Optimal Expression And Immobilization Of Linoleic Acid Isomerase PAI

Conjugated linoleic acid(CLA) is a generic term used to describe a mixture of positional and geometric isomers of linoleic acid,with conjugated double bonds. Studies show that CLA has multiple functions, such as anti-cancer, anti-aging, anti atherosclerosis, regulating body lipid metabolism, strengthening the body immunity and other important roles. In this study, Propionibacterium acnes Linoleate Isomerase(PAI)gene was expressed in E. coli BL21(DE3). The optimal fermentation of the recombinant bacteria was studied. The immobilization technique was also used to improve the capacity and stability of catalysis of the PAI, biosynthesis from LA( linoleic acid) to CLA.(1) Construction of prokaryotic expression system of PAI: The PAI gene was synthesized according to NCBI(Sequence number AX062088). And the recombinant plasmid pET24a-pai was transformed into competent cell of E. coli BL21(DE3). The PAI was successfully expressed by the recomninant stain BL21(DE3)- pET24a-pai.(2)Optimal fermentation of the recombinant bacteria: Auto-induction fermentation can increase PAI expression of catalytic biomass and enzyme activities. The conversion rate of self- induction medium ZYM-5052 fermentation catalytic synthesis of trans-10, cis-12 CLA is 2.15 times of IPTG induction. Optimization design of experimental results show that temperature,glycerol and arabinose are the main factors influence the expression of PAI. After optimization,auto-induction accordance to the over all solution, the conversion rate of the CLA increased from 28% to 35.5%.(3) Immobilization of PAI: The immobilization technology was used on PAI, and immobilized carrier is resin. The conversion rate of catalytic synthesis CLA from LA by immobilized enzyme is only 28%, which is 79.3% of crude enzyme. And the reuse activity of immobilized enzyme decreased rapidly, less than 10% of the orignal at the fourth cycle.