TY - JOUR
T1 - Codon optimization of 1,3-propanediol oxidoreductase expression in Escherichia Coli and enzymatic properties
AU - Li, Wei
AU - Ng, I. Son
AU - Fang, Baishan
AU - Yu, Jincong
AU - Zhang, Guangya
PY - 2011
Y1 - 2011
N2 - 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 colis 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 (3-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 oC 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 Mn 2+.
AB - 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 colis 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 (3-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 oC 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 Mn 2+.
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U2 - 10.2225/vol14-issue4-fulltext-9
DO - 10.2225/vol14-issue4-fulltext-9
M3 - Article
AN - SCOPUS:79960805235
SN - 0717-3458
VL - 14
SP - 7
JO - Electronic Journal of Biotechnology
JF - Electronic Journal of Biotechnology
IS - 4
ER -