Effects of Induction Starting Time and Ca~(2+)on Expression of Active Penicillin G Acylase in Escherichia coli

摘要

Formation of inclusion bodies is an important obstacle to the production of active recombinant protein in Escherichia coli.Thus,soluble expression of penicillin G acylase from Kluyvera citrophila was investigated in BL21(DE3).In this study,the yield of active enzyme was significantly enhanced by the composition of the medium and induction opportunity.When 0.5 mmol/L IPTG was added to complex medium at 15 h after incubation,the volumetric and specific activities of penicillin G acylase both achieved the highest values,respectively.However,aggravation of intracellular proteolysis and decline of enzyme expression were also observed if induction occurred too much later.Ca~(2+)ion was another critical factor in cell growth and protein expression.When 24 mmol/L Ca~(2+)ion was adding to the medium at the beginning of fermentation,a greater than 2-fold increase in cell density and a 7-fold increase in volumetric activity of penicillin G acylase were reached.Nevertheless,no significant benefit for recombination protein expression was found when excess Ca~(2+)was added after induction time.This study demonstrates that the induction starting time and Ca~(2+)ion are two critical factors for the expression of active penicillin G acylase.