Purification and Characterization of Recombinant Aeromonas punctata Prolyl Endopeptidase
10.3321/j.issn:1000-3061.2000.03.012
- VernacularTitle:重组点状产气单胞菌脯氨酰内肽酶的纯化和鉴定
- Author:
Min LI
1
;
Chang-Qing CHEN
;
De-Bao WANG
Author Information
1. Shanghai Research Center of Biotechnology, The Chinese Academy of Sciences
- Keywords:
Prolyle endopeptidase;
high cell-density fermentation;
purification;
specific activity;
characterization
- From:
Chinese Journal of Biotechnology
2000;16(3):345-348
- CountryChina
- Language:Chinese
-
Abstract:
The study of down-stream techniques of recombinant Aeromonas punctata proiyl endopeptidase (apPEP) was presented here. High cell-density fermentation of E. coli BL21/pKKH-PEP in NBS BioFlo 3000 5L fermentor was achieved, the final cell density was 22.5g (DCW)/L after 14h cultivation, the yield of apPEP expressed in soluble protein was 3.0g 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 65.5u/mg, apPEP yield reached 0.86g/L broth. Total recovery of enzyme protein was 8.2 % , actviity recovery was 24.4 %. The molecular weight of apPEP was 76464±30Da 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.
