[Purification and characterization of recombinant Aeromonas punctata prolyl endopeptidase].
The study of down-stream techniques of recombinant Aeromonas punctata prolyl endopeptidase (apPEP) was presented here. High cell-density fermentation of E. coli BL21/pKKH-PEP in NBS BioFlo 3000 5 L fermentor was achieved, the final cell density was 22.5 g (DCW)/L after 14 h cultivation, the yield of apPEP expressed in soluble protein was 3.0 g per litter broth. After sonication, the supernatant of free cell extract was purified by ammonium sulfate fractionation, High performance Q sepharose FF, Phenyl sepharose 6 FF, the purity of apPEP reached 96%, enzyme specific activity was 65.5 u/mg, apPEP yield reached 0.86 g/L broth. Total recovery of enzyme protein was 8.2%, actviity recovery was 24.4%. The molecular weight of apPEP was 76,464 +/- 30 Da measured by MS, N terminus amino acids sequence consistent with that deduced from DNA sequence. pI 6.0, which was similar with PEP from Aeromonas hydrophila.
Li
M
M
Shanghai Research Center of Biotechnology, Chinese Academy of Sciences.
Chen
C Q
CQ
Wang
D B
DB
D000333:Aeromonas / Q000201:enzymology*; D005285:Fermentation; D008970:Molecular Weight; D000085822:Prolyl Oligopeptidases; D011994:Recombinant Proteins / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases