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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 123 (1994), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4°C or −20°C and had a pH optimum at pH 9, and a pI of 4.7; the temperature optimum was at 37°C. As the enzyme was activated by Co2+ and Zn2+, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the α or β position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. Km values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 124 (1994), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract 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-β-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.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 122 (1994), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The transcriptional start points of ten Lactobacillus delbrückii ssp. lactis DSM7290 genes were determined by primer extension. The upstream located promoter regions, including potential −35 and −10 regions and the spacing between them were compared to the well-known Escherichia coli and Bacillus subtilis promoters. The Lb. delbrückii−35 consensus sequence (TTGACA) seems to be less conserved then the E. coli sequence. The nucleotides TGC were often found upstream of the −10 region (TATAAT). The most frequently observed spacing between the two core promoter regions was 17 nt and the main distance between the −10 region and the transcriptional start point was mostly determined to be 6 nt in contrast to 7 nt, as described for E. coli promoters. The preferred initiation nucleotides in Lb. delbrückii were shown to be definitely purines (A or G). The ribosome binding sites located downstream of the promoters revealed the consensus sequence 3′-UCCUCCU-5′, being the predicted 3′-OH end of the Lactobacillus 16S rRNA with a high degree of homology to known 16S rRNAs.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1617-4623
    Keywords: Branched-chain amino acid transport ; Lactobacillus delbrückii subsp. lactis ; Nucleotide sequence analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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 α-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.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1617-4623
    Keywords: Prolidase ; Metalloprotease ; Lactobacillus delbrueckii subsp. lactis ; Nucleotide sequence analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract From a genomic library of Lactobacillus delbrueckii subsp. lactis (DSM7290) DNA, in the low-copy-number vector pLG339, a recombinant clone was selected, which complemented a mutation in the prolidase gene (pepQ) of Escherichia coli UK173. Nucleotide sequence analysis revealed an open reading frame of 1104 nucleotides corresponding to a protein of 368 amino acids with a calculated pI of 4.64 and a molecular mass of 41087 Da. The start site of pepQ transcription was determined by primer extension analysis with mRNA prepared from L. delbrueckii. Based on homology of the gene product to various peptidases and on the substrate specificity determined, the peptidase was designated PepQ. The influence of various protease inhibitors and cations on peptidase activity indicated that PepQ is a metalloprotease. The absence of a membrane-spanning domain and a signal peptide sequence argues for a cytoplasmic localization of the enzyme.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1617-4623
    Keywords: pepD gene ; gpt gene ; Intergenic region ; lpcA locus ; Peptidase D purification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A cloned DNA fragment, carrying the gene for peptidase D (pepD) of Escherichia coli, was partially sequenced. By purification of peptidase D and sequence determination of an amino-terminal oligopeptide the reading frame of the pepD gene, starting with a GTG initiator codon, was unambiguously identified. An overlap of the established nucleotide sequence with the previously sequenced 5′ flanking region of the gpt gene allowed the exact distance between pepD and gpt to be calculated. The two genes are pointing towards each other and are separated by 260 bp. A search for open reading frames (ORFs) and the analysis of possible codon usage in the intercistronic region indicate the absence of an additional gene (lpcA) between pepD and gpt.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 230 (1991), S. 230-240 
    ISSN: 1617-4623
    Keywords: envCD operon ; Nucleotide sequence ; EnvC protein ; Membrane localization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The chromosomal DNA insert in plasmid pJK131, which complements the phenotypic defects associated with a mutation in the envC gene of Escherichia coli strain PM61, was sequenced. The analysis of the nucleotide sequence revealed two open reading frames (ORFs) coding for the proteins EnvC (41281 daltons) and EnvD (104415 daltons). The envC gene product is synthesized as a pre-protein and, after cleavage of a signal peptide, the mature protein is incorporated into the cytoplasmic membrane. The detection of a common transcript for both ORFs indicated the existence of an envCD operon. Deletion analysis and the generation of frameshifts demonstrated that simultaneous expression of both genes is required to complement the defects in strain PM61. Overproduction of EnvC protein appears to be lethal to Escherichia coli. The envD gene, however, could be cloned and expressed at high levels under control of the tac promotor without deleterious effects on the host.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1617-4623
    Keywords: Peptidase D ; pepD promoter ; Deletion analysis ; Phosphate limitation ; pepD regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A series of deletions removing progressively larger parts of the 5′ flanking region of the Escherichia coli pepD gene was constructed. After fusing the resulting promoter fragments to the chromosomal malPQ operon, their activities were determined by assaying for amylomaltase, the product of the malQ gene. Transcription from the pepD promoter region in exponentially growing cells was estimated to be about 5 times less efficient than transcription from the induced lac promoter. Approximately 115 by preceding the translation start site of the pepD gene are important for regular promoter functioning, whereas the more distal sequences could be deleted without any significant effects. In bacterial cultures containing limiting amounts of inorganic phosphate, the rate of de novo synthesis of peptidase D, simultaneously with the derepression of alkaline phosphatase, increased about fivefold as a consequence of phosphate starvation. This regulation was shown to occur at the transcriptional level by the use of chromosomal pepD promoter-malPQ fusions. The inducibility by phosphate limitation was conserved in all of the deletion clones in which the pepD promoter region was still functional. As demonstrated by the use of phoB, R, and M mutants, the modulation of pepD expression is independent of the genetic system controlling the pho regulon.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Current microbiology 21 (1990), S. 341-347 
    ISSN: 1432-0991
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract DNA fragments complementing theenvC mutation could be isolated by cloning chromosomal DNA in the vector pUH84. When the frequencies of transformation and the frequencies of restoring theenvC + phenotype were compared, the high copy number hybrid plasmids complemented with a frequency of 10−5. After subcloning theenvC-complementing DNA fragment into the low copy number plasmid pLG339, efficient complementation was achieved by spontaneous integration of the IS2 element ofEscherichia coli. By nucleotide sequence analysis, a potential promoter, a ribosome-binding site, and an unidentified reading frame were detected in the respective DNA fragment.
    Type of Medium: Electronic Resource
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