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

Vesanto, Erkki; Savijoki, Kirsi; Rantanen, Terhi; Steele, J. L.; Palva, Airi

doi:10.1099/13500872-141-12-3067

Cited by 64 publications

(49 citation statements)

References 36 publications

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“…The substrate showing the highest affinity for the recombinant DPP IV was Gly-Pro-pNa, with a K m of 0.26 mM and a V max of 7.8 mol mg Ϫ1 min Ϫ1 . This value is comparable to those reported for P. albensis (27) and L. helveticus (26). The recombinant DPP IV also had significant affinity for Lys-AlapNa (K m of 0.87 mM) and Ala-Pro-pNa (K m of 12 mM) with V max values of 5.9 and 30 mol mg Ϫ1 min Ϫ1 , respectively.…”

Section: S Suissupporting

confidence: 70%

Cloning, Purification, and Enzymatic Properties of Dipeptidyl Peptidase IV from the Swine Pathogen Streptococcus suis

et al. 2005

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In this study, the dipeptidyl peptidase IV (DPP IV) of the swine pathogen Streptococcus suis was cloned, overexpressed in Escherichia coli, and characterized. The coding region comprises 2,268 nucleotides containing an open reading frame that codes for a 755-amino-acid protein with a calculated molecular mass of 85 kDa. The amino acid sequence contained the sequence Gly-X-Ser-X-X-Gly, which is a consensus motif flanking the active-site serine shared by serine proteases. The recombinant DPP IV showed a high affinity for the synthetic peptide glycine-proline-p-nitroanilide and was strongly inhibited by Hg 2؉ and diprotin A.

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Section: S Suissupporting

confidence: 70%

Cloning, Purification, and Enzymatic Properties of Dipeptidyl Peptidase IV from the Swine Pathogen Streptococcus suis

et al. 2005

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“…The degree of purification of the enzyme preparation used was of 716.6-fold. with a subunit molecular mass of 80 to 95 kDa (3,11,15,16,32,33). However, the enzymes of Lactobacillus casei and of some strains of Lactobacillus helveticus and Lactobacillus delbrueckii have been found to be monomers or trimers (2,9,17,19,20).…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

Sanz

Toldrá

2001

Appl Environ Microbiol

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An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu 2؉ , Hg 2؉ , and Zn 2؉). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K m s for Gly-Pro-and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 M, respectively; those for Gly-Pro-and Ala-Pro-p-nitroanilide were 192 and 50 M, respectively. Among peptides, ␤-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.Lactic acid bacteria constitute one of the most important group of microorganisms used in food fermentation. In the last decades, the metabolic traits of these bacteria have been the object of exhaustive studies, which evidence promising applications (12). The proteolytic properties of dairy lactic acid bacteria are among the best characterized to date (5, 13). This is a consequence of the impact of proteolysis on the physiology of these organisms as well as on the development of texture and flavor of dairy products (6, 18). Proteolysis on milk proteins is initiated by a cell wall-associated proteinase, which hydrolyzes caseins into oligopeptides. The generated peptides are mainly translocated via the oligopeptide transport system and, further, hydrolyzed by a pool of intracellular peptidases, which include endopeptidases, aminopeptidases, dipeptidases, tripeptidases, and dipeptidyl peptidases (13).Lactobacillus sakei is the most competitive species in meat fermentation and therefore constitutes a frequently used starter culture. The proteolytic events that occur during meat processing also lead to the generation of small peptides and free amino acids, which are considered to be flavor compounds (1, 31). In fermented meat products, muscle as well as microbial enzymes are responsible for the proteolytic changes, but their roles remain elusive (21, 31). Particularly, the studies on the proteolytic system of meat lactobacilli are rather limited. It has been shown that several Lactobacillus spp. exhibit proteolytic activity on porcine muscle myofibrillar and sarcoplasmic proteins (7,8,28). These studies highlighted the potential role of L. sakei in amino acid and peptide generation especially from sarcoplasmic proteins. Attention has also been focused on intracellular peptidases of L. ...

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“…helveticus CNRZ 32 [38] and Lb. helveticus 53/7 [33] and 35% homology with PepX from Lb. delbrueckii ssp.…”

Section: Compoundsmentioning

confidence: 99%

“…L -1 . The inhibitory effect of Hg 2+ , Cd 2+ , Cu 2+ and Zn 2+ on PepXs characterized from other LAB has been reported [1,8,10,22,33].…”

Section: Compoundsmentioning

confidence: 99%

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

Cited by 64 publications

References 36 publications

Cloning, Purification, and Enzymatic Properties of Dipeptidyl Peptidase IV from the Swine Pathogen Streptococcus suis

Cloning, Purification, and Enzymatic Properties of Dipeptidyl Peptidase IV from the Swine Pathogen Streptococcus suis

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

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

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