Abstract
The recombinant Escherichia coli harboring pPT/proCPB (procarboxypeptidase B) gene was constructed for the overexpression of rat proCPB gene. The proCPB expression was controlled by the rrnB P2 promoter fused with lac operator. In the fed batch fermentations, the expression of proCPB was accelerated by the temperature shift from 30 to 37°C without lac operon inducers such as isopropyl-1-thio-β-d-galactoside (IPTG) and lactose. Fermentation strategies including the 3-step increase was optimized to harvest high titers of cell growth and ProCPB. The ideal results of optical density 80 and 66.7% of ProCPB content were obtained through the optimized 3-step shift fed-batch fermentation from 30 to 37oC for 6 hr. After refolding and activation of ProCPB to CPB by trypsin treatment, the CPB activity of 39,375 U/L with specific activity 135 U/mg was obtained in the culture broth. In the conversion reaction by ProCPB, preproinsulin was successfully transformed into insulin.
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