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Objective: To investigate the prolyl oligopeptidase inhibitory constituents from Glehnia littoralis., Methods: To determine the prolyl oligopeptidase inhibitory activity in vitro of ethanol extract of G. littoralis and its fractions with Yoshimoto's method, to separate the constituents of the active fractions by repeated silica gel column chromatogrphy., Results: Two ferulate compounds were isolated from EtOAc-soluble fractions of ethanol extract of G. littoralis, which (100 microg/ml) showed mile activity (inhibitory rate: 50%). Compound I, named glehnilate, was a new biphenyl ferulate. Compound II, identified as 2-(4'-hydroxyphenol)-glycol mono trans-ferulate, was isolated from this plant for the first time. Their structures were determined on the basis of their spectral data.

Prolyl endopeptidase gene was cloned from Aeromonas punctata subsp. Punctata(ST-78-3-3) using activity screening method and the 3.3 kb DNA fragment containing PEP gene was sequenced. DNA sequence from 20-2092 bp was ORF region coding 690 amino acids of prolyl endopeptidase. It was a new PEP gene through gene search. The genetic engineered E. coli BL21/pGEM-PEP overexpressing recombinant Aeromonas punctata prolyl endopeptidase(apPEP) was constructed. Cultured in YH medium, the expressed apPEP was about 30% of total cellular protein, the activity was 112 times more than that of wild strain. Expressed apPEP was mainly soluble intracellular protein, about 5% of apPEP was secreted to medium. Non-reduced SDS-PAGE analysis showed it's monomer with molecular weight about 76 kD, which corresponded with prediction by gene sequence. Recombinant apPEP was purified after tube culture, the purity reached 90% and specific activity was 67 U/mg.

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.

Engineered E. coli BL21/pGEM-PEP could constitutively express recombinant prolyl endopeptidase from Aeromonas punctata, which was extremely effected by culture conditions, so fermentation conditions were optimized to obtain it's high expression level. Firstly, the stability of BL21/pGEM-PEP was investigated, then, culture temperature, pH, time, medium were optimized in shake flaskd. The L9(3(4)) orthogonal experiment confirmed that shaker speed, pH, culture temperature, culture time had high degree statistical meaning. Based on these data, high cell density fermentation of E. coli BL21/pGEM-PEP on NBS BioFlo 3000 5 L fermentor was achieved, after 20 h cultivation, the final density(dwt) was 60OD600(22.5 g/L), the expressed PEP was about 28% of total cellular protein, the yield was 3.15 g per litter broth.