p-Nitrophenyl beta-D-glucopyranoside → (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal
Reaction equation
C1C([N+]([O-])=O)=CC=C([O:10][C@@H:11]2[O:16][C@H:15]([CH2:17][OH:18])[C@@H:14]([OH:19])[C@H:13]([OH:20])[C@H:12]2[OH:21])C=1>CS(C)=O.C([O-])(=O)C.[Na+].C1C([N+]([O-])=O)=CC=C(O[C@@H]2O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]2O)C=1>[O:10]=[CH:11][C@@H:12]([C@H:13]([C@@H:14]([C@@H:15]([CH2:17][OH:18])[OH:16])[OH:19])[OH:20])[OH:21]Participants
Procedure
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.
Workup
- customto produce a 100 mM stock solution
- additionEnzyme was diluted to a wide range of concentrations in the same buffer
- concentrationTo 20 μl of the enzyme dilutions (a range of concentration), 100 μl of the 1 mM pNPG solution
- additionwere quickly added
Sources & evidence
- ORD record
- ord-9658204289084a37a45df1f414d1e3aa
- Source dataset
- uspto-grants-2013_11
- Patent number
- US08581042B2
Reaction notes
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