Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica)
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Brazilian Archives of Biology and Technology |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132013000400002 |
Resumo: | A gene encoding NAD+-dependent sorbitol dehydrogenase (SDH) in peach fruit was cloned and expressed in Escherichia coli. Recombinant SDH protein with 6×His-tagged was localized exclusively in the cytoplasmic soluble fraction of E. coli when the strains were grown for 4-5 h at 37 ºC. Highly pure protein was isolated by Ni2+-resin chromatography with 150 mM imidazole in 50 mM Tris, pH 8.0, by elution. In order to ensure that the recombinant SDH could be used for further study, the fluorescence and ultraviolet spectrum of the recombinant SDH were detected. Recombinant SDH was confirmed to be capable of oxidizing sorbitol by enzymatic activity assay. The activity of the recombinant SDH was 2.73 U mg-1min-1, which was similar with that directly extracted from peach fruits. The activities of SDH extracted from the fruits in different periods (30, 60, 90 days after flowing) were 7.75, 5.95, 3.26 U mg-1min-1, respectively. |
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Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica)sorbitol dehydrogenaserecombinant SDHexpression and purificationNi-NTAenzyme assaypeach fruitA gene encoding NAD+-dependent sorbitol dehydrogenase (SDH) in peach fruit was cloned and expressed in Escherichia coli. Recombinant SDH protein with 6×His-tagged was localized exclusively in the cytoplasmic soluble fraction of E. coli when the strains were grown for 4-5 h at 37 ºC. Highly pure protein was isolated by Ni2+-resin chromatography with 150 mM imidazole in 50 mM Tris, pH 8.0, by elution. In order to ensure that the recombinant SDH could be used for further study, the fluorescence and ultraviolet spectrum of the recombinant SDH were detected. Recombinant SDH was confirmed to be capable of oxidizing sorbitol by enzymatic activity assay. The activity of the recombinant SDH was 2.73 U mg-1min-1, which was similar with that directly extracted from peach fruits. The activities of SDH extracted from the fruits in different periods (30, 60, 90 days after flowing) were 7.75, 5.95, 3.26 U mg-1min-1, respectively.Instituto de Tecnologia do Paraná - Tecpar2013-08-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132013000400002Brazilian Archives of Biology and Technology v.56 n.4 2013reponame:Brazilian Archives of Biology and Technologyinstname:Instituto de Tecnologia do Paraná (Tecpar)instacron:TECPAR10.1590/S1516-89132013000400002info:eu-repo/semantics/openAccessSun,ZhenMa,ChunmeiZhou,JieZhu,Shuhuaeng2013-09-02T00:00:00Zoai:scielo:S1516-89132013000400002Revistahttps://www.scielo.br/j/babt/https://old.scielo.br/oai/scielo-oai.phpbabt@tecpar.br||babt@tecpar.br1678-43241516-8913opendoar:2013-09-02T00:00Brazilian Archives of Biology and Technology - Instituto de Tecnologia do Paraná (Tecpar)false |
dc.title.none.fl_str_mv |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) |
title |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) |
spellingShingle |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) Sun,Zhen sorbitol dehydrogenase recombinant SDH expression and purification Ni-NTA enzyme assay peach fruit |
title_short |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) |
title_full |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) |
title_fullStr |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) |
title_full_unstemmed |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) |
title_sort |
Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica) |
author |
Sun,Zhen |
author_facet |
Sun,Zhen Ma,Chunmei Zhou,Jie Zhu,Shuhua |
author_role |
author |
author2 |
Ma,Chunmei Zhou,Jie Zhu,Shuhua |
author2_role |
author author author |
dc.contributor.author.fl_str_mv |
Sun,Zhen Ma,Chunmei Zhou,Jie Zhu,Shuhua |
dc.subject.por.fl_str_mv |
sorbitol dehydrogenase recombinant SDH expression and purification Ni-NTA enzyme assay peach fruit |
topic |
sorbitol dehydrogenase recombinant SDH expression and purification Ni-NTA enzyme assay peach fruit |
description |
A gene encoding NAD+-dependent sorbitol dehydrogenase (SDH) in peach fruit was cloned and expressed in Escherichia coli. Recombinant SDH protein with 6×His-tagged was localized exclusively in the cytoplasmic soluble fraction of E. coli when the strains were grown for 4-5 h at 37 ºC. Highly pure protein was isolated by Ni2+-resin chromatography with 150 mM imidazole in 50 mM Tris, pH 8.0, by elution. In order to ensure that the recombinant SDH could be used for further study, the fluorescence and ultraviolet spectrum of the recombinant SDH were detected. Recombinant SDH was confirmed to be capable of oxidizing sorbitol by enzymatic activity assay. The activity of the recombinant SDH was 2.73 U mg-1min-1, which was similar with that directly extracted from peach fruits. The activities of SDH extracted from the fruits in different periods (30, 60, 90 days after flowing) were 7.75, 5.95, 3.26 U mg-1min-1, respectively. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013-08-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132013000400002 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132013000400002 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S1516-89132013000400002 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Instituto de Tecnologia do Paraná - Tecpar |
publisher.none.fl_str_mv |
Instituto de Tecnologia do Paraná - Tecpar |
dc.source.none.fl_str_mv |
Brazilian Archives of Biology and Technology v.56 n.4 2013 reponame:Brazilian Archives of Biology and Technology instname:Instituto de Tecnologia do Paraná (Tecpar) instacron:TECPAR |
instname_str |
Instituto de Tecnologia do Paraná (Tecpar) |
instacron_str |
TECPAR |
institution |
TECPAR |
reponame_str |
Brazilian Archives of Biology and Technology |
collection |
Brazilian Archives of Biology and Technology |
repository.name.fl_str_mv |
Brazilian Archives of Biology and Technology - Instituto de Tecnologia do Paraná (Tecpar) |
repository.mail.fl_str_mv |
babt@tecpar.br||babt@tecpar.br |
_version_ |
1750318275671097344 |