Research On Improving Recombinant E.coli To Express Cholesterol Oxidase

The significant functions of cholesterol oxidase (EC 1.1.3.6, COD) in food processing, medical assay and biological pest-resistance were recognized gradually and showed potentially use. This research is mainly focused on optimizing fermentation of recombinant E.coli to improve the level of enzyme production.The production of cholesterol oxidase was studied using recombinant E.coli JM109, optimized the fermentation medium, inducing conditions and environmental conditions. Optimization of culture medium is glycerol 10g/L,tryptone 10g/L,Yeast power 5g/L, and the best induction time is intermediate cell density , optimization of IPTG concentration is 0.3mmol/L and the inducing time is 6h. The environmental conditions is incolation size 5%(V/V), intial pH 7.5 cultured at 37℃on rotary shaker at 180r/min and the suitable volume of medium was 100 mL in 500 mL flask. Under the optimized cultivation conditions, the enzyme activity reached 1686.57U/L, which is 2.4 times of 700 U / L before optimization.Through research on different substrates'fed-batch, recombinant E.coli's high density culture was realize. It is found that though pH-stat method can get relativelly high dry cell weight 42.68g/L(DCW),its fermentation cycle is long(36h), which is not beneficial for production intensity's improvement. Exponential feeding strategy that the specific growth rate was controlled at different times has realized short term high density culture, with DCW reaching 40.13g/L at 14th hour. It's also found that inducing recombinant E.coli to express Cholesterol oxidase at intermediate cell density is good. And the highest cholesterol oxidase activity (6436.35 U/L,which is 9.2 times of 700 U / L before optimization) and highest cholesterol oxidase productivity (459.75 U/(L?h)) were achieved.Through the experiment that cheap and innoxious lactose instead of dear and noxious IPTG induce recombinant E.coli to express COD. It was found that lactose could induce foreign protein expression and enhance cell growth during the induction period. It's also found that during the inducer of adding lactose, E.coli has about 10h's adaption period to lactose. Through research on compound interflow with carbon substrate (glycerine and lactose) to induce recombinant E.coli to express COD, it was found this method didn't eliminate E.coli's adaption period to lactose, while pH-stat method's fed-batch overcomed negative effect due to long term's starvation of adaption period. Its enzyme production reached 6005.66U/L, 93.3% of IPTG's inducing level; cholesterol oxidase productivity 200.19 U/(L·h),47.4% of IPTG's inducing level.