An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus.
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 90.6 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with delta G of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 90.6 kDa protein showed 49.3, 49.4 and 77.7% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lactobacillus delbrueckii subsp. lactis, 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 degrees C. 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 1,10-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding DTT, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.
Vesanto
E
E
Agricultural Research Centre of Finland, Food Research Institute, Jokioinen, Finland.
Savijoki
K
K
Rantanen
T
T
Steele
J L
JL
Palva
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018819:Dipeptidyl Peptidase 4