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Title: Discovery, characterisation and application of imine reductases for biocatalytic reductive amination
Author: Montgomery, Sarah
ISNI:       0000 0005 0290 1600
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2019
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A panel of known and novel imine reductases (IREDs) have been characterised in terms of their expression, stability and substrate scope, and the most active enzymes have been applied to more challenging industrially relevant reductive aminations, extending the scope of IRED reactions to include the use of aniline a nucleophile and the cellulose derivative Cyrene as a chiral electrophile. The novel IRED from Cystobacter ferrugineus (p-IR23) has also been applied on a preparative scale in the chemoenzymatic synthesis of 2,2-disubstituted azepanes. A combination of sequence and conversion data has been used to draw conclusions about the origin of certain types of activity, with several key residues being identified with the aid of computational tools. A single point mutation was shown to confer a threefold increase in conversion in the reductive amination of cyclohexanone with aniline, an important target for commercial applications. Additionally, a redox-neutral cascade has been developed which combines one of three alcohol dehydrogenases with the reductive aminase from Aspergillus oryzae to recycle the nicotinamide cofactor in a one-pot process for the alkylation of amines with primary and secondary alcohols. Complete conversion was obtained in the alkylation of amines with achiral alcohols, and well as moderate conversion and full selectivity in alkylation with at least one racemic substrate. This cascade was subsequently made more efficient by the application of a crude preparation of p- IR23 with a mutant of the thermostable alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus to achieve the same conversion with 80% less amine donor, at temperatures up to 50 degrees Celsius. An extension of the cascade employs a P450-catalysed hydroxylation of cycloalkanes to enable the direct conversion of hydrocarbon precursors to functionalised amines, combining two sequential oxidative steps and a reductive step in one pot.
Supervisor: Flitsch, Sabine ; Turner, Nicholas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: imine reductase ; biocatalysis ; reductive amination ; chiral amine ; synthesis