A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay

Moretti, Rocco; Thorson, Jon S.

doi:10.1016/j.ab.2008.03.018

Cited by 64 publications

(46 citation statements)

References 64 publications

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“…However, initial pHBH assays revealed that the presence of the Ni 2ϩ -NTA-agarose greatly reduced assay sensitivity. However, consistent with the well documented detrimental effect of metal ions (such as Ni 2ϩ ) on the pHBH reaction (36,56), the addition of 50 mM EDTA to the sodium hydroxide reagent was sufficient to restore sensitivity (Fig. 2C).…”

Section: Resultsmentioning

confidence: 50%

“…The one previous report of directed evolution for this enzyme class was restricted to native substrates and a substrate-inflexible biological readout (35). Herein we describe the application of a recently developed general high-throughput screen for sugar 1-phosphate toward RmlA directed evolution (36). In this study, epPCR and site-saturation techniques were combined to identify a number of improved variants for both the NTP and sugar 1-phosphate substrates.…”

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confidence: 99%

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Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

Moretti

Chang

Peltier‐Pain

et al. 2011

Journal of Biological Chemistry

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Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine-and purine-based NTPs as well as non-native D-and L-sugars (both ␣-and ␤-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.

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Section: Resultsmentioning

confidence: 50%

mentioning

confidence: 99%

Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

Moretti

Chang

Peltier‐Pain

et al. 2011

Journal of Biological Chemistry

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“…[14d] DNS is an aromatic compound that reacts with free sugars to form 3-amino-5-nitrosalicylic acid to show distinct colorimetric properties which can be detected at 575 nm. The DNS assay with the detection limit ranging from 500-5000 nmol, is rarely influenced by protein [32] and has been used in activity determination of other bacterial GalKs.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3

Hsiao

et al. 2014

Adv Synth Catal

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Herein, we report the first characterization of a novel galactokinase from Meiothermus taiwanensis sp. nov. WR-220 (MtGalK), which is overexpressed in E. coli and exhibits a k cat /K m value of 168.47 mM À1 s À1 toward Gal at 75 8C. The thermophilic MtGalK shows specific substrate recognition toward the Gal configuration with limited tolerance for modifications at the C-2 position to form products such as GalN-1-P, GalN 3 -1-P, and GalNAc-1-P. Due to its unique thermostability toward elevated reaction temperatures, MtGalK served as an ideal biocatalyst in the synthesis of useful sugar-1-phosphates and was further combined with glucose-1-phosphate thymidylyltransferase (RmlA) and a-1,4-galactosyltransferase (LgtC) to achieve the efficient preparative-scale synthesis of globotriose analogs (Gb3) using a one-pot three-enzyme system. In combination with a chemical synthetic strategy, a carbohydrate antigen from breast cancer stem cells, stage specific embryonic antigen-3 (SSEA-3) pentasaccharide, was synthesized from Gb3 in ten steps with a 23% overall yield. This flexible chemoenzymatic strategy for the synthesis of SSEA-3 also allowed us to further expand the inventory of valuable natural and non-natural glycans.

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“…These characteristics ultimately result in time-consuming measurements and consequently reduce throughput. Moreover, although the FPU [9] and DNS assays [10] have been adapted to the microscale level, there are more sensitive microscale assays available [11], including the p-hydroxybenzoic acid hydrazide (pHBAH) assay. More sensitive assays are especially useful for the realization of short hydrolysis times at low conversion levels with lignocellulosic substrates.…”

Section: Technical Reportmentioning

confidence: 99%

Sensitive high‐throughput screening for the detection of reducing sugars

Mellitzer

Glieder

Weis³

et al. 2011

Biotechnology Journal

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The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 μM. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity-based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei β-mannanase, or T. reesei cellobiohydrolase 2.

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A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay

Cited by 64 publications

References 64 publications

Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3

Sensitive high‐throughput screening for the detection of reducing sugars

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