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

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Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia coli and enzymatic properties.htm (421bytes)
Date
2011
Author
Li, Wei(Huaqiao Univ, Key Lab Ind Biotechnol Fujian Higher Educ)
Ng, I-Son
Fang, Baishan
方柏山
Yu, Jincong(Huaqiao Univ, Key Lab Ind Biotechnol Fujian Higher Educ)
Zhang, Guangya
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  • 化学化工-已发表论文 [14469]
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Abstract
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 beta-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 25 degrees C for 10 hrs. Then his-tagged enzyme was separated by using Ni-IDA column. The favorite environment for enzyme activity was at 5 degrees 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 Mn(2+).
Citation
ELECTRONIC JOURNAL OF BIOTECHNOLOGY,2011,14(4):
URI
http://dx.doi.org/doi:10.2225/vol14-issue4-fulltext-9
WOS:000293632600010
https://dspace.xmu.edu.cn/handle/2288/11576

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