Immobilization and Stabilization of Recombinant Multimeric Uridine and Purine Nucleoside Phosphorylases from Bacillus subtilis
We selected the PnpI/PupG (PNP) with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine and the Up/Pdp (UP) with specificity for uridine, thymidine, and deoxyuridine from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis. Then, an ext...
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| Veröffentlicht in: | Biomacromolecules 2004-11, Vol.5 (6), p.2195-2200 |
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| creator | Rocchietti, Silvia Ubiali, Daniela Terreni, Marco Albertini, Alessandra M Fernández-Lafuente, Roberto Guisán, José M Pregnolato, Massimo |
| description | We selected the PnpI/PupG (PNP) with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine and the Up/Pdp (UP) with specificity for uridine, thymidine, and deoxyuridine from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis. Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP−Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose. Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 °C) unlike the free enzymes which were promptly inactivated. The derivatives prepared were successfully used in the synthesis of 2‘-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2‘-deoxyuridine and guanine. |
| doi_str_mv | 10.1021/bm049765f |
| format | Article |
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Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP−Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose. Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 °C) unlike the free enzymes which were promptly inactivated. The derivatives prepared were successfully used in the synthesis of 2‘-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2‘-deoxyuridine and guanine.</description><identifier>ISSN: 1525-7797</identifier><identifier>EISSN: 1526-4602</identifier><identifier>DOI: 10.1021/bm049765f</identifier><identifier>PMID: 15530033</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Bacillus subtilis ; Bacillus subtilis - enzymology ; Biological and medical sciences ; Biophysical Phenomena ; Biophysics ; Biotechnology ; Cross-Linking Reagents - pharmacology ; Deoxyguanosine - chemistry ; Deoxyuridine - chemistry ; Enzymes, Immobilized - chemistry ; Fundamental and applied biological sciences. Psychology ; Glycosylation ; Guanine - chemistry ; Hydrogen-Ion Concentration ; Immobilization of enzymes and other molecules ; Immobilization techniques ; Macromolecular Substances ; Methods. Procedures. 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Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP−Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose. Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 °C) unlike the free enzymes which were promptly inactivated. The derivatives prepared were successfully used in the synthesis of 2‘-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2‘-deoxyuridine and guanine.</description><subject>Bacillus subtilis</subject><subject>Bacillus subtilis - enzymology</subject><subject>Biological and medical sciences</subject><subject>Biophysical Phenomena</subject><subject>Biophysics</subject><subject>Biotechnology</subject><subject>Cross-Linking Reagents - pharmacology</subject><subject>Deoxyguanosine - chemistry</subject><subject>Deoxyuridine - chemistry</subject><subject>Enzymes, Immobilized - chemistry</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glycosylation</subject><subject>Guanine - chemistry</subject><subject>Hydrogen-Ion Concentration</subject><subject>Immobilization of enzymes and other molecules</subject><subject>Immobilization techniques</subject><subject>Macromolecular Substances</subject><subject>Methods. Procedures. Technologies</subject><subject>Purine-Nucleoside Phosphorylase - chemistry</subject><subject>Recombinant Proteins - chemistry</subject><subject>Sepharose - chemistry</subject><subject>Temperature</subject><subject>Time Factors</subject><subject>Uridine - chemistry</subject><subject>Uridine Phosphorylase - chemistry</subject><issn>1525-7797</issn><issn>1526-4602</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0U1vFiEQB3BiNLZWD34BsxdNPGwdYAE52saXJm1t1J43LAwpDSyPsBzqoZ_dbfvEx4OJp5lMfswk_Al5SeGQAqPvpgSDVlL4R2SfCib7QQJ7fN-LXimt9sizWq8BQPNBPCV7VAgOwPk-uT1JKU8hhl9mCXnuzOy674v5a5J99w1tTlOYzbx0Zy0uIWEJtrsswYUZ799ctHLXnjcbMdfgsLu4ynVzlctNNBVr50tO3ZGxIcZWu9qmZT1Rn5Mn3sSKL7b1gFx--vjj-Et_-vXzyfGH095wxZaeIkzoUHA7MAHGUAESQQjQkgsFyJwyVnvDUA-gPHcKBqmY847qSSnOD8ibh72bkn82rMuYQrUYo5kxtzpKBYJypv4LqdbDeyHFCt8-QFtyrQX9uCkhmXIzUhjvUhn_pLLaV9ulbUrodnIbwwpeb4Gp1kRfzGxD3TnJ1hQ52zlj63idW5nXT_vHwd_Dh6Jk</recordid><startdate>20041101</startdate><enddate>20041101</enddate><creator>Rocchietti, Silvia</creator><creator>Ubiali, Daniela</creator><creator>Terreni, Marco</creator><creator>Albertini, Alessandra M</creator><creator>Fernández-Lafuente, Roberto</creator><creator>Guisán, José M</creator><creator>Pregnolato, Massimo</creator><general>American Chemical Society</general><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>7QL</scope><scope>7QO</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>20041101</creationdate><title>Immobilization and Stabilization of Recombinant Multimeric Uridine and Purine Nucleoside Phosphorylases from Bacillus subtilis</title><author>Rocchietti, Silvia ; Ubiali, Daniela ; Terreni, Marco ; Albertini, Alessandra M ; Fernández-Lafuente, Roberto ; Guisán, José M ; Pregnolato, Massimo</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a372t-1e0bede53c4250aa1506e0550963570e2d7ac9fa2e9407f3d704672dfd19b7733</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Bacillus subtilis</topic><topic>Bacillus subtilis - enzymology</topic><topic>Biological and medical sciences</topic><topic>Biophysical Phenomena</topic><topic>Biophysics</topic><topic>Biotechnology</topic><topic>Cross-Linking Reagents - pharmacology</topic><topic>Deoxyguanosine - chemistry</topic><topic>Deoxyuridine - chemistry</topic><topic>Enzymes, Immobilized - chemistry</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glycosylation</topic><topic>Guanine - chemistry</topic><topic>Hydrogen-Ion Concentration</topic><topic>Immobilization of enzymes and other molecules</topic><topic>Immobilization techniques</topic><topic>Macromolecular Substances</topic><topic>Methods. Procedures. Technologies</topic><topic>Purine-Nucleoside Phosphorylase - chemistry</topic><topic>Recombinant Proteins - chemistry</topic><topic>Sepharose - chemistry</topic><topic>Temperature</topic><topic>Time Factors</topic><topic>Uridine - chemistry</topic><topic>Uridine Phosphorylase - chemistry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rocchietti, Silvia</creatorcontrib><creatorcontrib>Ubiali, Daniela</creatorcontrib><creatorcontrib>Terreni, Marco</creatorcontrib><creatorcontrib>Albertini, Alessandra M</creatorcontrib><creatorcontrib>Fernández-Lafuente, Roberto</creatorcontrib><creatorcontrib>Guisán, José M</creatorcontrib><creatorcontrib>Pregnolato, Massimo</creatorcontrib><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>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</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>Biomacromolecules</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Rocchietti, Silvia</au><au>Ubiali, Daniela</au><au>Terreni, Marco</au><au>Albertini, Alessandra M</au><au>Fernández-Lafuente, Roberto</au><au>Guisán, José M</au><au>Pregnolato, Massimo</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Immobilization and Stabilization of Recombinant Multimeric Uridine and Purine Nucleoside Phosphorylases from Bacillus subtilis</atitle><jtitle>Biomacromolecules</jtitle><addtitle>Biomacromolecules</addtitle><date>2004-11-01</date><risdate>2004</risdate><volume>5</volume><issue>6</issue><spage>2195</spage><epage>2200</epage><pages>2195-2200</pages><issn>1525-7797</issn><eissn>1526-4602</eissn><abstract>We selected the PnpI/PupG (PNP) with specificity for ribo- and deoxyriboguanosine and ribo- and deoxyriboinosine and the Up/Pdp (UP) with specificity for uridine, thymidine, and deoxyuridine from the purine and pyrimidine salvage pathway of the Gram-positive bacterium Bacillus subtilis. Then, an extensive study of the UP (uridine phosphorylase) and PNP (purine nucleoside phosphorylase) immobilization and stabilization was carried out: optimal UP preparation was achieved by immobilization onto Sepabeads coated with poly(ethyleneimine) and finally cross-linked with aldehyde dextran (UP−Sep-PEI-Dx); optimal immobilized PNP was prepared onto glyoxyl-agarose. Both derivatives were highly stable and active even under drastic experimental conditions (pH 10, 45 °C) unlike the free enzymes which were promptly inactivated. The derivatives prepared were successfully used in the synthesis of 2‘-deoxyguanosine by enzymatic transglycosylation in aqueous solution between 2‘-deoxyuridine and guanine.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>15530033</pmid><doi>10.1021/bm049765f</doi><tpages>6</tpages></addata></record> |
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| subjects | Bacillus subtilis Bacillus subtilis - enzymology Biological and medical sciences Biophysical Phenomena Biophysics Biotechnology Cross-Linking Reagents - pharmacology Deoxyguanosine - chemistry Deoxyuridine - chemistry Enzymes, Immobilized - chemistry Fundamental and applied biological sciences. Psychology Glycosylation Guanine - chemistry Hydrogen-Ion Concentration Immobilization of enzymes and other molecules Immobilization techniques Macromolecular Substances Methods. Procedures. Technologies Purine-Nucleoside Phosphorylase - chemistry Recombinant Proteins - chemistry Sepharose - chemistry Temperature Time Factors Uridine - chemistry Uridine Phosphorylase - chemistry |
| title | Immobilization and Stabilization of Recombinant Multimeric Uridine and Purine Nucleoside Phosphorylases from Bacillus subtilis |
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