EFFECT OF PH AND AERATION ON PLASMID STABILITY AND PHYTASE EXPRESSION IN Escherichia coli BL21(DE3) DURING BATCH CULTIVATIONS IN SEMI-SCALE BIOREACTOR.

摘要

Phytase (myo-inositol hexakisphosphate) has become important feed additive for monogastric livestock. The enzyme action activity is by hydrolyzing phytic acid to myo-inositol derivates and inorganic phosphorus. Since the digestive tract of monogastric animal including pigs and poultry lack phytase, organic phytic acid that naturally exist in the feed cannot be utilized by these animals and thus inorganic phosphate has to be added to meet sufficient phosphate supply (11). Furthermore E. coli was widely used bacterial platform nowadays for expressing simple heterologous proteins such as phytase (8). E. coli is facultative anaerobic bacterium which capable to live either in the presence of oxygen or absence of oxygen (24). Unfortunately, under normal growth condition E. coli can only express very low phytase activity (6). Growing the E. coli to high cell density and improving the volumetric productivity is the major objective of any E. coli based process. Since expression of heterologus protein by recombinant E. coli is relative to the final cell density and the specific productivity of the host cells (4). One of the key factors in obtaining the aim is aeration supply. Scale-up processes for HCDC of recombinant E.coli for protein expression from shake flasks to fermentors generally have low protein expression great difference between shake flasks and fermentors is the supply and limitation of oxygen (15, 18, 23, 25). However, oxygen supply has been reported to have variable effects on heterogeneous gene expression in recombinant E.coli cells would increase or decrease the expression of heterogeneous proteins that depends on the strain and types of plasmid used.To obtain high cell density cultivation (HCDC) and recombinant phytase production, a high capacity of oxygen through aeration supply is required. The formation of acetate during aerobic growth is the main problem. Overproduction of acetate, also known as metabolism overflow eventhough cultivation is done under aerobic condition with excess glucose. This occurs when conversion of glucose to acetate is high compared to the conversion of glucose to biomass and carbon dioxide (18). Assimilation of acetate as low as 0.5 g L~(-1) in the fermentation broth is the main problem in the HCDC cultivation of recombinant E.coli or wildtype E.coli strain due to low cell growth and volumetric yield of the expression protein (10, 19,20,26). Previous researchers found that the suitable aeration supply to the bioreactor culture can improve the cultivation performance of E. coli and also the production of the expression. Oxygen supply can be controlled by manipulating the aeration rate and agitation speed (12).The main purpose of the investigations reported here was to study the growth kinetics of E. coli BL21(DE3) in different pH condition and aeration rate in the semi scale 16L bioreactor by analyzing cell growth, acetate accumulation, plasmid stability, glucose consumption and distribution of phytase.