反应详情

For the determination of transglycosylation catalytic activity the mixture of cell lysates containing both UPase and PNPase were prepared by mixing the lysates so as to have UPase:PNPase enzymatic-activity ratio of about 1:1, determined as in Examples 6 and 7. Transglycosylation reaction was carried out at analytical scale in the following conditions: 250 μl of cell lysates (equivalent to 14 units of each UPase and PNPase enzymatic activities) was added to 10 ml of a solution having the following composition: 4 mM 1-β-D-ribofuranosyluracil (uridine nucleoside), 4 mM adenine base, 30 mM potassium phosphate buffer pH7, thermostatically controlled at 60° C. After 1.5 hours at 60° C., the reaction was stopped by diluting the mixture 1:5 and cooling in ice. The percentage of bioconversion of adenine base to 9-β-D-ribofuranosyladenine (adenosine nucleoside) was determined by analyzing an aliquot of the reaction mixture by high performance liquid chromatography (HPLC) with the use of Kromasil 100-5C18 (Akzo Nobel) column of a size of 250×4.6 mm and eluted with a 4% methanol-water solution. The transglycosylation catalytic activity was expressed as units·ml−1 (μmoles of Ara-A formed in 1.5 hours·ml−1 of mixture of cell lysates) or in units·g−1 of moist resin (μmoles of D-ribofuranosyladenine formed in 1.5 hours·ml−1 of cell lysate) and was calculated relative to a standard D-ribofuranosyladenine solution eluted by HPLC in the same conditions. Under these conditions, about 55 percent of adenosine nucleoside was formed (approximately 9 units per ml of cell lysate).

1. additionFor the determination of transglycosylation catalytic activity the mixture of cell lysates
2. additioncontaining both UPase and PNPase
3. customwere prepared
4. additionby mixing the lysates so as
5. customTransglycosylation reaction
6. customthermostatically controlled at 60° C
7. additionby diluting the mixture 1:5
8. temperaturecooling in ice
9. washeluted with a 4% methanol-water solution