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Volume 141, Issue 12

An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Free

Abstract

The X-prolyl dipeptidyl aminopeptidase gene () of an industrially used 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 δG of −84.1 kJ mol 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 subsp. , Lc. subsp. and subsp. , 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 in Lb. was studied as a function of growth. Transcription of was typical of exponential growth phase expression. The gene has been cloned into pKK223-3 and expressed at a high level in JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6.5 and 45 °C. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu, Cd and Zn. EDTA and 1,10-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by -hydroxy-mercuribenzoate 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.

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Keyword(s): Lactobacillus belvetious , peptidases and X-prolyl dipeptidyl aminopeptidase
© Society for General Microbiology 1995
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1995-12-01
2024-04-25
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An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus
Microbiology 141, 3067 (1995); https://doi.org/10.1099/13500872-141-12-3067
/content/journal/micro/10.1099/13500872-141-12-3067
/content/journal/micro/10.1099/13500872-141-12-3067
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