Bioprocess strategies to optimize thermostable phytase production by Escherichia coli B121 (DE3) when induced with lactose

Abstract

The recombinant Escherichia coli BL21(DE3) that carries a thermostable phytase gene from Bacillussp. MD2 and lactose as an inducer was used in this study. This research aims to study the influenced of induction strategy to maximize expression of phytase by E. coli BL21(DE3) and leakage of phytase out of cell membrane in the shake flask cultivation by a statistical approach of response surface methodology (RSM) based on Box-Behnken design in shake flask cultures. The interaction of the variables that affects the induction strategy was studied between three different levels of induction components such as lactose, CaCl2 and glycine. E. coli BL21(DE3) was grown in the synthetic glycerol-minimal medium with 1mM of ampicillin. Finally, the optimal concentration of lactose, CaCl2 and glycine obtained using statistical optimization of induction strategy is approximately 25.68 mM, 11.32 mm and 0.65 % (w v-1), respectively. This resulted reduced of post-induction phase from 12 hours to 6 hours with the productivity of the total phytase production and total phytase activity was increased up to 116.09 % and 29.65 %, respectively. In this study, more than 90 % of the total phytse activity was excreted outside the cell membrane when compared to un- optimization induction strategy only 76.05 %. This work has demonstrated the use of the second order polynomial equation to determine conditions leading to the maximum total phytase activity, excretion of phytase outside the cell membrane and minimize acetic acid production. In conclusion, the factor that significantly influenced cell growth, phytase production and accumulation of acetic acid by E. coli BL21 (DE3) in the fermentation broth was lactose. Furthermore, the comparison studied of the induction phase with and without addition of ampicillin was studied. The cultures without the addition of ampicillin grew faster than cells with the selected plasmid with the specific growth rate of 0.16 h-1 which gave high acetic acid production (max=1.55 g L-1). This contributed to rapid declined of plasmid lost after 2 hours of induced with lactose. Finally, the new developed of medium composition and induction strategy can be used for the study in 16 L bioreactor without the addition of ampicillin since the productivity of phytase expression decreased approximately only 18.96 % with more than 92 % was excreted outside the cell membrane.