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Codon optimization of 1, 3-propanediol oxidoreductase expression in escherichia coli and enzymatic properties
Li, Wei; Ng, I-Son; Fang, Baishan; Yu, Jincong; Zhang, Guangya.
  • Li, Wei; Huaqiao University. The Key Laboratory for Industrial Biotechnology of Fujian Higher Education. Xiamen. CN
  • Ng, I-Son; Xiamen University. College of Chemistry and Chemical Engineering. Department of Chemical and Biochemical Engineering. Xiamen. CN
  • Fang, Baishan; Xiamen University. College of Chemistry and Chemical Engineering. Department of Chemical and Biochemical Engineering. Xiamen. CN
  • Yu, Jincong; Huaqiao University. The Key Laboratory for Industrial Biotechnology of Fujian Higher Education. Xiamen. CN
  • Zhang, Guangya; Xiamen University. College of Chemistry and Chemical Engineering. The Key Laboratory for Chemical Biology of Fujian Province. Xiamen. CN
Electron. j. biotechnol ; 14(4): 7-7, July 2011. ilus, tab
Article in English | LILACS | ID: lil-640502
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 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+.
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Full text: Available Index: LILACS (Americas) Main subject: Propylene Glycols / Alcohol Dehydrogenase / Codon / Escherichia coli Language: English Journal: Electron. j. biotechnol Journal subject: Biotechnology Year: 2011 Type: Article / Project document Affiliation country: China Institution/Affiliation country: Huaqiao University/CN / Xiamen University/CN

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Full text: Available Index: LILACS (Americas) Main subject: Propylene Glycols / Alcohol Dehydrogenase / Codon / Escherichia coli Language: English Journal: Electron. j. biotechnol Journal subject: Biotechnology Year: 2011 Type: Article / Project document Affiliation country: China Institution/Affiliation country: Huaqiao University/CN / Xiamen University/CN