Lactis DSM7290 Research Articles

Purification and characterization of an aminopeptidase from Lactobacillus curvatus DPC2024

Abstract An aminopeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024, a component of the non-starter lactic acid bacterial flora of Cheddar cheese. Optimal aminopeptidase activity on leucyl- p -nitroanilide (Leu–pNA) was observed at pH 7.0 and 40°C. Molecular masses of ∼64 and ∼32 kDa were estimated for the enzyme by gel permeation chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis, respectively, indicating that the native enzyme (64 kDa) exists as a dimer. The aminopeptidase was slightly activated by Ba 2+ , Ca 2+ and dithiothreitol but strongly inhibited by Hg 2+ , Ni 2+ , Cu 2+ , Cd 2+ , Zn 2+ , thiol-blocking agents (p-hydroxymercuribenzoate, p-chloromercuribenzoate and iodoacetic acid) at 1 m m and by EDTA, o-phenanthroline and phenylmethylsulphonyl fluoride at 10 m m . The enzyme was most active on Leu–pNA and a number of dipeptides but showed little or no activity against tri- and oligopeptides. The N-terminal amino acid sequence was determined for 20 residues and showed significant homology to a leucyl-aminopeptidase from Lb. delbrueckii subsp. lactis DSM7290 and prolinases from Lb. helveticus CNRZ32 and Lb. rhamnosus 1/6. Regarding its molecular mass, metal dependency and specificity, the enzyme was different, in most respects, from the general aminopeptidases (PepN and PepC) and glutamyl aminopeptidase (PepA) of lactic acid bacteria.

PepN-like aminopeptidase from Lactohacillus curvatus DPC2024: purification and characterization

An enzyme with a PepN-like aminopeptidase activity was purified 127-fold with 4.3 % recovery from a cell-free extract of Lactobacillus curvatus DPC2024, a component of the non-starter lactic acid bacterial flora of Cheddar cheese. The purified enzyme exhibited maximum activity on leucyl-p-nitroanilide (Leu-pNA) at pH 7.0 and 40 oc.The enzyme had a molecular mass of -98 kDa as estimated by gel permeation chromatography, and showed one band corresponding to a molecu- lar mass of -95 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), indicating that the native enzyme existed as a monomer. The aminopeptidase appeared to be a met- alloenzyme since it was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) and o-phenan- throline at 0.1 mmol-Lr ' concentration and was partially reactivated by Zn2+ and C02+. The enzyme was also partially inhibited by p-chloromercuribenzoate and iodoacetic acid, suggesting the involve- ment of su1phydryl groups in the reaction mechanism. The enzyme had a broad substrate specificity, hydrolysing a number of p-nitroanilide derivatives of amino acids and peptides and di-, tri-, tetra- and pentapeptides. The N-tenninal amino acid sequence of the first 20 amino acid residues was determined (AELMRFYQSFQPEHYQVFLD), and showed 40-55 % homology with Zn2+-dependent aminopep- tidases from Streptococcus thermophilus NCDu53, Lactobacillus delbrueckii subsp. lactis DSM7290, Lactococcus lactis subsp. lactis MG 1363 and Lactococcus lactis subsp. cremoris W g2. © Inra/Elsevier, Paris.

The arbZ gene from Lactobacillus delbrueckii subsp. lactis confers to Escherichia coli the ability to utilize the beta-glucoside arbutin.

From a genomic library of the industrially used strain Lactobacillus delbrueckii subsp. lactis DSM7290, a gene designated arbZ (869bp; encoding a 33.5kDa protein) was isolated by screening E. coli transformants for the ability to utilize the beta-glucoside arbutin. Out of 9000 transformants nine were able to ferment arbutin, whereas no utilization of the beta-glucosides salicin, esculin or cellobiose could be detected. Overexpression of arbZ using the T7-polymerase-T7-promoter-system resulted in the formation of insoluble, catalytically inactive protein aggregates (inclusion bodies). Accordingly, overexpression was not accompanied by an increase in ArbZ activity. Induction of arbZ controlled by the lac promoter under conditions that reduce protein aggregation resulted in a 12-fold increase in arbutin hydrolyzing activity of intact cells and a 13-fold increase in phospho-beta-glycosidase activity in cell-free extracts of the respective transformants. Nucleotide sequence analysis revealed a second gene upstream of arbZ that was designated arbX (830bp). ArbX (32.6kDa) shared similarity with several glycosyltransferases involved in the biosynthesis of lipopolysaccharides in Gram-negative bacteria. In Lb. delbrueckii subsp. lactis DSM7290 two transcripts, one covering arbX together with arbZ and one covering arbZ alone were detected by Northern blot analysis.

Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32.

An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.

Lactobacillus delbrueckii subsp. lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase

A number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp. lactis gene library capable of hydrolysing the chromogenic substrate Gly-Ala-beta-naphthylamide (Gly-Ala-beta NA). Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC. Nucleotide sequence analyses of the plasmid inserts of the remaining clones resulted in the identification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC. One gene, designated pepG, was overexpressed in E. coli and the crude extracts obtained were shown to be peptidolytically active both against chromogenic substrates and peptides, and in a Salmonella typhimurium growth test. PepC and PepG activities were compared using chromogenic beta NA and p-nitroanilide substrates and leucine or proline-containing peptides were applied in growth experiments of recombinant Sal. typhimurium. The results indicate that the enzymes, although structurally related, have different substrate preferences. No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system. Primer extension analysis, using mRNA isolated from Lb. delbrueckii subsp. lactis DSM7290 did establish that orfW was transcribed.

Characterization of pepR1, a gene coding for a potential transcriptional regulator of Lactobacillus delbrueckii subsp. lactis DSM7290

The gene designated pepR1, encoding a potential transcription regulator of Lactobacillus delbrueckii subsp. lactis DSM7290, was identified by sequence similarity of an open reading frame located upstream of the prolidase pepQ orientated in opposite direction. pepQ and pepR1 coding regions are spaced by 152 nucleotides. Upstream of the -35 region of pepQ, a 14-bp palindromic sequence, homologous to the catabolite responsive element, could be identified. The pepR1 gene has the potential to encode a protein of 333 amino acids with a calculated molecular mass of 36955 Da and a calculated p/ of 5.5. The deduced protein sequence shows significant identity to the catabolite control protein of Bacillus. Co-expression in Escherichia coli was studied with the pepR1-pepQ intergenic region fused to the promoterless β-galactosidase reporter gene. The pepQ-β-galactosidase hybrid displayed an enhanced expression in the presence of cloned pepR1.

Characterization of pepR1, a gene coding for a potential transcriptional regulator of Lactobacillus delbrueckii subsp. lactis DSM7290

The gene designated pepR1, encoding a potential transcription regulator of Lactobacillus delbrueckii subsp. lactis DSM7290, was identified by sequence similarity of an open reading frame located upstream of the prolidase pepQ orientated in opposite direction. pepQ and pepR1 coding regions are spaced by 152 nucleotides. Upstream of the -35 region of pepQ, a 14-bp palindromic sequence, homologous to the catabolite responsive element, could be identified. The pepRl gene has the potential to encode a protein of 333 amino acids with a calculated molecular mass of 36955 Da and a calculated pl of 5.5. The deduced protein sequence shows significant identity to the catabolite control protein of Bacillus. Co-expression in Escherichia coli was studied with the pepR1-pepQ intergenic region fused to the promoterless beta-galactosidase reporter gene. The pepQ-beta-galactosidase hybrid displayed an enhanced expression in the presence of cloned pepR1.

Cloning and characterization of brnQ, a gene encoding a low-affinity, branched-chain amino acid carrier in Lactobacillus delbrückii subsp. lactis DSM7290.

A gene (brnQ), encoding a carrier for branched-chain amino acids in Lactobacillus delbrückii subsp. lactis DSM7290 was cloned in the low-copy-number vector pLG339 by complementation of a transport-deficient Escherichia coli strain. The plasmid carrying the cloned gene restored growth of an E. coli strain mutated in 4 different branched-chain amino acid transport genes at low concentrations of isoleucine, and increased its sensitivity to valine. Transport assays showed that leucine, isoleucine and valine are transported by this carrier and that transport is driven by the proton motive force. Nucleotide sequence analysis revealed an open reading frame of 1338 bp encoding a hydrophobic protein of 446 amino acids with a calculated molecular mass of 47864 Daltons. The start site of brnQ transcription was determined by primer extension analysis using mRNA from Lactobacillus delbrückii subsp. lactis DSM7290. The hydropathy profile suggests the existence of at least 12 hydrophobic domains that probably form membrane-associated alpha-helices. Comparisons of the nucleotide sequence of brnQ from Lactobacillus delbrückii subsp. lactis DSM7290, the amino acid sequence of its product and the topology of the hydrophobic domains with those of the respective carrier genes and proteins of Salmonella typhimurium and Pseudomonas aeruginosa revealed extensive homology.

Molecular Cloning and DNA Sequence Analysis of Pepl, a Leucyl Aminopeptidase Gene from Lactobacillus delbrueckii Subsp. Lactis DSM7290

A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau3A partial digestion in the low-copy-number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine-beta-naphthylamide (Leu-NH-Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu-NH-Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34,541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie-blue-stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC.

A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-beta-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50,909 Da. The enzyme is functional and extremely overexpressed in E. coli.

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.