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Protein Eng Des Sel. 2016 Dec;29(12):557-562. Epub 2016 Aug 29.

Fusion of an alcohol dehydrogenase with an aminotransferase using a PAS linker to improve coupled enzymatic alcohol-to-amine conversion.

Author information

1
Munich Center for integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
2
Munich Center for integrated Protein Science (CiPSM) and Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany skerra@tum.de.

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

PMID:
27578886
DOI:
10.1093/protein/gzw039
[Indexed for MEDLINE]

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