[The bottleneck steps limiting maturation of penicillin G acylase in Escherichia coli].
We have identified the bottleneck steps limiting maturation of penicillin G acylase (PAC) through comparison of the maturation performance for various PAC-expression systems (Pac, Tac, T7, Vgb + T7) with different efficiencies of proteolysis, subunit folding and assembly. The maturation of PAC could be limited by various steps, such as translocation, periplasmic proteolysis, subunit folding and assembly depending on the host/vector systems. In BL21(pPA6) cells, maturation of PAC were limited by proteolysis and folding steps; the efficiency of proteolysis was 57.2%; the subunit folding and assembly capacity was 0.72. In BL21(pKKpacSP) cells, the stability and folding of alpha subunit was bottleneck steps. In T7 and dissolved-oxygen regulation expression systems, PAC proprecursor could be maturated efficiently. Results also indicate that the folding of alpha peptide plays a key role in folding of precursor for PAC in E. coli. Developing proper host/vector systems and fermentation technology with superior abilities on subunit folding and assembly of precursor for PAC could be plausible for enhancing production of PAC. In this study, pac could be expressed (transcribed, translated and maturated) efficiently under the control of T7 promoter.
Xu
J N
JN
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China. fanchao@npc.haplink.com.cn
Yang
Y G
YG
Gong
Y
Y
Yang
S L
SL
Yu
J T
JT
D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D010399:Penicillin Amidase; D010100:Oxygen