Cloning, DNA sequence analysis and partial characterization of pepN, a lysyl aminopeptidase from Lactobacillus delbrückii ssp. lactis DSM7290.
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.
Klein
J R
JR
Fachbereich Biologie, Abteilung Mikrobiologie, Universität Kaiserslautern, Germany.
Klein
U
U
Schad
M
M
Plapp
R
R
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D015032:Zinc / Q000378:metabolism
D008670:Metals; D000626:Aminopeptidases; C034113:peptidase N; D015032:Zinc