Improving Thermostable Phytase Production in Recombinant Escherichia Coli During Fed-Batch Cultivation in Semi-Industrial Scale Stirred Tank Bioreactor

The expression of thermophile phytase gene from Bacillus sp. MD2 under the control of lac-0 promoter by E. coli BL21 (DE3) was studied in 16L bioreactor. This is to investigate the expression of thermostable phytase in relation to the effect of continuous glycerol feeding in the post-induction phase for high cell density cultivation when one pulse induction with lactose was applied. The expression of recombinant protein was induced at the mid of exponential phase by one pulse induction strategies of 30 mM lactose. The challenge of a conventional single induction with lactose is decreased of cell growth after induction that effect lactose uptake as inducer for foreign protein expression. To overcome this problem the strategies should focusing on maintaining the exponential growth at the post-induction phase. Therefore the mid-optimum continuous glycerol feeding consisted of 46 g L^-1 hr^-1 glycerol  can maintained the exponential cell growth with the specific growth rate of 0.20 h^-1. The expression of total phytase activity reached 47.97 U mL^-1 with the specific phytase activity of 3,646.61 U g^-1. Moreover, the excretion as extracellular phytase activity was predominantly excreted outside the cell membrane with the maximum excretion after 7 hour of induction with the volumetric phytase production rate of 6,850.00 U L^-1 h^-1. When compared with maximum and low glycerol feeding rate during the post-induction phase, both showing 50 % decreased of cell growth immediately after induced with lactose and the expression level was very low with the volumetric total phytase production rate only 2,300.00 and 1,310.28 U L^-1 h^-1 respectively.

Keywords: fed-batch cultivation, lac promoter, phytase production, recombinant E. coli.