A Practical NMR-Based High-Throughput Assay for Screening Enantioselective Catalysts and Biocatalysts

Reetz, Manfred T.; Eipper, Andreas; Tielmann, Patrick; Mynott, Richard

doi:10.1002/1615-4169(200210)344:9<1008::aid-adsc1008>3.0.co;2-t

Cited by 70 publications

(23 citation statements)

References 76 publications

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“…Screening by high-performance liquid chromatography or thinlayer chromatography is also feasible but similarly requires sample pretreatment and is only high-throughout with parallel, automated instrumentation. Screening using nuclear magnetic resonance spectroscopy (NMR) has also been demonstrated (167). Up to 1400 samples per day could be analyzed by an 72 UMENO ET AL.…”

Section: Prospects and Challenges For Diversifying Other Pathways By supporting

confidence: 83%

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Umeno

Tobias

Arnold

2005

Microbiol Mol Biol Rev

195

148

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Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed

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Section: Prospects and Challenges For Diversifying Other Pathways By supporting

confidence: 83%

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Umeno

Tobias

Arnold

2005

Microbiol Mol Biol Rev

195

148

View full text Add to dashboard Cite

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“…Although chromatography and NMR techniques for highthroughput screening have been developed in recent years (22,23), they require expensive equipment, and data analysis is time-consuming. The methods shown in the present work are simple and inexpensive and do not require special equipment besides a multiplate reader.…”

Section: Discussionmentioning

confidence: 99%

“…The third class of enzymatic assays rely on product detection by instruments and include gas chromatography (GC), highpressure liquid chromatography (HPLC), mass spectrometry, nuclear magnetic resonance (NMR) spectrometry, and infrared radiation assays that have been adapted for high throughput (22,23,33). Such assays require expensive and sophisti-cated equipment, but they allow working directly with the substrate of interest and are rapidly adapted once the instrument is available (23).…”

mentioning

confidence: 99%

Rapid Methods for High-Throughput Detection of Sulfoxides

Shainsky

Derry

Leichtmann‐Bardoogo

et al. 2009

Appl Environ Microbiol

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Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho-monooxygenase in a saturation mutagenesis library, using methyl-p-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.The growing demand for green catalytic processes has increased the utilization of enzymes as industrial biocatalysts for the synthesis of fine chemicals (6,19,20). As a consequence, there is a continuous search for novel or improved biocatalysts. In order to find an appropriate candidate for a process, various sources of enzymes must be screened for activity (23). Therefore, a sensitive, reproducible, accurate, and simple highthroughput screening method is a key prerequisite for the development of biocatalytic processes on an industrial scale (32, 39).Screening systems are divided into three different classes. The first class contains assays applicable to testing growing or resting microbial colonies for enzymatic activity directly on agar plates (23), for example, detection of epoxide hydrolase activity on butane oxide by use of safranin O. Oxidation of the 1,2-diol product by Escherichia coli modified the membrane potential and led to accumulation of the red dye in the colonies producing active enzyme (34). In another study, the spontaneous oxidation of substituted catechols to brown-red quinones was used to screen random libraries of whole cells expressing toluene monooxygenases (TMOs) for regioselective oxidation of substituted phenols (12, 30). The positive cl...

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“…An elegant recent example uses a pseudo-prochiral 15 N-labelled 3-hydroxyglutaronitrile as substrate to assay enantioselective nitrilases ( Figure 6) [33 ]. Isotopically labelled pseudo-racemic substrates have also been analysed by high-throughput 1 H-NMR [34] and Fourier-transform infrared (FT-IR) spectroscopy [35 ] and these methods were used to screen libraries of enzyme mutants [36].…”

Section: Isotopic Labelsmentioning

confidence: 99%