A colorimetric assay for screening transketolase activity

Smith, Mark E.; Kaulmann, Ursula; Ward, John M.; Hailes, Helen C.

doi:10.1016/j.bmc.2006.06.008

Cited by 49 publications

(41 citation statements)

References 16 publications

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“…The D469 library displayed good activities with no inactive mutants generated, and single-point mutants that enhanced and reversed the stereoselectivities were identified. It should be noted that the GC assay was more sensitive (> 1-2% bioconversions detected) than the colorimetric assay (> 8% bioconversions detected [20] ), so mutants with low activities not readily detected due to a faint coloration the colorimetric assay, were identified in the GC assay. Mutants showing good ees and conversions to 4, from the intense coloration in the colorimetric screen, were investigated further and sequenced as Table 2).…”

Section: Stereoselectivities With Active-site Single-point Tk Mutantsmentioning

confidence: 98%

“…Reaction plates of the E. coli TK libraries obtained by saturation mutagenesis of H26, D469, and H100 were incubated with propanal and 1 prior to screening for product 4 with a tetrazolium-based colorimetric assay [20] and for product ee using the GC assay. Both assays were successfully applied to a 96-well format allowing for screening in a high-throughput manner (representative data for colorimetric assay in Figure 2 for D469).…”

Section: Stereoselectivities With Active-site Single-point Tk Mutantsmentioning

confidence: 99%

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Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations

et al. 2008

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Chiral auxiliary methodology and chiral assays have been developed to establish the enantiomeric purities of erythrulose and 1,3-dihydroxypentan-2-one generated using wild-type (WT) Escherichia coli transketolase (TK). l-Erythrulose was formed in 95% ee and (3S)-1,3-dihydroxypentan-2-one in 58% ee. Since the latter compound was formed in moderate ee, TK libraries were screened to identify higher performing mutants. A colorimetric screen and chiral assay were successfully applied to a 96-well format, and new active TK mutants were identified, which gave 1,3-dihydroxypentan-2-one in high stereoselectivities. Remarkably, activesite single-point mutants were identified that were able to both enhance and reverse the stereoselectivity of TK.

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Section: Stereoselectivities With Active-site Single-point Tk Mutantsmentioning

confidence: 98%

Section: Stereoselectivities With Active-site Single-point Tk Mutantsmentioning

confidence: 99%

Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations

et al. 2008

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“…The results are given in Table III. Several authors have contributed to the determination of the substrate specificity of TK enzymes from different sources (E. coli, S. cerevisiae, G. stearothermophilus) using different assay methods towards various phosphorylated and unphosphorylated aldoses or aldehydes. 4,[14][15][16][17][18][19][20] The activity profiles of TKs are very similar, owing to the strong homology of active sites. Our system yields the same results as those obtained with TK gst using other methods.…”

Section: Determination Of Tk Acceptor Specificitymentioning

confidence: 99%

“…Measurement of chiral product formation by an optical method 16,17 is highly dependent on the substrate structure, and so not of generic utility. Determination of HPA depletion by near-UV spectroscopic monitoring 18 or HPLC (High Performance Liquid Chromatography) analysis [14][15][16][17][18][19][20] is hampered by low sensitivity or low throughput. Colorimetric determination of ketose formation with tetrazolium red-based oxidation is restricted to non-hydroxylated aldehyde acceptors such as propanal.…”

Section: Introductionmentioning

confidence: 99%

“…Colorimetric determination of ketose formation with tetrazolium red-based oxidation is restricted to non-hydroxylated aldehyde acceptors such as propanal. 16 A major disadvantage of all methods that involve substrate/product determination is that they are often discontinuous, and do not allow direct measurement of enzyme kinetics. We have recently described an assay, independent of the substrate and the product, and based on direct monitoring of pH changes with phenol red as pH indicator.…”

Section: Introductionmentioning

confidence: 99%

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Droplet millifluidics for kinetic study of transketolase

et al. 2016

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We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme. The oxaloacetate formed is reduced to malate by NADH in the reaction catalysed by malate dehydrogenase as the second auxiliary enzyme. The extent of oxidation of NADH was measured by spectrophotometry at 340 nm. This system gives a direct, quantitative, generic method to evaluate the TK activity versus different substrates. We demonstrate the accuracy of this strategy to determine the enzymatic kinetic parameters and to study the substrate specificity of a thermostable TK from thermophilic microorganism Geobacillus stearothermophilus, offering promising prospects in biocatalysis. Millifluidic systems are useful in this regard as they can be used to rapidly evaluate the TK activity towards various substrates, and also different sets of conditions, identifying the optimal operating environment while minimizing resource consumption and ensuring high control over the operating conditions. Published by AIP Publishing. [http://dx

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Handbook of Biological Dyes and Stains

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Physical Form Bright orange-yellow powder Solubility Freely soluble in water; soluble in ethanol, cellosolve; insoluble in xylene Melting Point 300 C Absorption (l max ) 425 nm Synthesis Synthetic methods 1-25 ,91 dermal toxicity; 92 genotoxicity; 93-96 hyperactive behavior in children; 97 mutagenicity; 98-106 neurotoxicity; 107 pseudoallergic reactions; 108 reproductive toxicity 107 Ruoff, M. K.; Vittum, P. W. The formation of azomethine dyes from 4-anisylideneand 4,4 0 -anisylidenebispyrazolones. J. Am. Chem. Soc. 1950, 72, 4947-4949. 21. Freeman, K. A.; Jones, J. H.; Graichen, C. Coal-tar colors. VIII. FD&C yellow No. 5. J. Assoc. Off.

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A colorimetric assay for screening transketolase activity

Cited by 49 publications

References 16 publications

Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations

Enhancing and Reversing the Stereoselectivity of Escherichia coli Transketolase via Single‐Point Mutations

Droplet millifluidics for kinetic study of transketolase

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