Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum
Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of Penicillium chrysogenum 31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved...
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| Veröffentlicht in: | Molecular biotechnology 2015-06, Vol.57 (6), p.539-548 |
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| creator | Iwai, Marin Yamada, Hiroyuki Ikemoto, Takeshi Matsumoto, Shotaro Fujiwara, Daisuke Takenaka, Shigeo Sakamoto, Tatsuji |
| description | Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of
Penicillium chrysogenum
31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the
Aspergillus aculeatus
RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in
Escherichia coli
demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration. |
| doi_str_mv | 10.1007/s12033-015-9847-4 |
| format | Article |
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Penicillium chrysogenum
31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the
Aspergillus aculeatus
RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in
Escherichia coli
demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.</description><identifier>ISSN: 1073-6085</identifier><identifier>EISSN: 1559-0305</identifier><identifier>DOI: 10.1007/s12033-015-9847-4</identifier><identifier>PMID: 25666014</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Bacteria ; Biochemistry ; Biological Techniques ; Biotechnology ; Catalytic Domain ; Cations ; Cell Biology ; Chemistry ; Chemistry and Materials Science ; Chromatography, Ion Exchange ; Cloning, Molecular ; Crystallography ; Crystallography, X-Ray ; E coli ; Electrophoresis, Polyacrylamide Gel ; Enzymatic activity ; Enzymes ; Gene expression ; Human Genetics ; Penicillium chrysogenum - enzymology ; Polysaccharide-Lyases - chemistry ; Polysaccharide-Lyases - genetics ; Polysaccharide-Lyases - isolation & purification ; Polysaccharide-Lyases - metabolism ; Protein Science ; Sequence Alignment ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><ispartof>Molecular biotechnology, 2015-06, Vol.57 (6), p.539-548</ispartof><rights>Springer Science+Business Media New York 2015</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c508t-44b3ccbd69a05f04be5df3058d015f35a1f7301b7ec4698792769e5155b888693</citedby><cites>FETCH-LOGICAL-c508t-44b3ccbd69a05f04be5df3058d015f35a1f7301b7ec4698792769e5155b888693</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s12033-015-9847-4$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s12033-015-9847-4$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25666014$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Iwai, Marin</creatorcontrib><creatorcontrib>Yamada, Hiroyuki</creatorcontrib><creatorcontrib>Ikemoto, Takeshi</creatorcontrib><creatorcontrib>Matsumoto, Shotaro</creatorcontrib><creatorcontrib>Fujiwara, Daisuke</creatorcontrib><creatorcontrib>Takenaka, Shigeo</creatorcontrib><creatorcontrib>Sakamoto, Tatsuji</creatorcontrib><title>Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum</title><title>Molecular biotechnology</title><addtitle>Mol Biotechnol</addtitle><addtitle>Mol Biotechnol</addtitle><description>Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of
Penicillium chrysogenum
31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the
Aspergillus aculeatus
RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in
Escherichia coli
demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.</description><subject>Bacteria</subject><subject>Biochemistry</subject><subject>Biological Techniques</subject><subject>Biotechnology</subject><subject>Catalytic Domain</subject><subject>Cations</subject><subject>Cell Biology</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Chromatography, Ion Exchange</subject><subject>Cloning, Molecular</subject><subject>Crystallography</subject><subject>Crystallography, X-Ray</subject><subject>E coli</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzymatic activity</subject><subject>Enzymes</subject><subject>Gene expression</subject><subject>Human Genetics</subject><subject>Penicillium chrysogenum - enzymology</subject><subject>Polysaccharide-Lyases - chemistry</subject><subject>Polysaccharide-Lyases - genetics</subject><subject>Polysaccharide-Lyases - isolation & purification</subject><subject>Polysaccharide-Lyases - metabolism</subject><subject>Protein Science</subject><subject>Sequence Alignment</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><issn>1073-6085</issn><issn>1559-0305</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNp1kU2LFDEQhoMo7of-AC_S4MVLtNL56OSow-oKA-tBzyGdrp7J0p2MybQ4_nrTzCoieEqoPG9VvXkJecHgDQPo3hbWAucUmKRGi46KR-SSSWkocJCP6x06ThVoeUGuSrkHaJkU_Cm5aKVSCpi4JIf3Ifk9zsG7qdnsXXb-iDn8dMeQYuPi0Nx9x4w_DhlLWUtprNXmJg6J5r2bY9q5qWqWnGKtb0-uYDPmNDefMQYfpiksc-P3-VTSDuMyPyNPRjcVfP5wXpOvH26-bG7p9u7jp827LfUS9JEK0XPv-0EZB3IE0aMcxupKD9XtyKVjY8eB9R16oYzuTNspg7K677XWyvBr8vrc95DTtwXL0c6heJwmFzEtxTKlWauUgRV99Q96n5Yc63YrBabVxuhKsTPlcyol42gPOcwunywDu8Zhz3HYuqBd47Cial4-dF76GYc_it__X4H2DJT6FHeY_xr9366_AACylhY</recordid><startdate>20150601</startdate><enddate>20150601</enddate><creator>Iwai, Marin</creator><creator>Yamada, Hiroyuki</creator><creator>Ikemoto, Takeshi</creator><creator>Matsumoto, Shotaro</creator><creator>Fujiwara, Daisuke</creator><creator>Takenaka, Shigeo</creator><creator>Sakamoto, Tatsuji</creator><general>Springer US</general><general>Springer Nature B.V</general><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>3V.</scope><scope>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>Q9U</scope><scope>7X8</scope></search><sort><creationdate>20150601</creationdate><title>Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum</title><author>Iwai, Marin ; Yamada, Hiroyuki ; Ikemoto, Takeshi ; Matsumoto, Shotaro ; Fujiwara, Daisuke ; Takenaka, Shigeo ; Sakamoto, Tatsuji</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c508t-44b3ccbd69a05f04be5df3058d015f35a1f7301b7ec4698792769e5155b888693</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Bacteria</topic><topic>Biochemistry</topic><topic>Biological Techniques</topic><topic>Biotechnology</topic><topic>Catalytic Domain</topic><topic>Cations</topic><topic>Cell Biology</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Chromatography, Ion Exchange</topic><topic>Cloning, Molecular</topic><topic>Crystallography</topic><topic>Crystallography, X-Ray</topic><topic>E coli</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Enzymatic activity</topic><topic>Enzymes</topic><topic>Gene expression</topic><topic>Human Genetics</topic><topic>Penicillium chrysogenum - enzymology</topic><topic>Polysaccharide-Lyases - chemistry</topic><topic>Polysaccharide-Lyases - genetics</topic><topic>Polysaccharide-Lyases - isolation & purification</topic><topic>Polysaccharide-Lyases - metabolism</topic><topic>Protein Science</topic><topic>Sequence Alignment</topic><topic>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Iwai, Marin</creatorcontrib><creatorcontrib>Yamada, Hiroyuki</creatorcontrib><creatorcontrib>Ikemoto, Takeshi</creatorcontrib><creatorcontrib>Matsumoto, Shotaro</creatorcontrib><creatorcontrib>Fujiwara, Daisuke</creatorcontrib><creatorcontrib>Takenaka, Shigeo</creatorcontrib><creatorcontrib>Sakamoto, Tatsuji</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Engineering Collection</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><jtitle>Molecular biotechnology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Iwai, Marin</au><au>Yamada, Hiroyuki</au><au>Ikemoto, Takeshi</au><au>Matsumoto, Shotaro</au><au>Fujiwara, Daisuke</au><au>Takenaka, Shigeo</au><au>Sakamoto, Tatsuji</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum</atitle><jtitle>Molecular biotechnology</jtitle><stitle>Mol Biotechnol</stitle><addtitle>Mol Biotechnol</addtitle><date>2015-06-01</date><risdate>2015</risdate><volume>57</volume><issue>6</issue><spage>539</spage><epage>548</epage><pages>539-548</pages><issn>1073-6085</issn><eissn>1559-0305</eissn><abstract>Rhamnogalacturonan lyase (PcRGL4A) was purified from the culture supernatant of
Penicillium chrysogenum
31B. PcRGL4A optimal activity occurred between pH 7–8 and at 40 °C. Conserved Domain Search analysis identified PcRGL4A as a member of Polysaccharide Lyase family 4. PcRGL4A contains two conserved catalytic and four conserved substrate-binding residues as determined by X-ray crystallography of the
Aspergillus aculeatus
RG lyase. Recombinant PcRGL4A (rPcRGL4A) expressed in
Escherichia coli
demonstrated specific activity against rhamnogalacturonan (RG) but not homogalacturonan. Analysis of the RG reaction products by high-performance anion-exchange chromatography revealed that rPcRGL4A cleaved the substrate in an endo-manner and that the major final product was an RG tetrasaccharide with 4-deoxy-4,5-unsaturated galacturonic acid at the nonreducing end. Based on these results, PcRGL4A was classified as an endo-acting RG lyase (EC 4.2.2.23). Divalent cations were not essential for the enzymatic activity of rPcRGL4A, but addition of calcium ions to the reaction mixture increased enzymatic activity. rPcRGL4A demonstrated a preference for RG lacking galactose decoration.</abstract><cop>New York</cop><pub>Springer US</pub><pmid>25666014</pmid><doi>10.1007/s12033-015-9847-4</doi><tpages>10</tpages></addata></record> |
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| ispartof | Molecular biotechnology, 2015-06, Vol.57 (6), p.539-548 |
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| subjects | Bacteria Biochemistry Biological Techniques Biotechnology Catalytic Domain Cations Cell Biology Chemistry Chemistry and Materials Science Chromatography, Ion Exchange Cloning, Molecular Crystallography Crystallography, X-Ray E coli Electrophoresis, Polyacrylamide Gel Enzymatic activity Enzymes Gene expression Human Genetics Penicillium chrysogenum - enzymology Polysaccharide-Lyases - chemistry Polysaccharide-Lyases - genetics Polysaccharide-Lyases - isolation & purification Polysaccharide-Lyases - metabolism Protein Science Sequence Alignment Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization |
| title | Biochemical Characterization and Overexpression of an Endo-rhamnogalacturonan Lyase from Penicillium chrysogenum |
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