Purification and characterization of an aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus

Bockelmann, Wilhelm; Schulz, Yvonne; Teuber, Michael

doi:10.1016/0958-6946(92)90003-5

Cited by 48 publications

(21 citation statements)

References 24 publications

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“…C02+ and Zn 2 +. AlI of the PepN-type aminopeptidases characterized to date from LAB are metalIoenzymes, strongly inhibited by EDTA and o-phenanthroline and, in sorne cases, this inhibition may be fully or partially restored by different divalent metal ions [5,38]. The enzyme was also partially inhibited by thiol reducing or blocking agents, indicating that thiol groups are important for aminopeptidase activity.…”

Section: Discussionmentioning

confidence: 99%

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PepN-like aminopeptidase from Lactohacillus curvatus DPC2024: purification and characterization

Magboul

McSweeney

1999

Lait

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-An enzyme with a PepN-like aminopeptidase activity was purified 127-fold with 4.3 % recovery from a cell-free extract of Lactobacillus curvatus DPC2024, a component of the non-starter lactic acid bacterial flora of Cheddar cheese. The purified enzyme exhibited maximum activity on leucyl-p-nitroanilide (Leu-pNA) at pH 7.0 and 40 oc. The enzyme had a molecular mass of -98 kDa as estimated by gel permeation chromatography, and showed one band corresponding to a molecular mass of -95 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that the native enzyme existed as a monomer. The aminopeptidase appeared to be a metalloenzyme since it was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline at 0.1 mmol-Lr ' concentration and was partially reactivated by Zn 2 + and C02+.The enzyme was also partially inhibited by p-chloromercuribenzoate and iodoacetic acid, suggesting the involvement of su1phydryl groups in the reaction mechanism. The enzyme had a broad substrate specificity, hydrolysing a number of p-nitroanilide derivatives of amino acids and peptides and di-, tri-, tetra-and pentapeptides. The N-tenninal amino acid sequence of the first 20 amino acid residues was determined (AELMRFYQSFQPEHYQVFLD), and showed 40-55 % homology with semble être une métalloenzyme car elle est fortement inhibée par l' EDT A et l' o-phenanthroline à la concentration de

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Section: Discussionmentioning

confidence: 99%

“…The enzyme was also partially inhibited by thiol reducing or blocking agents, indicating that thiol groups are important for aminopeptidase activity. Inhibition by thiol reagents was observed for a number of PepN and PepNlike aminopeptidases [1,2,5,7,13,20,26,28].…”

Section: Discussionmentioning

confidence: 99%

PepN-like aminopeptidase from Lactohacillus curvatus DPC2024: purification and characterization

Magboul

McSweeney

1999

Lait

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“…delbrueckii subsp. bulgaricus enzyme was found to differ from that of the lactococcal aminopeptidase [154]. However, in view of the significant homology of lactococcal aminopeptidase N to both E. coil and mammalian lysyl aminopeptidases [76,77] conservation of sequence and structure across the range of lactic acid bacteria would seem highly probable.…”

Section: Peptidasesmentioning

confidence: 92%

“…The mctallo-aminopeptidases from Lb. delbrueckii subsp, bulgaricus [154], Lb. heh'eticus [155,156] probably also belongs to this type.…”

Section: Peptidasesmentioning

confidence: 99%

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

Pritchard

Coolbear²

1993

FEMS Microbiology Reviews

274

155

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The inability of lactic acid bacteria to synthesize many of the amino acids required for protein synthesis necessitates the active functioning of a proteolytic system in those environments where protein constitutes the main nitrogen source. Biochemical and genetic analysis of the pathway by which exogenous proteins supply essential amino acids for growth has been one of the most actively investigated aspects of the metabolism of lactic acid bacteria especially in those species which are of importance in the dairy industry, such as the lactococci. Much information has now been accumulated on individual components of the proteolytic pathway in lactococci, namely, the cell envelope proteinase(s), a range of peptidases and the amino acid and peptide transport systems of the cell membrane. Possible models of the proteolytic system in lactococci can be proposed but there are still many unresolved questions concerning the operation of the pathway in vivo. This review will examine current knowledge and outstanding problems regarding the proteolytic system in lactococci and also the extent to which the lactococcal system provides a model for understanding proteolysis in other groups of lactic acid bacteria.

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“…In contrast, the vast majority of aminopeptidases from lactic acid bacteria are metalloenzymes (Visser, 1993), with the notable exception of an aminopeptidase from Lactobacillus delbrueckii ssp bulgaricus B 14 (Wohlrab and Bockelmann, 1993), which was inhibited by thiol blocking agents. Of the metals assessed, Zn 2 + was the most inhibitory; aminopeptidases isolated from various lactic acid bacteria have also been shown to be extremely sensitive to Zn 2 + (Bockelmann et al, 1992;Midwinter and Pritchard, 1994;Rul et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Purification and characterisation of an intracellular aminopeptidase from Brevibacterium linens ATCC 9174

Rattray

Fox

1997

Lait

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Summary -An intracellular aminopeptidase from Brevibacterium linens ATCC 9174 was purified 4300-fold to homogeneity using ammonium sulphate fractionation, anion exchange chromatography, hydrophobie interaction chromatography and anion exchange chromatography. The pH and temperature optima were 8.5 and 35 "C, respectively. The purified aminopeptidase was stable over the range pH 8 to 10, and was thermally stable up to 20 "C at pH 8.5. The molecular mass of the enzyme was found to be 59 kOa by SOS-PAGE and 69 kOa by gel filtration, indicating that the native enzyme exists as a monomer. The aminopeptidase was strongly inhibited by the thiol blocking agent, p-hydroxymercuribenzoate, and by C02+ and Zn 2 +; activity was unaffected by metal chelators, reducing agents or phenylmethylsulphonyl fluoride. Km and k cat values for L-Ala-p-NA were 3.3 mmol/L and 4.3 s-I, respectively, while the corresponding values for L-Gly-p-NA were 0.2 mmol/L and 7.6 s-I, respectively. The aminopeptidase hydrolysed dipeptides with an alanine residue at the N-terminal but tripeptides were not hydrolysed. The sequence of the first 19 N-terminal amino acids was NHrPro-Phe-Asp-Gly-Pro-AspThr-Ala-Ala-I1e-I1e-Asp-Arg-Leu-?-Asn-Ala-?-Thr.

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Purification and characterization of an aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus

Cited by 48 publications

References 24 publications

PepN-like aminopeptidase from Lactohacillus curvatus DPC2024: purification and characterization

PepN-like aminopeptidase from Lactohacillus curvatus DPC2024: purification and characterization

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

Purification and characterisation of an intracellular aminopeptidase from Brevibacterium linens ATCC 9174

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