1887

Abstract

The gene for proline iminopeptidase from subsp. lactis DSM 7290 coding for an enzyme that hydrolyses the synthetic substrate -prolyl-β-naphthylamide (Pro-βNA) was cloned in . An enzymic plate assay was used to screen for positive clones. The gene, designated pepI, was subcloned into vector pUC18 and sequenced. The nucleotide sequence revealed an 882 bp open reading frame encoding 294 amino acids, coding for an enzyme with a calculated molecular mass of 32883 Da. By cloning under control of the lac promoter the peptidase was highly expressed. Sequence analysis showed that pepI is of a new sequence type, distinct from all peptidases so far sequenced. Amino acid homology to the active site of a putida esterase and inhibitor studies of the enzyme imply involvement of a serine residue in catalysis.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-140-5-1133
1994-05-01
2024-10-10
Loading full text...

Full text loading...

/deliver/fulltext/micro/140/5/mic-140-5-1133.html?itemId=/content/journal/micro/10.1099/13500872-140-5-1133&mimeType=html&fmt=ahah

References

  1. Atlan D., Gilbert C., Blanc B., Portalier R. Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from Lactobacillus delbrueckii subsp bulgaricus CNRZ 397. Microbiology 1994; 140:527–535
    [Google Scholar]
  2. Casey M.G., Meyer J. Presence of X-prolyl-dipeptidyl-peptidase in lactic acid bacteria. J Dairy Sci 1985; 58:3212–3215
    [Google Scholar]
  3. Dower W.J., Miller J.F., Ragsdale C.W. High efficiency transformation of E coli by high voltage electroporation. Nucleic Acids Rer 1988; 16:2127–2145
    [Google Scholar]
  4. Gilbert C., Atlan D., Blanc B., Portalier R. Proline iminopeptidase from Lactobacillus delbrueckii subsp bulgaricus CNRZ 397: purification and characterization. Microbiology 1994; 140:537–542
    [Google Scholar]
  5. Honjo M., Nakayama A., Fukazawa K., Kawamura K., Ando K., Hori M., Furutani Y. A novel Bacillus subtilis gene involved in negative control of sporulation and degradative-enzyme production. J Bacteriol 1990; 172:1783–1790
    [Google Scholar]
  6. Kitazono A., Yoshimoto T., Tsuru D. Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans. J Bacteriol 1992; 174:7919–7925
    [Google Scholar]
  7. Klein J.R., Klein U., Schad M., Plapp R. Cloning, DN A sequence analysis and partial characterization ofpepN, a lys-amino-peptidase from Lactobacillus delbriickii ssp lactis DSM7290. Eur J Biochem 1993; 217:105–114
    [Google Scholar]
  8. Kyte J., Doolittle R.F. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157:105–132
    [Google Scholar]
  9. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685
    [Google Scholar]
  10. Matsushima M., Inoue H., Ichinose M., Tsukada S., Miki K., Kuurokawa T., Takahashi T., Takahashi K. The nucleotide and deduced amino acid sequences of porcine liver proline-²-naphthylaminidase. FEBS Lett 1991; 293:37–41
    [Google Scholar]
  11. Menn F.M., Zylstra G.J., Gibson D.T. Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida FI. Gene 1991; 104:91–94
    [Google Scholar]
  12. Meyer-Barton E.C., Klein J.R., Imam M., Plapp R. Cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbriickii spp lactis DSM 7290. Appl Microbiol Biotechnol 1993; 40:82–89
    [Google Scholar]
  13. Miller C.G., Mackinnon K. Peptidase mutants of Salmonella typhimurium. J Bacteriol 1974; 120:355–363
    [Google Scholar]
  14. Miller C.G., Schwartz G. Peptidase deficient mutants of Escherichia coli. J Bacteriol 1978; 135:603–611
    [Google Scholar]
  15. Raleigh E.A., Murray N.E., Revel H., Blumenthal R.M., Westawy D., Reith A.D., Rigby P.W.J., Elhai J., Hanahan D. McrA and McrB restriction phenotypes of some E coli strains and implication for gene cloning. Nucleic Acids Res 1988; 16:1563–1575
    [Google Scholar]
  16. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd edn 1989 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  17. Sanger F., Nicklen S., Coulson A.R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463–5467
    [Google Scholar]
  18. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 1974; 71:1342–1346
    [Google Scholar]
  19. Stoker N.G., Fairweather N.F., Spratt B.G. Versatile low copy number plasmid vectors for cloning in Escherichia coli. Gene 1982; 18:335–341
    [Google Scholar]
  20. Tan P.S., T., Poolman B., Konings W.N. Proteolytic enzymes of Lactococcus lactis. J Dairy Res 1993; 60:269–286
    [Google Scholar]
  21. Van de Guchte M., Kok J., Venema G. Gene expression in Lactococcus lactis. FEMS Microbiol Rev 1992; 88:73–92
    [Google Scholar]
  22. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 1985; 33:103–119
    [Google Scholar]
  23. Yaron A., Naider F. Proline-dependent structural and biological properties of proteins. Grit Rev Biochem Mol Biol 1993; 28:31–81
    [Google Scholar]
/content/journal/micro/10.1099/13500872-140-5-1133
Loading
/content/journal/micro/10.1099/13500872-140-5-1133
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error