Comparison of enzymatic activity of two linoleic acid isomerases expressed in E. coli

Abstract

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.

Bacterial conjugated linoleic acid production and their applications

“…linoleate isomerase was also successfully hetero-expressed in E.coli [110]. All these trials would provide an effective product on process of CLA for the medical and nutritional purposes.…”

“…Baker's yea st. Saccharomuc es cerevisiae is anot her well studied organism for metabolic engineering. For example, it has been metabolically engineered to produce artemisinic acid and amorpha-4,11-diene [110]. Expression of PAI genes in S. cerevisiae has also been…”

Evidences and perspectives in the utilization of CLNA isomers as bioactive compounds in foods

“…In order to increase the CLA and CLNA production levels, the use of recombinant technologies has been tested during the last decade. The LAI gene has been successfully introduced in other microorganisms such as Saccharomyces cerevisiae (Hornung et al, 2005;He et al, 2015), Escherichia coli (Rosberg-Cody et al, 2007;Luo et al, 2013) and Bacillus spp. (Saengkerdsub, 2013).…”

Proteomics and Metabolomics Analysis Reveal the Regulation Mechanism of Linoleate Isomerase Activity and Function in Propionibacterium acnes

“…Since Propionibacterium acnes is a conditional pathogen, LAI is usually heterologously expressed in Escherichia coli, yeast , and Yarrowia lipolytica . However, the low expression of heterologous LAI has been acknowledged as a limiting factor in its industrial application. , The LAI of P. acnes can catalyze the production of t10, c12 CLA isomers from LA without the involvement of reducing compounds such as NADH or NADPH. , …”