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. 2016 Dec;29(12):557-562.
doi: 10.1093/protein/gzw039. Epub 2016 Aug 29.

Fusion of an Alcohol Dehydrogenase With an Aminotransferase Using a PAS Linker to Improve Coupled Enzymatic Alcohol-To-Amine Conversion

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Fusion of an Alcohol Dehydrogenase With an Aminotransferase Using a PAS Linker to Improve Coupled Enzymatic Alcohol-To-Amine Conversion

Alexandra Lerchner et al. Protein Eng Des Sel. .

Abstract

To facilitate biocatalytic conversion of the biotechnologically accessible dicyclic dialcohol isosorbide into its industrially relevant diamines, we have designed a fusion protein between two homo-oligomeric enzymes: the levodione reductase (LR) from Leifsonia aquatica and the variant L417M of the ω-aminotransferase from Paracoccus denitrificans (PDωAT(L417M)), mutually connected by a short Pro/Ala/Ser linker sequence. The hybrid protein was produced in Escherichia coli in correctly folded state, comprising a tetrameric LR moiety and presumably two dimers of PDωAT(L417M), as proven by SDS-PAGE and size exclusion chromatography. The bifunctional enzyme revealed beneficial kinetics over the two-component system, in particular at low substrate concentration.

Keywords: active site; chiral amine; coupled assay; fusion protein; substrate channeling.

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