SEQUENCE-ANALYSIS, DISTRIBUTION AND EXPRESSION OF AN AMINOPEPTIDASE N-ENCODING GENE FROM LACTOBACILLUS-HELVETICUS CNRZ32

Lactobacillus (Lb.) helveticus CNRZ32 possesses a 97-kDa metalloenzyme with aminopeptidase activity (PepN; EC 3.4.11.2). A 3.8-kb fragment e ncoding PepN was cloned into pIL253 and designated pSUW34. Transformat ion of Lactococcus (Lc.) lactis LM0230 with pSUW34 resulted in > 180-f old increase in general aminopeptidase (AP) activity using L-lysine-p- nitroanilide. Southern hybridization was conducted to determine the di stribution of homology to the CNRZ32 pepN gene among lactic-acid bacte ria (LAB). Hybridization was observed with strains of lactobacilli, pe diococci, leuconostoc, streptococci and lactococci. The pepN gene was sequenced and found to encode a protein containing 844 amino acid (aa) residues. A comparison of Lb. helveticus CNRZ32 pepN to Lb. delbrueck ii ssp. lactis DSM7290 pepN indicated 69.5% nucleotide (nt) identity a nd 71.8% aa identity, while comparison to pepN from Lc. lactis ssp. cr emoris MG1363 indicated 61.1% nt identity and 49.2% aa identity. Align ment of peptidase aa sequences of LAB, Escherichia coli, yeast and mam malian origin display homology in the zinc-binding domain, as well as a conserved region upstream from the putative active site.