Abstract: The culture of nucleoside phosphorylase forming E.coli cells in shaking flask and fermenter(5L) was studied.The effect of acetate and substrate inducing on the phosphorylase activity were also investivated.It was shown that the enzyme activity was highest when E.coli cells began to autolyse in the prolonging course of culture in shaking flask and increased by 7.7% or 11.0%,either by adding 10g/L sodium acetate or 0.001g/L dU inducer in fermentation medium respectively.In addition,there was not substrate dU inhibition in enzyme reaction because the obvious change of conversion rate was not observed in dU fed batch process.Diffusion control was appeared in biotransformation and the optimal shaker speed was 160r/min.