Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica)

Detalhes bibliográficos
Autor(a) principal: Sun,Zhen
Data de Publicação: 2013
Outros Autores: Ma,Chunmei, Zhou,Jie, Zhu,Shuhua
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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spelling 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
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