Ultrahigh-throughput screening enables efficient single-round oxidase remodelling
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Date
2019-09
Publication Type
Journal Article
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yes
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Abstract
Biocatalysis provides a potentially sustainable means of chemical manufacturing. However, the tailoring of enzymes to industrial processes is often laborious and time consuming, which limits the broad implementation of this approach. High-throughput screening methods can expedite the search for suitable catalysts, but are often constrained by the need for labelled substrates. The generalization of such techniques would therefore significantly expand their impact. Here we have established a versatile ultrahigh-throughput microfluidic assay that enables isolation of functional oxidases from libraries that contain up to 107 members. The increased throughput over prevalent methods led to complete active-site remodelling of cyclohexylamine oxidase in one round of directed evolution. A 960-fold increase in catalytic efficiency afforded an enzyme with wild-type levels of activity for a non-natural substrate, allowing biocatalytic synthesis of a sterically demanding pharmaceutical intermediate with complete stereocontrol. The coupled enzyme assay is label free and can be easily adapted to re-engineer any oxidase.
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published
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Journal / series
Volume
2 (9)
Pages / Article No.
740 - 747
Publisher
Nature
Event
Edition / version
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Software
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Date collected
Date created
Subject
Asymmetric catalysis; Biocatalysis; High-throughput screening; Oxidoreductase
Organisational unit
03492 - Hilvert, Donald (emeritus) / Hilvert, Donald (emeritus)
Notes
It was possible to publish this article open access thanks to a Swiss National Licence with the publisher.