Optimization Of Expression Conditions Of Recombinant Linoleic Acid Isomerase In Kluyveromyces Lactis And-microcapsulation

In recent years, conjugated linoleic acid take more and more attention of people because of its physiological functions. Linoleic acid isomerase is very important in the process of formation of conjugated linoleic acid enzymes. The faults of using the enzyme of microbial fermentation or enzyme are:complicated, difficult, unstable.This experiment improved the stability and activity of the enzyme by con-struction of a genetically engineered K. lacti bacterium which expresses linoleic acid isomerase. We used two ways to microcapsulate LAI:(1) polyelectrolyte microcapsules(2) sodium alginate microcapsules. The experiment could be a foundation for further study of producting CLA by LAI..The LAI amplified by PCR from Lactobacillus plantarum P-8, then the gene was inserted into the pKLAC1. The pKLACl-LAI was transformed into CICC. After optimizing the conditions for expression, we purfied the recombinant LAI. At last we used two ways to microcapsulate linoleic acid isomerase, and compared these two ways on three’aspects:particle size, activity and stability of the enzyme. An genetically engineered bacterium was successfully constructed. Optimal conditions of recombinant LAI expression were:4 days of induction,30℃, pH=5, and using lactose as carbon source in 90 mL of culture in 250 mL flask the culture was diluted in 2%, at 200 r/min, the enzyme activity was 3.43±0.12 ×10HU/mL. After purificating, only a strip detected by SDS-PAGE electrophoresis. The enzyme activity was 9.10±0.01 ×103U/mL.The enzyme activity of sodium alginate microcapsules was 1.74±0.08×103U/mg, the enzyme activity of polyelectrolyte microcapsules was 2.14±0.10×103U/mg; sodium alginate microcapsules were stable in 20～40℃, polyelectrolyte microcapsules were stable in 20～50℃; sodium alginate microcapsules were stable in pH 4 to 5, polyelectrolyte microcapsules were stable in pH 4 to 6. Polyelectrolyte microcapsules was better than sodium alginate microcapsules on enzyme activity and stability.