Cell age is considered to be one of important factors that highly influence the protein over-expression (30). It has been reported that younger cell gives a higher yield of phytase from recombinant E. coli BL21(DE3) by using the response surface methodology (17). As a consequence of it, it was compared to start phytase induction at exponential or stationery stage in two-stage fermentation. The specific growth rate in the growth phase was increased to 0.48 h1 but phytase yield decreased by 42% in fermentation III. The overexpression of phytase began rapidly at induction time of 2 h in fermentation II. This demonstrated that the cell growth at stationary phasewas ripe for achieving high yield in the present study. The plasmid stability decreased as the phytase activity increased in fermentation I, II and III (see Figs. 4 and 5), which may be attributed to the metabolic stress arising from the rapid and massive production
of foreign protein under the stronger promoter. The medium cost evaluation was found to be economic in two-stage fermentation than in one-stage fermentation although an extra medium was required (i.e., 0.66105 compared to 1.25105 US$ U1; see Table 2). The phytase activity staining in SDS-PAGE shown in Fig. 6 confirms the higher activity of phytase production in fermentation II and III.