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Title: Efforts toward the directed evolution of a bacterial molybdenum-dependent aldehyde oxidase for use in synthesis
Author: Riley, C.
ISNI:       0000 0004 7428 6806
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Molybdenum-dependent xanthine oxidoreducatases (XOR) are a well-known family of enzymes, which oxidise purines, imines and aldehydes in cellular metabolism. Despite many studies relating to drug metabolism, application to chemical synthesis has only recently begun to be explored. Herein we report the first use of XORs in the cascade synthesis of lactams from cyclic amines by the combination of the mutant monoamine oxidase (MAO) MAO-N D9 and XOR (E.coli XDH & R. capsulatus XDH E232V). A number of single step imine oxidations were also performed in excellent conversions with the methodology comparing well to current chemical oxidation methods. The reactions were performed in water at ambient temperature and pH using oxygen as the terminal oxidant with the only by-product being H2O2. A solid-phase colorimetric assay has been developed to detect aldehyde oxidase activity from membrane-bound bacterial colonies of TP1000 x PaoABC using a coupled peroxidase system. The screen was used to assay a library of mutants generated at the Moco-containing sub unit of PaoABC with phenylacetaldehyde used as a model substrate. This resulted in the discovery of an improved mutant L443Q, which exhibited improved solid-phase activity in addition to improved kinetics. A saturation mutagenesis approach has also been employed on Ec PaoABC at analogous positions responsible for substrate binding in Rc XDH to search for a novel “imine oxidase”. An interesting variant L246G was isolated with a switch in substrate specificity toward 3,4-dihydroisoquinoline.
Supervisor: Carnell, Andrew J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral