Effects of induction starting time and Ca2+ on expression of active penicillin G acylase in Escherichia coli.
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. Ca2+ ion was another critical factor in cell growth and protein expression. When 24 mmol/L Ca2+ 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 Ca2+ was added after induction time. This study demonstrates that the induction starting time and Ca2+ ion are two critical factors for the expression of active penicillin G acylase.
Jiang
Yong Mei
YM
State Key Laboratory of Bioreactor Engineering, Institute of Newworld Biotechnology, East China University of Science and Technology, Shanghai 200237, China.
Tong
Wang Yu
WY
Wei
Dong Zhi
DZ
D019149:Bioreactors / Q000382:microbiology*; D002118:Calcium / Q000008:administration & dosage*; D018929:Cell Culture Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D044086:Kluyvera / Q000201:enzymology* / Q000235:genetics; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors