Biocatalytic Redox Cascades Involving ω‐Transaminases

w-Transaminases are av aluablec lass of enzymesf or the production of chiral amines with either (R)-or (S)-configuration in high optical purity and 100% yield by the biocatalytic reductivea mination of prochiral ketones.Aversatile new assay was developed to quantify w-transaminase activity for the kinetic characterization and enantioselectivity typing of novel or engineered enzymesb ased on the conversion of 1-(6-methoxynaphth-2-yl)alkylamines. Thea ssociated releaseo ft he acetonaphthone product can be monitoredb yt he development of its bright fluorescence at 450 nm with very high sensitivity and selectivity.T he assay principle can be used to quantify w-transaminase catalysis over av ery broad range of enzyme activity.B ecause of its simplicity and low substrate consumption in microtiter plate format the assay seems suitable for liquid screening campaigns with large library sizes in the directed evolution of optimized transaminases.F or assay substrates that incorporate structural variations,a ne fficient modular synthetic route was developed. This includesr acemate resolution by lipase-catalyzed transacylation to furnish enantiomericallyp ure (R)-and (S)-configured amines.T he latter are instrumental for the rapid enantioselectivity typing of w-transaminases. This method was used to characterize two novel( S )-selective taurine-pyruvate transaminases of the subtype 6a from thermophilic Geobacillus thermodenitrificans and G. thermoleovorans.

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Fluorescence‐Based Kinetic Assay for High‐Throughput Discovery and Engineering of Stereoselective ω‐Transaminases

Scheidt

Land

Anderson

et al. 2015

Adv Synth Catal

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Biocatalytic Redox Cascades Involving ω‐Transaminases

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Fluorescence‐Based Kinetic Assay for High‐Throughput Discovery and Engineering of Stereoselective ω‐Transaminases

Fluorescence‐Based Kinetic Assay for High‐Throughput Discovery and Engineering of Stereoselective ω‐Transaminases

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