1. acib GmbH, Petersgasse 14, 8010 Graz, Austria;2. Institute of Molecular Biotechnology, Graz University of Technology, NAWI Graz, Petersgasse 14, 8010 Graz, Austria;3. Institute of Organic Chemistry, Graz University of Technology, NAWI Graz, Stremayrgasse 9, 8010 Graz, Austria;4. Institute of Applied Synthetic Chemistry, TU Wien, Getreidemarkt 9/OC−163, 1060 Vienna, Austria;5. (+43)‐316‐873‐9333 | Fax: (+43)‐316‐873‐9302
Abstract:
The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate‐reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a Tm of 45 °C and inhibition by the co‐product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole‐cell biotransformation using the Escherichia coli K‐12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.
