escherichia coli cells to provide high productivity (0.79 g epsilon-caprolactone/L/h = 18 micromol epsilon-caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non-growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.Biotechnology Progress 09/2008; 18(2):262-8. · 1.85 Impact Factor
Article: Oxidizing enzymes as biocatalysts.[show abstract] [hide abstract]
ABSTRACT: This article describes oxidising enzymes used for biocatalytic applications. Redox biocatalysts are highly sought after because of the selectivity, controllability and economy of their reactions, in comparison with conventional chemical reactions. Increasing numbers of oxidative biotransformations are being reported, indicating wide variability in the biocatalyst characteristics and a range of potential and established applications. Several limitations apply to oxidative biotransformations, including the requirement for cofactor regeneration, and low stability and activities. Recent advances in addressing these problems include molecular and reaction engineering approaches.Trends in Biotechnology 01/2004; 21(12):543-9. · 9.66 Impact Factor
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ABSTRACT: Whole cells of an Escherichia coli strain that overexpresses Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase have been used for the Baeyer−Villiger oxidations of a variety of 4-mono- and 4,4-disubstituted cyclohexanones. In cases where comparisons were possible, this new biocatalytic reagent provided lactones with chemical yields and optical purities that were comparable to those obtained from the purified enzyme or a strain of bakers' yeast that expresses the same enzyme. The efficient production of cyclohexanone monooxygenase in the E. coli expression system (ca. 30% of total soluble protein) allowed these oxidations to reach completion in approximately half the time required for the engineered bakers' yeast strain. Surprisingly, 4,4-disubstituted cyclohexanones were also accepted by the enzyme, and the enantioselectivities of these oxidations could be rationalized by considering the conformational energies of bound substrates along with the enzyme's intrinsic enantioselectivity. The enzyme expressed in E. coli cells also oxidized several 4-substituted cyclohexanones bearing polar substituents, often with high enantioselectivities. In the case of 4-iodocyclohexanone, the lactone was obtained in >98% ee and its absolute configuration was assigned by X-ray crystallography. The crystal belongs to the monoclinic P21 space group with a = 5.7400(10), b = 6.1650(10), c = 11.377(2) Å, b = 99.98(2)°, and Z = 2. Taken together, these results demonstrate the utility of an engineered bacterial strain in delivering useful chiral building blocks in an experimentally simple manner.The Journal of Organic Chemistry 01/2001; 66(3). · 4.56 Impact Factor
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