High cell density culture of E.coli and high expression of recombinant alginate lyase

In order to find the optimized condition to produce alginate lyase, using fed-batch culture technology, the engineered E.coli BL21 was cultured in 5L fermented at high density.The use of single-factor test to optimize the concentration of carbon source in the feed medium, single factor tests and simplex optimization method to optimize the induction time and IPTG concentration, to obtain optimal high-density fermentation conditions:fermentation medium glucose 10g/L, yeast extract 5g/L, peptone 20g/L.Feed medium was glucose 150g/L, peptone 20g/L, yeast extract 10g/L.The stream acceleration rate was set to 100mL/h form 4h to 10h.The stream acceleration rate was set to 200mL/h form 10h to16h.Induction time was at 4.5h, and the IPTG was final concentration to 0.60mmol/L.Dissolved oxygen in the fermentation process wad controlled in the 30% to 40%, and pH was controlled at 7.0～7.2.The results showed that in un-induced condition, the OD₆₀₀ was 0.696 when final fermentation liquid were diluted 200 times.The engineered E.coli BL21 concentration was 65.38g/L.After induce with IPTG, the value of OD₆₀₀ was 0.457 when fermentation liquid were diluted 200 times.The concentration of the engineered E.coli BL21 was 60.15g/L, which was 8.43 time compared with the value from the batch fermentation.Finally, the crude enzyme was extracted from the high density fermentation strain by ultrasonication.The enzyme activity was 26.37U/mL, which was up to 5.48 time compared with the batch fermentation.