Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties

The gene dhaT from Klebsiella pneumoniae encoding 1,3-propanediol oxidoreductase (PDOR) was de novo synthesized by splicing overlap extension polymerase chain reaction (SOE-PCR) primarily according to Escherichia coli’s codon usage, as well as mRNA secondary structure. After optimization, Codon Adap...

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Autores principales: Li,Wei, Ng,I-Son, Fang,Baishan, Yu,Jincong, Zhang,Guangya
Lenguaje:English
Publicado: Pontificia Universidad Católica de Valparaíso 2011
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Acceso en línea:http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582011000400007
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spelling oai:scielo:S0717-345820110004000072012-04-17Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic propertiesLi,WeiNg,I-SonFang,BaishanYu,JincongZhang,Guangya 1,3-propanediol oxidoreductase codon optimization enzymatic properties Escherichia coli overlap extension PCR The gene dhaT from Klebsiella pneumoniae encoding 1,3-propanediol oxidoreductase (PDOR) was de novo synthesized by splicing overlap extension polymerase chain reaction (SOE-PCR) primarily according to Escherichia coli’s codon usage, as well as mRNA secondary structure. After optimization, Codon Adaptation Index (CAI) value was improved from 0.75 to 0.83, meanwhile energy of mRNA secondary structure was increased from -400.1 to -86.8 kcal/mol. This synthetic DNA was under control by phage T7 promoter in the expression vector pET-15b and transformed into the E. coli BL21 (DE3) strain. Inducers such as isopropyl β-D-thiogalactoside (IPTG) and lactose were compared by activity at different inducing time. The activity of PDOR after codon optimized was 385.4 ± 3.6 U/mL, which was almost 5-fold higher than wild type (82.3 ± 1.5 U/ml) under the flask culture at 25ºC for 10 hrs. Then his-tagged enzyme was separated by using Ni-IDA column. The favorite environment for enzyme activity was at 5°C and pH 10.0, PDOR showed a certainly stability in potassium carbonate buffer for 2 hrs at diverse temperatures, enzyme activity was significantly improved by Mn2+.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.14 n.4 20112011-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582011000400007en
institution Scielo Chile
collection Scielo Chile
language English
topic 1,3-propanediol oxidoreductase
codon optimization
enzymatic properties
Escherichia coli
overlap extension PCR
spellingShingle 1,3-propanediol oxidoreductase
codon optimization
enzymatic properties
Escherichia coli
overlap extension PCR
Li,Wei
Ng,I-Son
Fang,Baishan
Yu,Jincong
Zhang,Guangya
Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties
description The gene dhaT from Klebsiella pneumoniae encoding 1,3-propanediol oxidoreductase (PDOR) was de novo synthesized by splicing overlap extension polymerase chain reaction (SOE-PCR) primarily according to Escherichia coli’s codon usage, as well as mRNA secondary structure. After optimization, Codon Adaptation Index (CAI) value was improved from 0.75 to 0.83, meanwhile energy of mRNA secondary structure was increased from -400.1 to -86.8 kcal/mol. This synthetic DNA was under control by phage T7 promoter in the expression vector pET-15b and transformed into the E. coli BL21 (DE3) strain. Inducers such as isopropyl β-D-thiogalactoside (IPTG) and lactose were compared by activity at different inducing time. The activity of PDOR after codon optimized was 385.4 ± 3.6 U/mL, which was almost 5-fold higher than wild type (82.3 ± 1.5 U/ml) under the flask culture at 25ºC for 10 hrs. Then his-tagged enzyme was separated by using Ni-IDA column. The favorite environment for enzyme activity was at 5°C and pH 10.0, PDOR showed a certainly stability in potassium carbonate buffer for 2 hrs at diverse temperatures, enzyme activity was significantly improved by Mn2+.
author Li,Wei
Ng,I-Son
Fang,Baishan
Yu,Jincong
Zhang,Guangya
author_facet Li,Wei
Ng,I-Son
Fang,Baishan
Yu,Jincong
Zhang,Guangya
author_sort Li,Wei
title Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties
title_short Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties
title_full Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties
title_fullStr Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties
title_full_unstemmed Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties
title_sort codon optimization of 1,3-propanediol oxidoreductase expression in escherichia coli and enzymatic properties
publisher Pontificia Universidad Católica de Valparaíso
publishDate 2011
url http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582011000400007
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AT ngison codonoptimizationof13propanedioloxidoreductaseexpressioninescherichiacoliandenzymaticproperties
AT fangbaishan codonoptimizationof13propanedioloxidoreductaseexpressioninescherichiacoliandenzymaticproperties
AT yujincong codonoptimizationof13propanedioloxidoreductaseexpressioninescherichiacoliandenzymaticproperties
AT zhangguangya codonoptimizationof13propanedioloxidoreductaseexpressioninescherichiacoliandenzymaticproperties
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