Cloning, DNA sequence analysis and partial characterization of <i>pepN</i>, a lysyl aminopeptidase from <i>Lactobacillus delbrückii</i> ssp. <i>lactis</i> DSM7290

Abstract

In cell extracts of Lactobacillus delbrückii ssp. lactis DSM7290 a peptidase with the ability to hydrolyse Phe-beta-naphthylamide (Phe-beta-NA) and His-beta-NA could be detected. Escherichia coli lacking the enzyme activity in an enzymic plate assay was used to screen high-copy-number and low-copy-number plasmid libraries of size-fractionated Lactobacillus DNA. Clones with the desired phenotype were detected, and the gene, designated pepN, was further subcloned and sequenced. A large open reading frame of 2529 nucleotides is predicted to encode a protein of 843 amino acids (95358 Da). Comparison of the pepN gene from Lb. delbrückii ssp. lactis DSM7290 indicates that it is homologous to genes of the family of Zn(2+)-metallohydrolases and PepN shows identity with the active centre Zn(2+)-binding motif of these enzymes. The substrate Lys-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 efficiently overexpressed in E. coli and subcloning of the gene in Lactobacillus 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-NH-Ph) in the assay procedure. In a four-step procedure including streptomycin sulfate precipitation, anion-exchange chromatography and gel filtration the peptidase was purified to electrophoretic homogeneity.