Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection

Gawronski, Jeffrey D.; Benson, David R.

doi:10.1016/j.ab.2003.12.024

Cited by 97 publications

(98 citation statements)

References 21 publications

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“…Their phosphatase activity on both 30 mM αGlc1P and βGlc1P in 30 mM MOPS buffer pH 7 was tested at 37°C in 1.7 mL Eppendorf tubes in a heating block. Samples were inactivated for 10 min at 95°C and subsequently analyzed for their phosphate concentration using the phosphomolybdate method [19]. All reactions were performed in triplicate.…”

Section: Purification and Crystallization Of β β β βGlc1pmentioning

confidence: 99%

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

Borght¹,

Desmet²,

Soetaert³

2010

Biotechnology Journal

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beta-D-Glucose-1-phosphate (beta Glc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of beta Glc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60 C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, beta Glc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what ist observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline beta Glc1P is now available from the cheap substrates trehalose and inorganic phosphate

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Section: Purification and Crystallization Of β β β βGlc1pmentioning

confidence: 99%

Enzymatic production of β‐D‐glucose‐1‐phosphate from trehalose

Borght¹,

Desmet²,

Soetaert³

2010

Biotechnology Journal

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“…The determination of inorganic phosphate in the presence of acid-labile organic phosphate was based on the method of Gawronski and Benson [12], but had to be modified because molybdate was already present as phosphatase inhibitor in the reaction mixture. …”

Section: Determination Of Inorganic Phosphatementioning

confidence: 99%

“…Initial reaction velocities were determined by measuring the amount of released inorganic phosphate using the original phospomolybdate method [12]. The k cat value was calculated assuming a molecular mass of 91.4 kDa for the catalytic unit (one monomer).…”

Section: Characterization Of Enzyme Variantsmentioning

confidence: 99%

“…In this method, a phosphomolybdate complex is reduced under acidic conditions resulting in the formation of a molybdenum blue compound which can be measured spectrophotometrically. The original method could be improved using ascorbic acid as a reducing agent instead of ferrous sulfate and by adding stop solution to stop color development which is useful in the presence of acidlabile organic phosphates [12]. The assay is cheap, fast, sensitive and robust, and thus convenient for high-throughput screening.…”

Section: Introductionmentioning

confidence: 99%

“…It appeared necessary to inhibit endogenous E. coli phosphatase activity in crude cell extracts because it causes high background values and consumes the glycosyl phosphate substrate. For the measurement of inorganic phosphate, a modified phosphomolybdate method was developed based on the method of Gawronski and Benson [12] and the screening procedure was evaluated using C. uda CP as a model. CP uses α-glucose 1-phosphate (αGlc1P) as glycosyl donor and glucose as acceptor.…”

Section: Introductionmentioning

confidence: 99%

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Development and application of a screening assay for glycoside phosphorylases

Groeve

Tran

Hoorebeke

et al. 2010

Analytical Biochemistry

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Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They only require a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here, we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen an E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity towards alkyl β-glucosides and phenyl β-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.

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