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...
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| Veröffentlicht in: | Journal of Bacteriology 1997-04, Vol.179 (8), p.2529-2533 |
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| description | 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. |
| doi_str_mv | 10.1128/jb.179.8.2529-2533.1997 |
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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.</description><identifier>ISSN: 0021-9193</identifier><identifier>EISSN: 1098-5530</identifier><identifier>EISSN: 1067-8832</identifier><identifier>DOI: 10.1128/jb.179.8.2529-2533.1997</identifier><identifier>PMID: 9098049</identifier><identifier>CODEN: JOBAAY</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Acids ; actividad enzimatica ; activite enzymatique ; Amino Acid Sequence ; Bacteria ; Bacteriology ; Base Sequence ; casein ; caseina ; caseine ; chemical composition ; chlorure de sodium ; Cloning, Molecular ; cloruro sodico ; composicion quimica ; composition chimique ; Cysteine - physiology ; Endopeptidases - chemistry ; Endopeptidases - genetics ; Endopeptidases - isolation & purification ; Endopeptidases - metabolism ; Enkephalin, Methionine - metabolism ; Enzyme Inhibitors - pharmacology ; Enzyme Stability ; enzymic activity ; gene ; Genes ; Genes, Bacterial - genetics ; Hydrogen-Ion Concentration ; Lactobacillus - enzymology ; Lactobacillus - genetics ; Lactobacillus helveticus ; Milk ; Molecular Sequence Data ; Molecular Weight ; nucleotide sequence ; peptidasas ; peptidase ; peptidases ; peptide ; peptides ; peptidos ; Protease Inhibitors ; Restriction Mapping ; secuencia nucleotidica ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; sequence nucleotidique ; sodium chloride ; Substrate Specificity ; temperatura ; Temperature</subject><ispartof>Journal of Bacteriology, 1997-04, Vol.179 (8), p.2529-2533</ispartof><rights>Copyright American Society for Microbiology Apr 1997</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c518t-e8e4ef5a29dd20c42ab909159ab414c862f580407a70541e96024c9997eb4923</citedby><cites>FETCH-LOGICAL-c518t-e8e4ef5a29dd20c42ab909159ab414c862f580407a70541e96024c9997eb4923</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC179000/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC179000/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,724,777,781,882,27905,27906,53772,53774</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9098049$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fenster, K M</creatorcontrib><creatorcontrib>Parkin, K L</creatorcontrib><creatorcontrib>Steele, J L</creatorcontrib><creatorcontrib>University of Wisconsin-Madison, Madison, WI</creatorcontrib><creatorcontrib>Selskostopanska Akademiya, Sofia (Bulgaria)</creatorcontrib><title>Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32</title><title>Journal of Bacteriology</title><addtitle>J Bacteriol</addtitle><description>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.</description><subject>Acids</subject><subject>actividad enzimatica</subject><subject>activite enzymatique</subject><subject>Amino Acid Sequence</subject><subject>Bacteria</subject><subject>Bacteriology</subject><subject>Base Sequence</subject><subject>casein</subject><subject>caseina</subject><subject>caseine</subject><subject>chemical composition</subject><subject>chlorure de sodium</subject><subject>Cloning, Molecular</subject><subject>cloruro sodico</subject><subject>composicion quimica</subject><subject>composition chimique</subject><subject>Cysteine - physiology</subject><subject>Endopeptidases - chemistry</subject><subject>Endopeptidases - genetics</subject><subject>Endopeptidases - isolation & purification</subject><subject>Endopeptidases - metabolism</subject><subject>Enkephalin, Methionine - metabolism</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Enzyme Stability</subject><subject>enzymic activity</subject><subject>gene</subject><subject>Genes</subject><subject>Genes, Bacterial - genetics</subject><subject>Hydrogen-Ion Concentration</subject><subject>Lactobacillus - enzymology</subject><subject>Lactobacillus - genetics</subject><subject>Lactobacillus helveticus</subject><subject>Milk</subject><subject>Molecular Sequence Data</subject><subject>Molecular Weight</subject><subject>nucleotide sequence</subject><subject>peptidasas</subject><subject>peptidase</subject><subject>peptidases</subject><subject>peptide</subject><subject>peptides</subject><subject>peptidos</subject><subject>Protease Inhibitors</subject><subject>Restriction Mapping</subject><subject>secuencia nucleotidica</subject><subject>Sequence Analysis, DNA</subject><subject>Sequence Homology, Amino Acid</subject><subject>sequence nucleotidique</subject><subject>sodium chloride</subject><subject>Substrate Specificity</subject><subject>temperatura</subject><subject>Temperature</subject><issn>0021-9193</issn><issn>1098-5530</issn><issn>1067-8832</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkk2r1DAUhoMo1_HqT1CrC3et-ZwkCxcy-AWDgl5B3IQ0PZ1maJuatFf015syw8XrxtUJnOc9vOe8QegpwRUhVL081hWRulIVFVSXVDBWEa3lHbQhWKtSCIbvog3GlJSaaHYfPUjpiDHhXNALdKEzhLneoG-7zkbrZoj-t519GIvQFraYOx_6soEJxgbGucglTDDNvrEJijaGodhnVait832_pKKD_hpm7_Jz9_Hzd0Yfonut7RM8OtdLdPX2zdXufbn_9O7D7vW-dIKouQQFHFphqW4aih2nts7eiNC25oQ7taWtyE6xtBILTkBvMeVO51Wh5pqyS_TqNHZa6gEal81G25sp-sHGXyZYb253Rt-ZQ7g2-XoY46x_cdbH8GOBNJvBJwd9b0cISzJSaSY1Y_8Fs-UtUWoFn_8DHsMSx3wDQ6nEkjNGMiRPkIshpQjtjWOCzRqwOdarRaPMGrBZAzZrwFn5-O-Fb3TnRHP_2anf-UP300cwNg23p2XmyYlpbTD2EH0yX7-s0zFhapu_yx9xULZg</recordid><startdate>19970401</startdate><enddate>19970401</enddate><creator>Fenster, K M</creator><creator>Parkin, K L</creator><creator>Steele, J L</creator><general>American Society for Microbiology</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19970401</creationdate><title>Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32</title><author>Fenster, K M ; Parkin, K L ; Steele, J L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c518t-e8e4ef5a29dd20c42ab909159ab414c862f580407a70541e96024c9997eb4923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><topic>Acids</topic><topic>actividad enzimatica</topic><topic>activite enzymatique</topic><topic>Amino Acid Sequence</topic><topic>Bacteria</topic><topic>Bacteriology</topic><topic>Base Sequence</topic><topic>casein</topic><topic>caseina</topic><topic>caseine</topic><topic>chemical composition</topic><topic>chlorure de sodium</topic><topic>Cloning, Molecular</topic><topic>cloruro sodico</topic><topic>composicion quimica</topic><topic>composition chimique</topic><topic>Cysteine - physiology</topic><topic>Endopeptidases - chemistry</topic><topic>Endopeptidases - genetics</topic><topic>Endopeptidases - isolation & purification</topic><topic>Endopeptidases - metabolism</topic><topic>Enkephalin, Methionine - metabolism</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Enzyme Stability</topic><topic>enzymic activity</topic><topic>gene</topic><topic>Genes</topic><topic>Genes, Bacterial - genetics</topic><topic>Hydrogen-Ion Concentration</topic><topic>Lactobacillus - enzymology</topic><topic>Lactobacillus - genetics</topic><topic>Lactobacillus helveticus</topic><topic>Milk</topic><topic>Molecular Sequence Data</topic><topic>Molecular Weight</topic><topic>nucleotide sequence</topic><topic>peptidasas</topic><topic>peptidase</topic><topic>peptidases</topic><topic>peptide</topic><topic>peptides</topic><topic>peptidos</topic><topic>Protease Inhibitors</topic><topic>Restriction Mapping</topic><topic>secuencia nucleotidica</topic><topic>Sequence Analysis, DNA</topic><topic>Sequence Homology, Amino Acid</topic><topic>sequence nucleotidique</topic><topic>sodium chloride</topic><topic>Substrate Specificity</topic><topic>temperatura</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fenster, K M</creatorcontrib><creatorcontrib>Parkin, K L</creatorcontrib><creatorcontrib>Steele, J L</creatorcontrib><creatorcontrib>University of Wisconsin-Madison, Madison, WI</creatorcontrib><creatorcontrib>Selskostopanska Akademiya, Sofia (Bulgaria)</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of Bacteriology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fenster, K M</au><au>Parkin, K L</au><au>Steele, J L</au><aucorp>University of Wisconsin-Madison, Madison, WI</aucorp><aucorp>Selskostopanska Akademiya, Sofia (Bulgaria)</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32</atitle><jtitle>Journal of Bacteriology</jtitle><addtitle>J Bacteriol</addtitle><date>1997-04-01</date><risdate>1997</risdate><volume>179</volume><issue>8</issue><spage>2529</spage><epage>2533</epage><pages>2529-2533</pages><issn>0021-9193</issn><eissn>1098-5530</eissn><eissn>1067-8832</eissn><coden>JOBAAY</coden><abstract>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.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>9098049</pmid><doi>10.1128/jb.179.8.2529-2533.1997</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Acids actividad enzimatica activite enzymatique Amino Acid Sequence Bacteria Bacteriology Base Sequence casein caseina caseine chemical composition chlorure de sodium Cloning, Molecular cloruro sodico composicion quimica composition chimique Cysteine - physiology Endopeptidases - chemistry Endopeptidases - genetics Endopeptidases - isolation & purification Endopeptidases - metabolism Enkephalin, Methionine - metabolism Enzyme Inhibitors - pharmacology Enzyme Stability enzymic activity gene Genes Genes, Bacterial - genetics Hydrogen-Ion Concentration Lactobacillus - enzymology Lactobacillus - genetics Lactobacillus helveticus Milk Molecular Sequence Data Molecular Weight nucleotide sequence peptidasas peptidase peptidases peptide peptides peptidos Protease Inhibitors Restriction Mapping secuencia nucleotidica Sequence Analysis, DNA Sequence Homology, Amino Acid sequence nucleotidique sodium chloride Substrate Specificity temperatura Temperature |
| title | Characterization of a thiol-dependent endopeptidase from Lactobacillus helveticus CNRZ32 |
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