MOLECULAR-CLONING AND DNA-SEQUENCE ANALYSIS OF PEPL, A LEUCYL AMINOPEPTIDASE GENE FROM LACTOBACILLUS-DELBRUECKII SUBSP LACTIS DSM7290

A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 D NA fragments from a Sau3A partial digestion in the low-copy-number vec tor pLG339, was used to screen Escherichia coli for the presence of pe ptidases. Using the chromogenic substrate leucine-beta-naphthylamide ( Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme a ctivity in an enzymic plate assay, allowed the isolation of two peptid ase genes; the newly described pepL and the recently cloned and sequen ced pepN. Clones could be distinguished not only by the restriction pa ttern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identi fied to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, toge ther with 200 bp upstream of the proposed AUG initiation codon, was fu rther subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids ( 34541 Da). Searching the EMBL database revealed similarity to the prol inase of Lactobacillus helveticus (45.8% identity), to the iminopeptid ases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulga ricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active s ites. The product encoded by the pepL gene was functional but could no t be visualized on Coomassie-blue-stained polyacrylamide gels. High le vel expression of peptidase L in E. coli was achieved by placing the g ene under the control of the T7 promoter.