LACTOBACILLUS-DELBRUECKII SUBSP LACTIS DSM7290 PEPG GENE ENCODES A NOVEL CYSTEINE AMINOPEPTIDASE

A number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp. lactis gene library capable of hydrolysing the chr omogenic substrate Gly-Ala-beta-naphthylamide (Gly-Ala-beta NA). Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC. Nucleotide sequence ana lyses of the plasmid inserts of the remaining clones resulted in the i dentification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC. One ge ne, designated pepG, was overexpressed in E. coli and the crude extrac ts obtained were shown to be peptidolytically active both against chro mogenic substrates and peptides, and in a Salmonella typhimurium growt h test. PepC and PepG activities were compared using chromogenic beta NA and p-nitroanilide substrates and leucine or proline-containing pep tides were applied in growth experiments of recombinant Sal. typhimuri um. The results indicate that the enzymes, although structurally relat ed, have different substrate preferences. No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system. Primer extension analysis, using mRNA isolated from Lb. delbrueckii subsp. lactis DSM7290 did est ablish that orfW was transcribed.