EXPRESSION OF HYPER-THERMOPHILE GENE IN HIGH-DENSITY CULTURE OF RECOMBINANT E.coli BY PH-STAT STRATEGY

Three methods of glucose determination during high density-culture of recombinant E.coli were compared. The 3,5-Dinitrosalicylic acid method was proved to be interfered seriously in this experiment. The strategy of fed-batch culture in 40L fermentator was based on the result of GOD-POD method. Feeding solution was added when the pH rose to a value greater than its set point (pH6.80) by 0.08 or 0.04, 0.02, 0.01. The pH-Stat strategy was used based on the higher biomass produced by the pH6.80+0.01. The Pyrococcus furiosus gene coding for a-Amylase was expressed in E.coli BL21 (DE3) plysS. In order to produce a-Amylase in large amounts, pH-Stat fed-batch culture was carried out in 40L Biostat Fermentator. The glucose concentration was lower than 0.6g ?L"1 during fed-batch culture. After 12h feeding, the cell optical density at 600nm (ODsoo) was 105, the dry cell weight (DCW) reached as high as 51.5g ?L"'and the acetic acid was only 6.26g ?L"1. When cells were induced by IPTG at OD6oo of 65.1, the optimal activity of a-Amylase was 972U ?ml"1. The expression of the recombinant a-Amylase was accounted for 5.4% of total soluble proteins.The expressions between BL21 (DE3) plysS and BL21-Codon Plus (DE3) -RIL which had rare codon tRNA genes were compared. The recombinant a-Amylase level of BL21-Codon Plus (DE3) -RIL was twice as high as that of conventional BL21 (DE3) strain.The expressions between IPTG induction and lactose induction were compared. The recombinant a-Amylase level of lactose induction was 56% of thatof IPTG induction.