Enzymatic production and purification of an RNA intermediate, a key chemical in manufacture of cancer drugs, were carried out using a recombinant Escherichia coli as a whole cell catalyst.
When the RNA intermediate was produced by enzymatic reaction, its conversion yield was significantly affected by reaction conditions. The optimal pH and temperature was pH 5.0 and 36℃ (conversion yield of 70.9%). When the substrate A was added to the reactor at a rate of 0.05 to 0.6 g/min (per unit volume of reaction mixture), there wasn’t significant difference in conversion yield. The addition of 1% (w/v) enzyme was enough to obtain the highest conversion yield. However, faster adding rates of substrate A and lower loading amounts of enzyme were preferred in order to reduce the degradation of the RNA intermediate and the reverse reaction.
A strong cation ion-exchange resin was used to separate the RNA intermediate. Under the condition of zero ionic strength, it was separated with a high selectivity from substrate B and by-product Q.
In conclusion, the RNA intermediate can be produced with the conversion yield of 70.9% by a whole cell enzyme system. Also, an ion-exchange resin showed a high selectivity in separation of the RNA intermediate.