Enzymatic production of β-D-glucose-1-phosphate from trehalose
Résumé
β-D-glucose-1-phosphate (βGlc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This paper provides an efficient production method of βGlc1P 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 that way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, i.e. either anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast followed by chemical phosphate precipitation. In a next step, βGlc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is 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 βGlc1P is now available from the cheap substrates trehalose and inorganic phosphate.
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