Comparison of enzymatic activity of two linoleic acid isomerases expressed in E. coli.
Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of octadecadienoic fatty acid with conjugated double bonds. CLA possesses many important physiological functions and it can be produced from linoleic acid (LA) by LA isomerases. In this report, we first cloned the genes encoding LA isomerases: C12 isomerases and C9 isomerase, then transformed the recombinant plasmids into Escherichia coli TOP10 and induced E. coli with IPTG (isopropylthio-β-D-galactoside) to express the recombinant proteins. Next, we purified the isomerases using a HisTrap™ HP column, followed with the analysis by SDS-PAGE or Western blot. Finally, we compared their enzymatic activity by biotransformation of LA into CLA. Plasmids containing LA isomerase genes were successfully constructed. LA isomerases were found expressed in E. coli, and the molecular weight was 64 KD for C12 LA isomerase and 55 KD for C9 LA isomerase. The enzyme activity (9.93 ± 0.01 U/ml for C12 LA isomerase and 8.12 ± 0.02 U/ml for C9 LA isomerase) of both LA isomerases reached the highest when IPTG concentration is 0.2 mM and the induction time is 18 h. After purification, C9 LA isomerase was enriched in peak 4 and C12 LA isomerase was enriched in peak 3. Optimum pH for C9 LA and C12 LA isomerases were 7.5 and 7.0 separately, and optimum temperatures was 37 °C for highest concentration of CLA. The work may provide theoretical significance for an effective production process of CLA for the medical and nutritional purposes.
School of Food Science and Engineering, Harbin Institute of Technology, No. 92 Xidazhi Street, Nangang District, Harbin, 150090, Heilongjiang, China.
Xue
X
Luo
Lanwei
L
Zhang
Hongbo
H
Li
Shuang
S
Zhang
Yuehua
Y
Jiao
Shumei
S
Wang
Chaohui
C
Xue
Rongbo
R
Fan
D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D019787:Linoleic Acid / Q000378:metabolism*; D011424:Propionibacterium / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
D019787:Linoleic Acid; D007535:Isomerases