Nitriles have a wide range of uses as building blocks, solvents, and alternative fuels, but also as intermediates and components of flavors and fragrances. The enzymatic synthesis of nitriles by aldoxime dehydratase (Oxd) is an emerging process with significant advantages over conventional approaches. Here we focus on the immobilization of His-tagged Oxds on metal affinity resins, an approach that has not been used previously for these enzymes. The potential of the immobilized Oxd was demonstrated for the synthesis of phenylacetonitrile (PAN) and E-cinnamonitrile, compounds applicable in the fragrance industry. A comparison of Talon and Ni-NTA resins showed that Ni-NTA with its higher binding capacity was more suitable for the immobilization of Oxd. Immobilized Oxds were prepared from purified enzymes (OxdFv from Fusarium vanettenii and OxdBr1 from Bradyrhizobium sp.) or the corresponding cell-free extracts. The immobilization of cell-free extracts reduced time and cost of the catalyst production. The immobilized OxdBr1 was superior in terms of recyclability (22 cycles) in the synthesis of PAN from 15 mM E/Z-phenylacetaldoxime at pH 7.0 and 30 °C (100% conversion, 61% isolated yield after product purification). The volumetric and catalyst productivity was 10.5 g/L/h and 48.3 g/g of immobilized protein, respectively.

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague CZ 142 00 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague CZ 142 00 Czech Republic; Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 Prague CZ 128 44 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 Prague CZ 142 00 Czech Republic; Faculty of Food and Biochemical Technology University of Chemistry and Technology Prague Technická 5 Prague CZ 166 28 Czech Republic

Institute of Molecular Biotechnology Faculty of Technical Chemistry Chemical and Process Engineering Biotechnology Graz University of Technology Petersgasse 14 Graz A 8010 Austria; Austrian Center of Industrial Biotechnology GmbH Krenngasse 37 Graz A 8010 Austria

Laboratory of Photosynthesis Centre Algatech Institute of Microbiology of the Czech Academy of Sciences Novohradská 237 Třeboň CZ 37981 Czech Republic

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Aldoxime dehydratases: production, immobilization, and use in multistep processes