Enhancement of phytase production in recombinant Escherichia coli by medium components modification and fed-batch...

This study focused on phytase overproduction in recombinant E. coli BL21(DE3)/pET-appA expressing E. coli phytase(APPA). A modified mineral salt medium was investigated for the strain growth and phytase production in shake-flask, which included 30 mM NH₄OH and 17 g L^-1 KH₂PO₄ as sole nitrogen(N) and phosphorous(P) sources, respectively. After that, fed-batch cultivation process, especially induction strategy feeding lactose intermittently was optimized in 30L bioreactor by Taguchi orthogonal experiments (L₁₈(2¹ × 3⁶)) at an induction cell density of OD₆₀₀ 25, followed by research on effect of induction cell densities. The optimized fed-batch cultivation process parameters included glucose starvation time of 15 min just before induction, induction cell density of OD₆₀₀ 45, induction temperature of 25 °C, predetermined specific growth rate of 0.04 h^-1, DO level of 15%, lactose concentration of 60 mM, lactose feed times of 6 times, and pH of 7.2-7.6. In the optimized condition, the highest soluble phytase activity of 2 152U mL^-1 ever reported was obtained at 33.0 h, indicating the enhancements of 31.6-fold and 3.7-fold compared to batch cultivation process in shake flask and pre-optimized fed-batch cultivation process in the bioreactor, respectively. We propose that the defined medium, the induction strategy and fed-batch process parameters could be applied to overproduce soluble proteins in E. coli.