Directed evolution of enzymes: new biocatalysts for asymmetric synthesis

Alexeeva, Marina; Carr, Reuben; Turner, Nicholas J.

doi:10.1039/b311055a

Cited by 73 publications

(26 citation statements)

References 18 publications

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“…Since the wild-type enzyme showed insufficient activity, directed evolution of this biocatalyst was performed to furnish a novel amine oxidase possessing not only a wider substrate spectrum, but also enhanced activity and enantioselectivity (Figure 2). [97][98][99] The Asn336Ser variant of the amine oxidase showed highest activity towards substrates bearing a methyl substituent and a bulky alkyl/aryl group adjacent to the amino-carbon atom. For instance, its activity on l-a-methylbenzylamine was 47-fold higher than that of the wild-type enzyme, [97] and it also accepted secondary amines.…”

Section: Combining An Oxidase With a Chemical Reducing Agentmentioning

confidence: 99%

From a Racemate to a Single Enantiomer: Deracemization by Stereoinversion

et al. 2006

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Abstract:The stereoinversion of one enantiomer into its mirror-image counterpart within a racemate furnishes a single stereoisomeric product in 100 % theoretical yield. This extremely efficient type of deracemization, whereby substrate and product possess an identical chemical structure, can be achieved by using bio-or chemo-catalysts or combinations thereof and is applicable to secondary alcohols, amines and a-substituted carboxylic acids. Special emphasis is devoted to the theoretical background of the onepot, single-step deracemization of sec-alcohols.

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Section: Combining An Oxidase With a Chemical Reducing Agentmentioning

confidence: 99%

From a Racemate to a Single Enantiomer: Deracemization by Stereoinversion

et al. 2006

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“…These methods generally use microbial colonies expressing the protein of interest to screen for activity directly in situ . 816–818 Advantages to this include the ability to use enzyme-coupled assays (like HRP) 819,820 or substrates of poor solubility or viscosity. 821 …”

Section: Screeningmentioning

confidence: 99%

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

et al. 2015

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“…For direct functional screening, cells were spread onto a Hybond-N transfer membrane, which enabled the E. coli transformants to be transferred to an agar plate containing 1 mM isopropyl-beta- d -thiogalactopyranoside to induce protein expression. For MAO-N activity assays, the induced bacterial colonies were analysed for oxidase activity through the production of hydrogen peroxide by the colorimetric assay outlined by Alexeeva et al (2002, 2003) using α-methylbenzylamine as the substrate.…”

Section: Methodsmentioning

confidence: 99%

SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution

Currin

Swainston

Day

et al. 2014

Protein Engineering, Design and Selection

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The de novo synthesis of genes is becoming increasingly common in synthetic biology studies. However, the inherent error rate (introduced by errors incurred during oligonucleotide synthesis) limits its use in synthesising protein libraries to only short genes. Here we introduce SpeedyGenes, a PCR-based method for the synthesis of diverse protein libraries that includes an error-correction procedure, enabling the efficient synthesis of large genes for use directly in functional screening. First, we demonstrate an accurate gene synthesis method by synthesising and directly screening (without pre-selection) a 747 bp gene for green fluorescent protein (yielding 85% fluorescent colonies) and a larger 1518 bp gene (a monoamine oxidase, producing 76% colonies with full catalytic activity, a 4-fold improvement over previous methods). Secondly, we show that SpeedyGenes can accommodate multiple and combinatorial variant sequences while maintaining efficient enzymatic error correction, which is particularly crucial for larger genes. In its first application for directed evolution, we demonstrate the use of SpeedyGenes in the synthesis and screening of large libraries of MAO-N variants. Using this method, libraries are synthesised, transformed and screened within 3 days. Importantly, as each mutation we introduce is controlled by the oligonucleotide sequence, SpeedyGenes enables the synthesis of large, diverse, yet controlled variant sequences for the purposes of directed evolution.

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Directed evolution of enzymes: new biocatalysts for asymmetric synthesis

Abstract: Directed evolution has been employed to generate new enzymes for the deracemisation of chiral amines.

Cited by 73 publications

References 18 publications

From a Racemate to a Single Enantiomer: Deracemization by Stereoinversion

From a Racemate to a Single Enantiomer: Deracemization by Stereoinversion

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution

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