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Coupled Enzymatic Alcohol-to-Amine Conversion of Isosorbide using Engineered Transaminases and Dehydrogenases

Authors

  • Dr. Alexandra Lerchner,

    1. Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan (Germany), Fax: (+49) 8161-71-4352
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  • Stefan Achatz,

    1. Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan (Germany), Fax: (+49) 8161-71-4352
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  • Dr. Christian Rausch,

    1. Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan (Germany), Fax: (+49) 8161-71-4352
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  • Dr. Thomas Haas,

    1. Creavis Technologies & Innovation, Evonik Industries AG, 45772 Marl (Germany)
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  • Prof. Arne Skerra

    Corresponding author
    1. Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan (Germany), Fax: (+49) 8161-71-4352
    • Lehrstuhl für Biologische Chemie, Technische Universität München, 85350 Freising-Weihenstephan (Germany), Fax: (+49) 8161-71-4352

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Abstract

A matching dehydrogenase and transaminase pair was engineered with regard to substrate recognition, catalytic activity, and cofactor specificity with the final goal to convert the bicyclic dialcohol isosorbide into a diamine by a multistep biocatalytic process. Individual catalytic turnover rates as well as coupled conversion to the amine were investigated for the enzymes in analytical assays that used the substrate isosorbide and different intermediates along the multiple pathway reaction cascade, in particular, stereoisomers of hydroxy ketones, amino alcohols, and amino ketones. For parallel screening and evaluation of mutant enzymes with regard to catalytic activities and optimal reaction conditions, a robotic platform was established that comprised all steps from bacterial protein expression to the enzymatic assay. As a result, we present a three-enzyme system composed of L. aquatica levodione reductase, an engineered P. denitrificans ω-aminotransferase, and B. subtilis alanine dehydrogenase that catalyzes formation of the isosorbide monoamine with a yield of up to ≈7 % under our analytical assay conditions. After further optimization and adaptation to whole-cell catalysis, this enzyme system may open a biotechnologically attractive route to a structurally rigid diamine for the production of biosynthetic polymer materials.

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