RESUMO
Two intracellular oligopeptide-preferring endopeptidases have been detected in Lactococcus lactis. A neutral thermolysin-like oligoendopeptidase (NOP) has been purified to homogeneity and an alkaline oligoendopeptidase has been partially purified. The specificity of the oligoendopeptidases towards important intermediary cheese peptides, produced by chymosin action on the caseins, clearly differs from that of the cell-envelope proteinase (CEP). NOP is active under conditions prevailing in cheese and contributes to initial proteolysis in a young cheese. It probably plays a crucial role in the degradation of an important bitter peptide in cheese, the beta-casein 193-209 fragment. The relatively low activity of the alkaline endopeptidase is further suppressed in cheese by the highly competitive actions of NOP and CEP.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Queijo/microbiologia , Endopeptidases/isolamento & purificação , Lactococcus lactis/enzimologia , Proteínas do Leite/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Endopeptidases/metabolismo , Concentração de Íons de Hidrogênio , Líquido Intracelular/metabolismo , Lactococcus lactis/genética , Dados de Sequência Molecular , Especificidade por SubstratoAssuntos
Proteínas de Transporte/genética , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/metabolismo , Trealose/metabolismo , Transporte Biológico , Etanol/metabolismo , Genes Fúngicos/genética , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Trealase/metabolismoRESUMO
The chromosomal pepN gene encoding lysyl-aminopeptidase activity in Lactococcus lactis has been identified in a lambda EMBL3 library in Escherichia coli by using an immunological screening with antiserum against a purified aminopeptidase fraction. The pepN gene was localized and subcloned in E. coli on the basis of its expression and hybridization to a mixed-oligonucleotide probe for the previously determine N-terminal amino acid sequence of lysyl-aminopeptidase (P. S. T. Tan and W. N. Konings, Appl. Environ. Microbiol. 56:526-532, 1990). The L. lactis pepN gene appeared to complement an E. coli strain carrying a mutation in its pepN gene. High-level expression of the pepN gene in E. coli was obtained by using the T7 system. The overproduction of the 95-kDa aminopeptidase N could be visualized on sodium dodecyl sulfate-polyacrylamide gels and immunoblots. Cloning of the pepN gene on a multicopy plasmid in L. lactis resulted in a 20-fold increase in lysyl-aminopeptidase activity that corresponded to several percent of total protein. Nucleotide sequence analysis of the 5' region of the pepN gene allowed a comparison between the deduced and determined amino-terminal primary sequences of aminopeptidase N. The results show that the amino terminus of PepN is not processed and does not possess the characteristics of consensus signal sequences, indicating that aminopeptidase N is probably an intracellular protein. The intracellular location of aminopeptidase N in L. lactis was confirmed by immunogold labeling of lactococcal cells.
