CLONING AND NUCLEOTIDE-SEQUENCE ANALYSIS OF THE LACTOBACILLUS-DELBRUECKII SSP LACTIS DSM7290 CYSTEINE AMINOPEPTIDASE GENE PEPC

A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of pe ptidase genes. Using the chromogenic substrate gly-ala-beta-naphthylam ide, which is not a substrate for any of the recently cloned peptidase s of DSM7290, and the multiple peptidase deficient Escherichia coli st rain CM89, allowed the isolation of clones, which contained the equiva lent hydrolytic activity. To identify genes encoding the conserved cat alytic active site of cysteine proteases, partial nucleotide sequencin g with a degenerate oligonucleotide was performed on recombinant plasm ids isolated from such clones. This allowed to identify two out of nin e clones to carry the Lactobacillus pepC gene. A total of 2026 nucleot ides were determined, and sequence analysis revealed a gene with stron g homology to the recently cloned Lb. helveticus (73.2%) and Lactococc us lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The pre dicted open reading frame consists of 449 codons, coding for a protein of 50909 Da. The enzyme is functional and extremely overexpressed in E. coli.