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High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

Rios-Solis, L; Mothia, B; Yi, S; Zhou, Y; Micheletti, M; Lye, GJ; (2015) High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation. Journal of Molecular Catalysis B: Enzymatic , 120 pp. 100-110. 10.1016/j.molcatb.2015.07.001. Green open access

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Abstract

Full kinetic models provide insight into enzyme mechanism and kinetics and also support bioconversion process design and feasibility assessment. Previously we have established automated microwell methods for rapid data collection and hybrid kinetic modelling techniques for quantification of kinetic constants. In this work these methods are applied to explore the substrate selectivity and kinetics of monoamine oxidase, MAO-N-D5, from Aspergillus niger. In particular we examine the MAO-N-D5 variant Ile246Met/Asn336Ser/Met348Lys/Thr384Asn to allow the oxidation of secondary amines Initial screening showed that MAO-N-D5 enabled the selective oxidation of secondary amines in 8 and 9 carbon rings, as well as primary ethyl and propyl amines attached to secondary amines of indolines and pyrrolidines. Subsequently we developed a first kinetic model for the MAO-N-D5 enzyme based on the ping-pong bi-bi mechanism (similar to that for the human MAO-A enzyme). The full set of kinetic parameters were then established for three MAO-N-D5 substrates namely; 3-azabicyclo[3,3,0]octane, 1-(2 amino ethyl) pyrrolidine and 3-(2,3-dihydro-1H-indole-1-yl)propan-1-amine. The models for each amine substrate showed excellent agreement with experimentally determined progress curves over a range of operating conditions. They indicated that in each case amine inhibition was the main determinant of overall reaction rate rather than oxygen or imine (product) inhibition. From the perspective of larger scale bioconversion process design, the models indicated the need for fed-batch addition of the amine substrate and to increase the dissolved oxygen levels in order to maximize bioconversion process productivity.

Type: Article
Title: High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation
Open access status: An open access version is available from UCL Discovery
DOI: 10.1016/j.molcatb.2015.07.001
Publisher version: http://dx.doi.org/ 10.1016/j.molcatb.2015.07.001
Language: English
Additional information: Copyright © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Monoamine oxidase; Aspergillus niger; Microscale kinetic modelling; Substrate screening
UCL classification: UCL
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/1495715
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