CLONING, DNA-SEQUENCE ANALYSIS AND PARTIAL CHARACTERIZATION OF PEPN, A LYSYL AMINOPEPTIDASE FROM LACTOBACILLUS-DELBRUCKII SSP LACTIS DSM7290

In cell extracts of Lactobacillus delbruckii ssp. lactis DSM7290 a pep tidase with the ability to hydrolyse Phe-beta-naphthylamide (Phe-beta- NA) and His-beta-NA could be detected. Escherichia coli lacking the en zyme activity in an enzymic plate assay was used to screen high-copy-n umber and low-copy-number plasmid libraries of size-fractionated Lacto bacillus DNA. Clones with the desired phenotype were detected, and the gene, designated pepN, was further subcloned and sequenced. A large o pen reading frame of 2529 nucleotides is predicted to encode a protein of 843 amino acids (95358 Da). Comparison of the pepN gene from Lb. d elbruckii ssp. lactis DSM7290 indicates that it is homologous to genes of the family of Zn2+-metallohydrolases and PepN shows identity with the active centre Zn2+-binding motif of these enzymes. The substrate L ys-beta-NA is more effectively cleaved than Phe-beta-NA or His-beta-NA which were used for screening in E. coli. The cloned pepN gene was ef ficiently overexpressed in E. coli and subcloning of the gene in Lacto bacillus casei resulted in a moderate overexpression of approximately 20-fold. The pepN gene product was purified from the pepN-deficient E. coli strain CM89, using the substrate Lys-p-nitroanilide (Lys-NHPh) i n the assay procedure. In a four-step procedure including streptomycin sulfate precipitation, anion-exchange chromatography and gel filtrati on the peptidase was purified to electrophoretic homogeneity.