Characterization of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. lactis

Lloyd, Richard; Pritchard, G. G.

doi:10.1099/00221287-137-1-49

Cited by 74 publications

(37 citation statements)

References 27 publications

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“…lactis subsp. lactis were, however, not inhibited by sulfhydryl inhibitors such as iodoacetate or pHMB, indicating that no functional sulfhydryl group was involved in catalysis in these enzymes (Habibi-Najafi & Lee, 1994;Zevaco et al, 1990;Lloyd & Pritchard, 1991). EDTA at concentration of 1 mM had no effect on PepX activity, while 1 ,lo-phenanthroline, another potential metallo-protease inhibitor reduced the activity slightly ( …”

Section: Characterization Of the Purified Pepx Proteinmentioning

confidence: 99%

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus

Vesanto¹,

Savijoki²,

Rantanen³

et al. 1995

Microbiology

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The X-prolyl dipeptidyl aminopeptidase gene (pepx) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 906 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with AG of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 906 kDa protein showed 49*3,494 and 777% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Ladobacillus delbnreckii subsp. Iactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6 5 and 45 OC. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and l,l0-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding Dll, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.

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Section: Characterization Of the Purified Pepx Proteinmentioning

confidence: 99%

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus

Vesanto¹,

Savijoki²,

Rantanen³

et al. 1995

Microbiology

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“…lactis ssp. lactis H1 [15]. Also, this enzyme was able to hydrolyse Ala-Ala-pNA at a slower rate but not Ala-Ala-Ala.…”

Section: Compoundsmentioning

confidence: 84%

“…acidophilus 357 [2]. Dimeric PepXs with molecular masses between 117-175 kDa were also reported for some strains of Lactococcus and Lactobacillus [1,3,5,15,22,36]. In addition to dimeric PepXs, there are also reports of monomeric PepXs [8,10,24,27] and a trimeric PepX isolated from Lb.…”

Section: Compoundsmentioning

confidence: 97%

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Purification and characterization of an X-prolyl-dipeptidyl aminopeptidase from Lactobacillus curvatus DPC2024

Magboul

McSweeney

2000

Lait

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-An X-prolyl-dipeptidyl aminopeptidase (PepX) was purified ~176-fold from the cell-free extract of Lactobacillus curvatus DPC2024. The native enzyme appeared to be a dimer with a subunit molecular mass of ~97.4 kDa as determined by sodium dodecyl sulphate gel electrophoresis. Optimal activity of the purified enzyme on Ala-Pro-p-nitroanilide (pNA) was at pH 7.5 and 45 °C. The enzyme retained more than 60% of its activity after pre-incubation for 30 min at 45 °C but the activity decreased sharply following pre-incubation at temperatures above 45 °C. The enzyme was activated by Co 2+ , Mn 2+ , Ni 2+ at 0.1 and 1.0 mmol . L -1 and by Cu 2+ , Cd 2+ and Zn 2+ at 0.1 mmol . L -1 but it was strongly inhibited by 1.0 mmol . L -1 phenylmethylsulphonyl fluoride, Hg 2+ , Cu 2+ , Cd 2+ and Zn 2+ and partially by ethylenediaminetetraacetic acid, o-phenanthroline and p-chloromercuribenzoate. The enzyme hydrolysed Ala-Pro-pNA, Arg-Pro-pNA, Gly-Pro-Arg, Val-Pro-Leu at a faster rate and slowly hydrolysed Ala-Ala-pNA but it was not active on Ala-Ala-Ala, Ala-Leu-Ala, Pro-Pro-Pro, ArgPro-Pro, dipeptides and N-terminally blocked p-nitroanilide derivatives and other peptides. The sequence of the first 20 amino acid residues was determined and showed 40% homology to X-prolyl-dipeptidyl aminopeptidases from Lactobacillus helveticus CNRZ 32, Lactobacillus helveticus 53/7, Lactococcus lactis ssp. cremoris P8-2-47 and Lactococcus lactis ssp. lactis NCDO 763 and 35% homology to a PepX from Lactobacillus delbrueckii ssp. lactis DSM 7290. Lactobacillus curvatus / X-prolyl-dipeptidyl aminopeptidase / purificationRésumé -Purification et caractérisation d'une X-prolyl-dipeptidyl aminopeptidase isolée à partir de Lactobacillus curvatus DPC2024. Une X-prolyl-dipeptidyl aminopeptidase (PepX) a été purifiée environ 176 fois à partir d'un extrait intracellulaire de Lactobacillus curvatus DPC2024. L'enzyme native est apparue être de la forme dimère avec des sous-unités de masse moléculairẽ 97,4 kDa déterminée par électrophorèse SDS-PAGE. L'activité optimale de l'enzyme purifiée, déterminée sur le fragment Ala-Pro-p-nitroanilide (pNA), a été obtenue à pH 7,5 et à 45 °C. L'enzyme Lait 80 (2000) 385-396 385

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“…cremoris SK11 was obtained from the culture collection of the New Zealand Dairy Research Institute, Palmerston North, New Zealand (isolate number 5221). Bacterial cultures for enzyme purification were grown in a 40 litre fermenter in a lactose/peptone/yeast extract medium following the procedure described by Lloyd & Pritchard (1991). Bacterial cells were harvested by centrifugation at the end of the exponential phase (approximately 6 h after inoculation), washed twice with cold 50 mM phosphate buffer (pH 6-4), and the washed pellets were stored frozen at -15 OC until required.…”

Section: G G P R I T C H a R D A D F R E E B A I R N A N D T Cmentioning

confidence: 99%

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11

Pritchard

Freebairn²,

Coolbear³

1994

Microbiology

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An endopeptidase has been purified from Lactococcus lactis subsp. crernoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,l O-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor plchloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl f luoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. T w o of the oligopeptides produced during the early stages of & casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic s ys tem .

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Characterization of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. lactis

Cited by 74 publications

References 27 publications

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus

Purification and characterization of an X-prolyl-dipeptidyl aminopeptidase from Lactobacillus curvatus DPC2024

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11

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