Purification and characterization of a Pseudomonas sp. lipase and its properties in non-aqueous media
An extracellular lipase from Pseudomonas sp. was purified to homogeneity by extraction, Bio‐gel P‐10 chromatography and Superose 12B chromatography, and a 37‐fold purification was attained. The purified enzyme showed a single band when it was subjected to SDS/PAGE and isoelectric focusing. The SDS/P...
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| Veröffentlicht in: | Biotechnology and applied biochemistry 1999-12, Vol.30 (3), p.251-256 |
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| creator | Dong, Huan Gao, Shujuan Han, Si-ping Cao, Shu-gui |
| description | An extracellular lipase from Pseudomonas sp. was purified to homogeneity by extraction, Bio‐gel P‐10 chromatography and Superose 12B chromatography, and a 37‐fold purification was attained. The purified enzyme showed a single band when it was subjected to SDS/PAGE and isoelectric focusing. The SDS/PAGE electrophoresis indicated a molecular mass of 30 kDa for this lipase. Its isoelectric point was 4.5. The optimum pH and temperature for hydrolysis were 7.0–9.0 and 45–60 °C, respectively. The enzyme was stable between pHs 6 and 12 and below 60 °C. In the presence of Ca2+ and Bi3+, the lipase activity was dramatically enhanced by 250% and 154%, respectively. Fe3+, Fe2+, Al3+, Zn2+ and Mn2+ could inhibit this lipase, but Ag+ and Pb2+ showed no influence on hydrolysis activity. Properties of purified lipase for lactonization in organic solvent were also determined. The purified lipase displayed the characteristic of ‘pH memory’ in organic media. This lipase was also thermostable in organic solvent with an optimum temperature range from 45 to 60 °C. Salt dramatically affected the lactonization activity of this lipase. |
| doi_str_mv | 10.1111/j.1470-8744.1999.tb00778.x |
| format | Article |
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The purified enzyme showed a single band when it was subjected to SDS/PAGE and isoelectric focusing. The SDS/PAGE electrophoresis indicated a molecular mass of 30 kDa for this lipase. Its isoelectric point was 4.5. The optimum pH and temperature for hydrolysis were 7.0–9.0 and 45–60 °C, respectively. The enzyme was stable between pHs 6 and 12 and below 60 °C. In the presence of Ca2+ and Bi3+, the lipase activity was dramatically enhanced by 250% and 154%, respectively. Fe3+, Fe2+, Al3+, Zn2+ and Mn2+ could inhibit this lipase, but Ag+ and Pb2+ showed no influence on hydrolysis activity. Properties of purified lipase for lactonization in organic solvent were also determined. The purified lipase displayed the characteristic of ‘pH memory’ in organic media. This lipase was also thermostable in organic solvent with an optimum temperature range from 45 to 60 °C. Salt dramatically affected the lactonization activity of this lipase.</description><identifier>ISSN: 0885-4513</identifier><identifier>EISSN: 1470-8744</identifier><identifier>DOI: 10.1111/j.1470-8744.1999.tb00778.x</identifier><identifier>PMID: 10574695</identifier><identifier>CODEN: BABIEC</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>Biological and medical sciences ; Biotechnology ; Chromatography, Ion Exchange ; Culture Media ; Electrophoresis, Polyacrylamide Gel ; Enzyme engineering ; Enzyme Stability ; Fundamental and applied biological sciences. Psychology ; Hydrogen-Ion Concentration ; Isoelectric Focusing ; Lipase - isolation & purification ; Lipase - metabolism ; Methods. Procedures. 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The purified enzyme showed a single band when it was subjected to SDS/PAGE and isoelectric focusing. The SDS/PAGE electrophoresis indicated a molecular mass of 30 kDa for this lipase. Its isoelectric point was 4.5. The optimum pH and temperature for hydrolysis were 7.0–9.0 and 45–60 °C, respectively. The enzyme was stable between pHs 6 and 12 and below 60 °C. In the presence of Ca2+ and Bi3+, the lipase activity was dramatically enhanced by 250% and 154%, respectively. Fe3+, Fe2+, Al3+, Zn2+ and Mn2+ could inhibit this lipase, but Ag+ and Pb2+ showed no influence on hydrolysis activity. Properties of purified lipase for lactonization in organic solvent were also determined. The purified lipase displayed the characteristic of ‘pH memory’ in organic media. This lipase was also thermostable in organic solvent with an optimum temperature range from 45 to 60 °C. Salt dramatically affected the lactonization activity of this lipase.</description><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Chromatography, Ion Exchange</subject><subject>Culture Media</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzyme engineering</subject><subject>Enzyme Stability</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hydrogen-Ion Concentration</subject><subject>Isoelectric Focusing</subject><subject>Lipase - isolation & purification</subject><subject>Lipase - metabolism</subject><subject>Methods. Procedures. 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Psychology</topic><topic>Hydrogen-Ion Concentration</topic><topic>Isoelectric Focusing</topic><topic>Lipase - isolation & purification</topic><topic>Lipase - metabolism</topic><topic>Methods. Procedures. Technologies</topic><topic>Miscellaneous</topic><topic>Pseudomonas</topic><topic>Pseudomonas - enzymology</topic><topic>Temperature</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Dong, Huan</creatorcontrib><creatorcontrib>Gao, Shujuan</creatorcontrib><creatorcontrib>Han, Si-ping</creatorcontrib><creatorcontrib>Cao, Shu-gui</creatorcontrib><collection>Istex</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>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Biotechnology and applied biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Dong, Huan</au><au>Gao, Shujuan</au><au>Han, Si-ping</au><au>Cao, Shu-gui</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of a Pseudomonas sp. lipase and its properties in non-aqueous media</atitle><jtitle>Biotechnology and applied biochemistry</jtitle><addtitle>Biotechnol Appl Biochem</addtitle><date>1999-12</date><risdate>1999</risdate><volume>30</volume><issue>3</issue><spage>251</spage><epage>256</epage><pages>251-256</pages><issn>0885-4513</issn><eissn>1470-8744</eissn><coden>BABIEC</coden><abstract>An extracellular lipase from Pseudomonas sp. was purified to homogeneity by extraction, Bio‐gel P‐10 chromatography and Superose 12B chromatography, and a 37‐fold purification was attained. The purified enzyme showed a single band when it was subjected to SDS/PAGE and isoelectric focusing. The SDS/PAGE electrophoresis indicated a molecular mass of 30 kDa for this lipase. Its isoelectric point was 4.5. The optimum pH and temperature for hydrolysis were 7.0–9.0 and 45–60 °C, respectively. The enzyme was stable between pHs 6 and 12 and below 60 °C. In the presence of Ca2+ and Bi3+, the lipase activity was dramatically enhanced by 250% and 154%, respectively. Fe3+, Fe2+, Al3+, Zn2+ and Mn2+ could inhibit this lipase, but Ag+ and Pb2+ showed no influence on hydrolysis activity. Properties of purified lipase for lactonization in organic solvent were also determined. The purified lipase displayed the characteristic of ‘pH memory’ in organic media. This lipase was also thermostable in organic solvent with an optimum temperature range from 45 to 60 °C. Salt dramatically affected the lactonization activity of this lipase.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>10574695</pmid><doi>10.1111/j.1470-8744.1999.tb00778.x</doi><tpages>6</tpages></addata></record> |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Biological and medical sciences Biotechnology Chromatography, Ion Exchange Culture Media Electrophoresis, Polyacrylamide Gel Enzyme engineering Enzyme Stability Fundamental and applied biological sciences. Psychology Hydrogen-Ion Concentration Isoelectric Focusing Lipase - isolation & purification Lipase - metabolism Methods. Procedures. Technologies Miscellaneous Pseudomonas Pseudomonas - enzymology Temperature |
| title | Purification and characterization of a Pseudomonas sp. lipase and its properties in non-aqueous media |
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