For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.Lactic acid bacteria (LAB) play an important role in dairy fermentation processes and have a great influence on the quality and preservation of end products. The primary roles of LAB are to produce lactic acid from lactose, resulting in a pH decrease, and, by proteolysis, to liberate short peptides and free amino acids affecting the flavor and texture of dairy products.Since the concentration of free amino acids and small peptides is insufficient to support the growth of LAB to high cell densities in milk, these bacteria are dependent on a proteolytic system to liberate free amino acids from milk proteins. The proteolytic system of LAB consists of a cell envelope-associated proteinase, membrane-bound transport systems, and several cytoplasmic peptidase classes. The proteolytic system is particularly important in the development of flavor and texture of cheeses (9). Since Lactococcus strains, along with those of Lactobacillus, are widely used as starters in cheese manufacture, substantial effort has been directed in the last two decades toward elucidating the proteolytic mechanism of Lactococcus lactis. More recently, the proteolytic system of lactobacilli has also been extensively examined.Over 10 different peptidase types have been identified in various LAB strains, and a large number of peptidase genes have been cloned from different Lactococcus and Lactobacillus species and characterized (reviewed ...
A dipeptidase gene (pepD) from an industrial Lactobacillus helveticus strain was isolated by colony hybridization. An open reading frame (ORF) of 1422 base pairs (bp) with a coding capacity for a 53.5-kDa protein (PepD) was identified. The ORF was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -51.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 53.5-kDa protein revealed no marked homologies when compared to the data bases of EMBL and SWISS-PROT. The 5'end of the 1.6-kb pepD transcript was determined both by a conventional primer extension method and using an automated sequencer. pepD was found to be maximally expressed at late exponential growth. The pepD gene was cloned into an expression vector to over-produce PepD in Escherichia coli JM105. Purification of PepD to homogeneity was achieved using three chromatographic steps. PepD was able to hydrolyze a number of dipeptides with the exception of those containing a proline residue. Optimal PepD activity was observed at pH 6.0 and 55 degrees C. The enzyme was inhibited by p-hydroxymercuribenzoate and reactivated by dithiothreitol whereas ethylenediaminetetraacetate had no inhibitory effect on PepD. The enzymatic properties of PepD suggest that it represents a novel dipeptidase type among lactic acid bacteria.
The operon of the putative lactobacillar oligopeptide transport system (Opp) from Lactobacillus delbrueckii subsp. bulgaricus B14 was cloned and characterized. The opp operon was found to consist of five genes, oppD, oppF, oppB, oppC and oppA (1). In addition, an oppA (1) homolog, oppA (2), was found downstream of the operon. Sequence comparisons of the L. delbrueckii subsp. bulgaricus Opp system with other bacterial transport systems revealed the highest similarity to the oligopeptide transport system of Lactococcus lactis. Northern analyses of oppmRNAs revealed 6.1-kb and 2.1-kb transcripts, confirming that, in addition to the operon structure oppDFBCA (1), the oppA (1) gene was also expressed as a monocistronic transcript. The oppA (2) gene was expressed as a separate 2.1-kb monocistronic transcript with a low expression level. Primer-extension mapping of the 5'end of oppDFBCA (1) mRNA revealed two adjacent transcriptional start sites, and primer extension analyses of oppA (1) and oppA (2) mRNAs confirmed the location of the predicted promoters of these genes. For complementation analysis, oppA (1) alone and the operon constructs oppDFBCA (1) and oppDFBCA (2) were fused with the nisA promoter and expressed in Lactococcus lactisNZ9000Delta oppA strain. Only the L. delbrueckii subsp. bulgaricus oppDFBCA (1)genes were able to complement the L. lactis oppA mutation.
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