β-Galactosidase from Bifidobacterium adolescentis DSM20083 prefers β-(1,4)-galactosides over lactose
A β-galactosidase gene (β-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (-) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. β-Gal II had a native molecular...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2004-12, Vol.66 (3), p.276-284 |
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| creator | Hinz, S.W.A Broek, L.A.M. van den Beldman, G Vincken, J.P Voragen, A.G.J |
| description | A β-galactosidase gene (β-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (-) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. β-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that β-Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl-β-D-galactopyranoside as a substrate. The K(m) and V(max) for Gal(β1-4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant β-Gal II was highly active towards Gal(β1-4)Gal and Gal(β1-4)Gal-containing oligosaccharides; only low activity was observed towards Gal(β1-3)Gal, lactose, and Gal(β1-3)GalOMe. No activity was found towards Gal(β1-6)Gal, Gal(β1-4)Man, Gal(β1-4)Gal, Gal(β1-3)Gal(β1-4)Gal, cellobiose, maltose and sucrose. β-Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(β1-4)](2)Gal peak area ratio was 9:1. |
| doi_str_mv | 10.1007/s00253-004-1745-9 |
| format | Article |
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The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. β-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that β-Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl-β-D-galactopyranoside as a substrate. The K(m) and V(max) for Gal(β1-4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant β-Gal II was highly active towards Gal(β1-4)Gal and Gal(β1-4)Gal-containing oligosaccharides; only low activity was observed towards Gal(β1-3)Gal, lactose, and Gal(β1-3)GalOMe. No activity was found towards Gal(β1-6)Gal, Gal(β1-4)Man, Gal(β1-4)Gal, Gal(β1-3)Gal(β1-4)Gal, cellobiose, maltose and sucrose. β-Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(β1-4)](2)Gal peak area ratio was 9:1.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-004-1745-9</identifier><identifier>PMID: 15480628</identifier><identifier>CODEN: AMBIDG</identifier><language>eng</language><publisher>Berlin: Springer</publisher><subject>Amino Acid Sequence ; beta-(1,4)-galactosides ; beta-galactosidase ; beta-Galactosidase - genetics ; beta-Galactosidase - isolation & purification ; beta-Galactosidase - metabolism ; Bifidobacterium ; Bifidobacterium adolescentis ; Biological and medical sciences ; Biology of microorganisms of confirmed or potential industrial interest ; Biotechnology ; Escherichia coli ; Fundamental and applied biological sciences. Psychology ; galactosides ; Galactosides - metabolism ; intestinal microorganisms ; Lactose - metabolism ; Miscellaneous ; Mission oriented research ; Molecular Sequence Data ; Substrate Specificity</subject><ispartof>Applied microbiology and biotechnology, 2004-12, Vol.66 (3), p.276-284</ispartof><rights>2005 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c384t-47c173738cd3fc4271c6f8e75d269cdb54f886dc9396952ed4724620613b5bd3</citedby><cites>FETCH-LOGICAL-c384t-47c173738cd3fc4271c6f8e75d269cdb54f886dc9396952ed4724620613b5bd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=16439406$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15480628$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Hinz, S.W.A</creatorcontrib><creatorcontrib>Broek, L.A.M. van den</creatorcontrib><creatorcontrib>Beldman, G</creatorcontrib><creatorcontrib>Vincken, J.P</creatorcontrib><creatorcontrib>Voragen, A.G.J</creatorcontrib><title>β-Galactosidase from Bifidobacterium adolescentis DSM20083 prefers β-(1,4)-galactosides over lactose</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><description>A β-galactosidase gene (β-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (-) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. β-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that β-Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl-β-D-galactopyranoside as a substrate. The K(m) and V(max) for Gal(β1-4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant β-Gal II was highly active towards Gal(β1-4)Gal and Gal(β1-4)Gal-containing oligosaccharides; only low activity was observed towards Gal(β1-3)Gal, lactose, and Gal(β1-3)GalOMe. No activity was found towards Gal(β1-6)Gal, Gal(β1-4)Man, Gal(β1-4)Gal, Gal(β1-3)Gal(β1-4)Gal, cellobiose, maltose and sucrose. β-Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(β1-4)](2)Gal peak area ratio was 9:1.</description><subject>Amino Acid Sequence</subject><subject>beta-(1,4)-galactosides</subject><subject>beta-galactosidase</subject><subject>beta-Galactosidase - genetics</subject><subject>beta-Galactosidase - isolation & purification</subject><subject>beta-Galactosidase - metabolism</subject><subject>Bifidobacterium</subject><subject>Bifidobacterium adolescentis</subject><subject>Biological and medical sciences</subject><subject>Biology of microorganisms of confirmed or potential industrial interest</subject><subject>Biotechnology</subject><subject>Escherichia coli</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>galactosides</subject><subject>Galactosides - metabolism</subject><subject>intestinal microorganisms</subject><subject>Lactose - metabolism</subject><subject>Miscellaneous</subject><subject>Mission oriented research</subject><subject>Molecular Sequence Data</subject><subject>Substrate Specificity</subject><issn>0175-7598</issn><issn>1432-0614</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtuFDEQhq0IlAwTDsAGegMiEoby215CQgJSIhYJa8vtR9SoezzYM5G4Vg7CmeJRj5JlNlWq0le_qupH6A2BzwRAfakAVDAMwDFRXGBzgBaEM4pBEv4CLYAogZUw-gi9qvUPAKFaykN0RATXIKleoPT_Hl-40flNrkNwNXap5Kn7NqQh5L61Yxm2U-dCHmP1cbUZand2fUUBNOvWJaZYatc0PpJP_ATfPirF2uW7WLq5jsfoZXJjja_3eYluzr_fnP7Al78ufp5-vcSeab7BXHmimGLaB5Y8p4p4mXRUIlBpfOgFT1rL4A0z0ggaA1eUS9rOZb3oA1uiD7PsuuS_21g3dhra1uPoVjFvq5WKKkaJeBZsjwOx22SJyAz6kmtt99p1GSZX_lkCdmeCnU2wzQS7M8GaNvN2L77tpxieJvZfb8D7PeCqd2MqbuWH-sRJzgwH2bh3M5dctu62NOb3NQXCgLTADGEPIziX4A</recordid><startdate>20041201</startdate><enddate>20041201</enddate><creator>Hinz, S.W.A</creator><creator>Broek, L.A.M. van den</creator><creator>Beldman, G</creator><creator>Vincken, J.P</creator><creator>Voragen, A.G.J</creator><general>Springer</general><scope>FBQ</scope><scope>IQODW</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>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20041201</creationdate><title>β-Galactosidase from Bifidobacterium adolescentis DSM20083 prefers β-(1,4)-galactosides over lactose</title><author>Hinz, S.W.A ; Broek, L.A.M. van den ; Beldman, G ; Vincken, J.P ; Voragen, A.G.J</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c384t-47c173738cd3fc4271c6f8e75d269cdb54f886dc9396952ed4724620613b5bd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Amino Acid Sequence</topic><topic>beta-(1,4)-galactosides</topic><topic>beta-galactosidase</topic><topic>beta-Galactosidase - genetics</topic><topic>beta-Galactosidase - isolation & purification</topic><topic>beta-Galactosidase - metabolism</topic><topic>Bifidobacterium</topic><topic>Bifidobacterium adolescentis</topic><topic>Biological and medical sciences</topic><topic>Biology of microorganisms of confirmed or potential industrial interest</topic><topic>Biotechnology</topic><topic>Escherichia coli</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>galactosides</topic><topic>Galactosides - metabolism</topic><topic>intestinal microorganisms</topic><topic>Lactose - metabolism</topic><topic>Miscellaneous</topic><topic>Mission oriented research</topic><topic>Molecular Sequence Data</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hinz, S.W.A</creatorcontrib><creatorcontrib>Broek, L.A.M. van den</creatorcontrib><creatorcontrib>Beldman, G</creatorcontrib><creatorcontrib>Vincken, J.P</creatorcontrib><creatorcontrib>Voragen, A.G.J</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Applied microbiology and biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hinz, S.W.A</au><au>Broek, L.A.M. van den</au><au>Beldman, G</au><au>Vincken, J.P</au><au>Voragen, A.G.J</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>β-Galactosidase from Bifidobacterium adolescentis DSM20083 prefers β-(1,4)-galactosides over lactose</atitle><jtitle>Applied microbiology and biotechnology</jtitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2004-12-01</date><risdate>2004</risdate><volume>66</volume><issue>3</issue><spage>276</spage><epage>284</epage><pages>276-284</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><coden>AMBIDG</coden><abstract>A β-galactosidase gene (β-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (-) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. β-Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that β-Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl-β-D-galactopyranoside as a substrate. The K(m) and V(max) for Gal(β1-4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant β-Gal II was highly active towards Gal(β1-4)Gal and Gal(β1-4)Gal-containing oligosaccharides; only low activity was observed towards Gal(β1-3)Gal, lactose, and Gal(β1-3)GalOMe. No activity was found towards Gal(β1-6)Gal, Gal(β1-4)Man, Gal(β1-4)Gal, Gal(β1-3)Gal(β1-4)Gal, cellobiose, maltose and sucrose. β-Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(β1-4)](2)Gal peak area ratio was 9:1.</abstract><cop>Berlin</cop><pub>Springer</pub><pmid>15480628</pmid><doi>10.1007/s00253-004-1745-9</doi><tpages>9</tpages></addata></record> |
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| subjects | Amino Acid Sequence beta-(1,4)-galactosides beta-galactosidase beta-Galactosidase - genetics beta-Galactosidase - isolation & purification beta-Galactosidase - metabolism Bifidobacterium Bifidobacterium adolescentis Biological and medical sciences Biology of microorganisms of confirmed or potential industrial interest Biotechnology Escherichia coli Fundamental and applied biological sciences. Psychology galactosides Galactosides - metabolism intestinal microorganisms Lactose - metabolism Miscellaneous Mission oriented research Molecular Sequence Data Substrate Specificity |
| title | β-Galactosidase from Bifidobacterium adolescentis DSM20083 prefers β-(1,4)-galactosides over lactose |
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