p-Nitrophenyl beta-D-glucopyranoside → (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal

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Reaction Steps

Specific activity. 4-Nitrophenyl-beta-D-glucopyranoside (pNPG) was dissolved in DMSO to produce a 100 mM stock solution. Right before assay, the 100 mM pNPG stock solution was diluted 100-fold in 50 mM sodium acetate pH 5 with 0.01% TWEEN® 20 to 1 mM pNPG. Enzyme was diluted to a wide range of concentrations in the same buffer. To 20 μl of the enzyme dilutions (a range of concentration), 100 μl of the 1 mM pNPG solution were quickly added. The reaction was incubated at 40° C. for 20 minutes before the reaction was stopped with 50 μl of 1 M sodium carbonate pH 10, and the absorbance at 405 nm was measured for 4-nitrophenolate production. The specific activity of the Penicillium oxalicum GH3 beta-glucosidase on pNPG was determined to be 72.3±1.7 IU/mg, where one mg of enzyme produce 1 μmole of glucose per minute at 40° C., pH 5.

Provenance & Evidence