Abstract
Lactobacillus delbrückii ssp. lactis DSM7290 possesses an X-prolyl-dipeptidyl-aminopeptidase, designated PepX, which catalyses the hydrolytic removal of N-terminal dipeptidyl residues from peptides containing proline in the penultimate position. Using the specific substrate L-Ala-L-Pro-p-nitroanilide, PepX was purified by a four-step procedure including ammonium sulphate fractionation, hydrophobic interaction chromotography, ion exchange chromotography, and affinity chromotography. The N-terminus of the purified protein was sequenced. Screening of a gene library of chromosomal Lactobacillus delbrückii ssp. lactis DSM7290 DNA in the low-copy-number vector pLG339 resulted in the identification of the pepX gene in Escherichia coli using a specific plate assay with Gly-L-Pro-β-naphthalamide as substrate. Nucleotide sequence analysis revealed an open reading frame of 2376 bp, coding for a protein of 792 amino acids with a molecular mass of 88449 Da.
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References
Abo-Elnaga IG, Plapp R (1987) Peptidases of Lactobacillus casei and Lactobacillus plantarum. J Basic Microbiol 27:123–130
Atlan D, Laloi P, Portalier R (1990) X-Prolyl-dipeptidyl-amino-peptidase of Lactobacillus delbrückii subsp. bulgaricus, characterization of the enzyme and isolation of deficient mutants. Environ Microbiol 56:2174–2179
Bertani G (1951) Studies of lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Bockelmann W, Fobker M, Teuber M (1991) Purification and characterization of the X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus delbrückii subsp. bulgaricus and Lactobacillus acidophilus. Int Dairy J 1:51–66
Booth M, Fhaolain D, Jennings PV, O'Cuinn G (1990) Purification and characterization of a proline dipeptidyl aminopeptidase from Streptococcus cremoris AM2. J Dairy Res 57:89–99
Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Casey MG, Meyer J (1985) Presence of X-prolyl-dipeptidyl-peptidase in lactic acid bacteria. J Dairy Sci 58:3212–3215
Collins MD, Rodrigues U, Ash C, Aguirre M, Farrow JAE, Martinez-Murcia A, Phillips BA, Williams AM, Wallbanks S (1991) Phylogenetic analysis of the genus Lactobacillus and related lactic acid bacteria as determined by reverse transcriptase sequencing of 16S rRNA. FEMS Microbiol Lett 77:5–12
Dower JW, Miller JW, Ragsdale W (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
Eckerskorn C, Mewes W, Goretzki F, Lottspeich F (1988) A new siliconized glass-fiber as support for protein-chemical analysis of electrobotted proteins. Eur J Biochem 176:509–519
Fukasawa KM, Harada M (1982) Purification and properties of dipeptidyl peptidase IV from Streptocyces mitis ATCC 9811. Arch Biochem Biophys 210:230–237
Guchte M van de, Kok J, Venema G (1992) Gene expression in Lactococcus lactis. FEMS Microbiol Rev 88:72–92
Harley CB, Reynolds RP (1987) Analysis of E. coli promotor sequences. Nucleic Acids Res 15:2343–2361
Khalid NM, Marth EH (1990) Purification and partial characterization of a prolydipeptidylaminopeptidase from Lactobacillus helveticus CNRZ 32. Appl Environ Microbiol 56:381–388
Kiefer-Partsch B, Bockelmann W, Geis A, Teuber M (1989) Purification of an X-prolyl-dipeptidyl-aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp. cremoris. Appl Microbiol Biotechnol 31:75–78
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Law BA, Kolstadt J (1983) Proteolytic systems in lactic acid bacteria. Antonie van Leeuwenhoek 49:225–245
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Lloyd RJ, Pritchard GG (1991) Characterization of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. lactis. J Gen Microbiol 137:49–55
Man JD de, Rogosa M, Sharpe ME (1960) A medium for cultivation of lactobacilli. J Appl Bacteriol 23:130–135
Mayo B, Kok J, Venema K, Bockelmann W, Teuber M, Reinke H, Venema G (1991) Molecular cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene from Lactococcus lactis subsp. cremoris. Appl Environ Microbiol 57:38–44
Meyer J, Jordi R (1987) Purification and characterization of X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus lactis and from Streptococcus thermophilus. J Dairy Sci 70:7358–7450
Miller CG, Mackinon K (1974) Peptidase mutants of Salmonella typhimurium. J Bacteriol 120:355–363
Miyakawa H, Kobayashi S, Shimamura S, Tomita M (1991) Purification and characterization of an X-prolyl dipeptidyl aminopeptidase from Lactobacillus delbrückii ssp. bulgaricus LBU-147. J Dairy Sci 74:2375–2381
Moran CP, Lang N, LeGrice SFJ, Lee G, Stephens M, Sonenshein AL, Pero J, Losick R (1982) Nucleotide sequences that signal initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet 186:339–346
Nardi M, Chopin MC, Chopin A, Cals MM, Gripon JC (1991) Cloning and DNA-sequence analysis of an X-prolyl-dipeptidyl-aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763. Appl Environ Microbiol 57:45–50
Queen C, Korn LJ (1984) A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res 12:581–599
Raleigh EA, Murray NE, Revel H, Blumenthal RM, Westaway D, Reith AD, Rigby PWJ, Elhai J, Hanahan D (1988) McrA and McrB restriction phenotypes of some E. coli strains and implication for gene cloning. Nucleic Acids Res 16:1563–1575
Sambrook J, Fritsch EJ, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Sanger F, Nicklen S, Coulsen AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Stoker NG, Fairweather NF, Spratt BG (1982) Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene 18:335–341
Tan PST, Chapot-Chartier MP, Pos KM, Rousseau M, Boquien JY, Gripon JC, Konings W (1992) Localization of peptidases in lactococci. Appl Environ Microbiol 58:285–290
Thomas TD, Pritchard GG (1987) Proteolytic enzymes of dairy starter cultures. FEMS Microbiol Rev 46:245–268
Weiss N, Schillinger U, Kandler O (1983) Lactobacillus lactis, Lactobacillus leichmannii and Lactobacillus bulgaricus, subjective synonyms of Lactobacillus delbrückii subsp. lactis comb. nov. and Lactobacillus delbrückii subsp. bulgaricus comb. nov. Syst Appl Microbiol 4:552–557
Yan TR, Ho SC, Hou CL (1992) Catalytic properties of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris nTR. Biosci Biotechnol Biochem 56:704–707
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33:103–119
Zevaco C, Monnet V, Gripon JC (1990) Intracellular X-prolyl dipeptidyl peptidase from Lactococcus lactis spp. lactis: purification and properties. J Appl Bacteriol 68:357–366
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Correspondence to: E. C. Meyer-Barton
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Meyer-Barton, E.C., Klein, J.R., Imam, M. et al. Cloning and sequence analysis of the X-Prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbrückii ssp. lactis DSM7290. Appl Microbiol Biotechnol 40, 82–89 (1993). https://doi.org/10.1007/BF00170433
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DOI: https://doi.org/10.1007/BF00170433