| The activity isomers of conjugated linoleic acid(CLA)have many physiological functions, such as anti-cancer, delaying organism senescence and so on. Conjugated linoleic acid synthesized by biotransformation have less amount of isomers and higher ratio of isomer which has physicalogical activeties. In this paper, the biotransformation of LA to CLA by linoleic acid isomerase (LAI) from Propionibacterium freudenreichii ssp. freudenreichii CGMCC1.2236 were studied.Firstly, parallel detection of the CLA concentration through the GC and UV methods were conducted, results showed that since UV detection mehod would be strongly interfered by media components and the substrate impurities, GC was precise and dependable method in CLA concentration determination. The growth curve of P. freudenreichii ssp. freudenreichii CGMCC1.2236 drawing by turbidity method, showed that the lag phase of the strain was from 0 to 4 h, logarithm phase 4 to 16 h, and stationary phase 16 to 20 h, after which the strain growth entered its decline phase. Furthermore, the addition of 0.1% (v/v) linoleic acid into the medium at the beginning of fermentation had strong inhibition to the growth of the strain. By determining the enzyme activities in the fermentation broth, cell contents and cell fragment respectively, we concluded that, the linoleic acid isomerase of P. freudenreichii ssp. freudenreichiiCGMCC1.2236 was a membrane-associated enzyme. Futher study of the enzyme activity showed that, under low-oxygen conditions, the enzyme activity was about 3.7 times as high as under aerobic conditions. Adding certain amount of antioxidants could significantly improve the enzyme activity. With addition of 200μL of 1% VC solution, linoleic acid isomerase activity reached 51.6 U/mL, which was 3.2 times as high as the one in micro-oxygen conditions.Secondly, the fermentation conditions of transformation of linoleic acid into CLA via P. freudenreichii ssp. freudenreichii were studied. Through single factor experiments and the Box-Behnken central composite design, the optimum fermentation conditions were obtained as follows: 2.11% glucose, 0.95% urea, and the initial pH6.52, Under which condition the CLA production reached 3.18μg /mL, which was 8.4 times as high as before optimization. CLA production has been greatly enhanced.Thirdly, the transformation of linoleic acid to CLA via dry cell powder in organic system were studied. Through orthogonal experiment, the optimum conditions were as follows: 10 mL n-heptane, 0.05 g cell dry powder, 100μL linoleic acid, and 70μL water added in 50 mL stoppered flask, the air in which was explled with high-purity nitrogen before reaction, on the water bath shaker, 35℃, 250 rpm. After 24 h, the CLA production reached 6.23μg/mL, which was 3.2 times as high as before optimization and was twice the value in the optimal fermentation. After 5 cycles of usage, the activity of the cell dry powder maintained 52% of the first cycle. This was the first studies of the optimization about the influencing factors of cell dry powder catalyzing LA into CLA in organic phase.Finally, considering the feasibility of industrial process, the immobilized cell is convenient in catalyst reusage, immobilization method was screened preliminarily. The results showed that cellulose acetate embedding was the best method, and the yield of CLA was 8.00μg/mL, which was higher than the one in whole-cell catalysis by 1.77μg/mL. After 5 cycles, the immobilized cells maintained 61% of its initial activity, moreover, after the third cycle the activety remained 92% of the beginning.
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