A rapid, sensitive colorimetric assay for the high-throughput screening of transaminases in liquid or solid-phase

Baud, Damien; Ladkau, Nadine; Moody, Thomas S.; Ward, John M.; Hailes, Helen C.

doi:10.1039/c5cc06817g

Cited by 64 publications

(66 citation statements)

References 35 publications

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“…Wells A1‐3: Ad2‐TAm in a freshwater‐based reaction medium; wells B1‐3: negative control in a freshwater‐based reaction medium; wells C1‐3: Ad2‐TAm in a seawater‐based reaction medium; wells D1‐3: negative control in a seawater‐based reaction medium. Assay results are shown alongside the reaction schematic for the transaminase‐catalyzed amination of furfural using NPEA as amino donor, adapted from a previously described assay …”

Section: Resultsmentioning

confidence: 99%

“…Freshwater and seawater systems were buffered at pH 9 using a universal buffer described previously . Colorimetric assays using 2‐(4‐nitrophenyl)ethan‐1‐amine (NPEA) as amino donor were adapted from those described previously . Screening assays were set up with the following conditions: buffered freshwater or buffered seawater (100 µL) was added to a 96‐well deepwell plate.…”

Section: Methodsmentioning

confidence: 99%

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Biocatalysis in seawater: Investigating a halotolerant ω‐transaminase capable of converting furfural in a seawater reaction medium

Kelly

Moody

Gilmore

2019

Engineering in Life Sciences

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The increasing demand for freshwater and the continued depletion of available resources has led to a deepening global water crisis. Significant water consumption required by many biotechnological processes contributes to both the environmental and economic cost of this problem. Relatively few biocatalytic processes have been developed to utilize the more abundant supply of seawater, with seawater composition and salinity limiting its use with many mesophilic enzymes. We recently reported a salt tolerant ω‐transaminase enzyme, Ad2‐TAm, isolated from the genome of a halophilic bacterium, Halomonas sp. CSM‐2, from a Triassic period salt mine. In this study we aimed to demonstrate its applicability to biocatalytic reactions carried out in a seawater‐based medium. Ad2‐TAm was examined for its ability to aminate the industrially relevant substrate, furfural, in both seawater and freshwater‐based reaction systems. Furfural was aminated with 53.6% conversion in a buffered seawater system, displaying improved function versus freshwater. Ad2‐TAm outperformed the commonly employed commercial ω‐TAms from Chromobacterium violaceum and Vibrio fluvialis, both of which showed decreased conversion in seawater. Given the increasingly precarious availability of global freshwater, such applications of enzymes from halophiles have the ability to reduce demand for freshwater in large‐scale industrial processes, delivering considerable environmental and economic benefits.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Biocatalysis in seawater: Investigating a halotolerant ω‐transaminase capable of converting furfural in a seawater reaction medium

Kelly

Moody

Gilmore

2019

Engineering in Life Sciences

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“…As in their case, this observation can potentially lead to a sensitive and operationally simple colorimetric assay to identify TA activity. [11,15] The formation of pyrrole was also demonstrated by NMR experiments performing the enzymatic reactions in deuterium oxide (see SI for more details). Surprisingly, when we studied the transformation using trans-2 as amine donor, it was also observed the presence of pyrrole in the reaction crude.…”

Section: Resultsmentioning

confidence: 99%

But‐2‐ene‐1,4‐diamine and But‐2‐ene‐1,4‐diol as Donors for Thermodynamically Favored Transaminase‐ and Alcohol Dehydrogenase‐Catalyzed Processes

et al. 2016

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Abstract. cis-and trans-But-2-ene-1,4-diamine have been prepared and efficiently applied as sacrificial cosubstrates in enzymatic transamination reactions. The best results were obtained with the cis-diamine. The thermodynamic equilibrium of the stereoselective transamination process is shifted to the amine formation due to tautomerization of 5H-pyrrole into 1H-pyrrole, achieving high conversions (78-99%) and enantiomeric excess (up to >99%) by using a small excess of the amine donor. Furthermore, when the reaction proceeded, a strong coloration was observed due to polymerization of 1H-pyrrole.A structurally related compound, cis-but-2-ene-1,4-diol, has been utilized as cosubstrate in different alcohol dehydrogenase (ADH)-mediated bioreductions. In this case high conversions (91-99%) were observed due to a lactonization process. Both strategies are convenient from both synthetic and atom economy points of view in the production of valuable optically active products.

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“…The designed sensing mechanism was based on nucleophilic addition by the three equivalent amino groups of the dye to the carbonyl moiety of cyclohexanone in the formation of an imine species [46][47][48][49], leading to changes in the UV-vis absorption band that allows for the direct identification of low or high concentration by the naked eye (Figure 3).…”

Section: Sensor Responses Of Gaseous Cyclohexanonementioning

confidence: 99%

Nanoporous Silica-Dye Microspheres for Enhanced Colorimetric Detection of Cyclohexanone

2018

Chemosensors

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Forensic detection of non-volatile nitro explosives poses a difficult analytical challenge. A colorimetric sensor comprising of ultrasonically prepared silica-dye microspheres was developed for the sensitive gas detection of cyclohexanone, a volatile marker of explosives 1,3,5-trinitro-1,3,5-triazinane (RDX) and 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX). The silica-dye composites were synthesized from the hydrolysis of ultrasonically sprayed organosiloxanes under mild heating conditions (150 • C), which yielded microspherical, nanoporous structures with high surface area (~300 m 2 /g) for gas exposure. The sensor inks were deposited on cellulose paper and given sensitive colorimetric responses to trace the amount of cyclohexanone vapors even at sub-ppm levels, with a detection limit down to~150 ppb. The sensor showed high chemical specificity towards cyclohexanone against humidity and other classes of common solvents, including ethanol, acetonitrile, ether, ethyl acetate, and ammonia. Paper-based colorimetric sensors with hierarchical nanostructures could represent an alternative sensing material for practical applications in the detection of explosives.

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A rapid, sensitive colorimetric assay for the high-throughput screening of transaminases in liquid or solid-phase

Cited by 64 publications

References 35 publications

Biocatalysis in seawater: Investigating a halotolerant ω‐transaminase capable of converting furfural in a seawater reaction medium

Biocatalysis in seawater: Investigating a halotolerant ω‐transaminase capable of converting furfural in a seawater reaction medium

But‐2‐ene‐1,4‐diamine and But‐2‐ene‐1,4‐diol as Donors for Thermodynamically Favored Transaminase‐ and Alcohol Dehydrogenase‐Catalyzed Processes

Nanoporous Silica-Dye Microspheres for Enhanced Colorimetric Detection of Cyclohexanone

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