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

Scheidt, Thomas; Land, Henrik; Anderson, Mattias; Chen, Yujie; Berglund, Per; Dong, Yi; Fessner, Wolf‐Dieter

doi:10.1002/adsc.201500215

Cited by 25 publications

(18 citation statements)

References 58 publications

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“…Cv ‐ATA is one of the best known ATAs and characteristics such as substrate scope, reaction kinetics, 3D‐structure and stability have all been studied in detail. The active site of Cv ‐ATA has also been the subject of several engineering studies .…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we developed a fluorogenic assay for measuring ATA activity based on the transamination of 1‐(6‐methoxynaphth‐2‐yl)alkylamines (Scheme , 1 ). This reaction produces an acetonaphthone product ( 2 ) that can be measured with high sensitivity due to its strong blue fluorescence . A modular synthetic route towards this group of assay substrates was also developed, allowing the incorporation of structural variations such as the desired benzyl moiety.…”

Section: Introductionmentioning

confidence: 99%

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Engineering the Active Site of an (S)‐Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

et al. 2019

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A protein engineering approach for expanding the substrate scope of the (S)‐selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl‐ and benzyl‐substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2‐diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)‐1,2‐diphenylethylamine with ee>99% under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20‐30% (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)‐selective amine transaminase showing activity towards benzyl‐substituted primary amines.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering the Active Site of an (S)‐Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

et al. 2019

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“…The formed polymer results in a clearly visible precipitate, indicating conversion in the screening [ 26 ]. The o -xyl screening-assay was chosen because other screening methods either use the ketone as a signaling molecule or were not compatible with the selected reaction conditions [ 27 , 28 , 29 , 30 ].…”

Section: Resultsmentioning

confidence: 99%

β-Phenylalanine Ester Synthesis from Stable β-Keto Ester Substrate Using Engineered ω-Transaminases

et al. 2018

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The successful synthesis of chiral amines from ketones using ω-transaminases has been shown in many cases in the last two decades. In contrast, the amination of β-keto acids is a special and relatively new challenge, as they decompose easily in aqueous solution. To avoid this, transamination of the more stable β-keto esters would be an interesting alternative. For this reason, ω-transaminases were tested in this study, which enabled the transamination of the β-keto ester substrate ethyl benzoylacetate. Therefore, a ω-transaminase library was screened using a coloring o-xylylenediamine assay. The ω-transaminase mutants 3FCR_4M and ATA117 11Rd show great potential for further engineering experiments aiming at the synthesis of chiral (S)- and (R)-β-phenylalanine esters. This alternative approach resulted in the conversion of 32% and 13% for the (S)- and (R)-enantiomer, respectively. Furthermore, the (S)-β-phenylalanine ethyl ester was isolated by performing a semi-preparative synthesis.

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“…However, despite the elegance of many of these approaches, the use of multiple enzymes can complicate assays and limit their practical application. Non‐enzymatic assays have also been developed, which rely on spectroscopic, fluorescent or colorimetric detection of (co)products. One of the most established and successful assays relies on the spectrophotometric detection of acetophenone, when the widely accepted amine donor, α‐methylbenzylamine, is employed .…”

Section: Methodsmentioning

confidence: 99%

A Comprehensive Quantitative Assay for Amine Transaminases

et al. 2019

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The development of effective high‐throughput screening assays has contributed greatly to the wealth of designer enzymes available, by enabling rapid identification of desired variants from large mutant libraries. Here, we report a general and operationally simple end‐point assay for transaminases that enables the screening of both amine donors and acceptors in liquid phase. The spectrophotometric‐based screen exploits the amine donor 2‐aminoethylaniline (2‐AEA) and relies on reaction of in situ generated indole with Ehrlich's reagent. The assay has also been adapted to allow screening in the reverse direction by addition of indole and subsequent spectrophotometric analysis. Importantly, the screen provides qualitative information on the enantio‐preference of the individual biocatalysts. To increase the assay throughput, an engineered expression strain (E. coli BL21(DE3) ΔtnaA) lacking tryptophanase activity, was generated to enable reliable and direct evaluation of individual colonies arrayed on agar plates.

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

Cited by 25 publications

References 58 publications

Engineering the Active Site of an (S)‐Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

Engineering the Active Site of an (S)‐Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

β-Phenylalanine Ester Synthesis from Stable β-Keto Ester Substrate Using Engineered ω-Transaminases

A Comprehensive Quantitative Assay for Amine Transaminases

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