| Conjugated linoleic acid is a collection of octadecadienoic acid isomers which have the conjugated double bonds. It is generally believed that CLA has 16 isomers because of its double bond's location and structure,but only c9, t11-CLA and t10, c12-CLA has physiological activity and closely related to human and animal's nutrition. In addition to having a strong anti-cancer function, the new dietary supplements CLA also can inhibit the deposition of fat, lower cholesterol, prevent atherosclerosis, improve human metabolism, regulate blood sugar, control diabetes, enhance immunity, improve bone density, anti-oxidation, improve sleep and so on.Natural sources of CLA is very limited, biological synthesis of CLA uses Linoleic acid isomerase to convert LA or its derivative into CLA, people always use costly linoleic acid, castor oil, sunflower oil, corn germ oil and some expensive materials as the substrate to generate CLA. As the big rapeseed planting's country, our annual yield of rapeseed oil can reach more than 1,000 tons. Rapeseed oil sediment is the waste of rapeseed oil's phospholipid hydration procession. As 1.5% estimated by the amount of rapeseed oil,we can produce more than 15 million tons of rapeseed oil sediment every year. The linoleic acid in it can be used by linoleic acid isomerase to covert to physiologically active fatty acid-CLA. The application of it will solve the problem of toxic byproducts by chemical high temperature and pressure, improve the quality of products, solve the problem of low conversion rate, long production cycle, high cost, high pollution and many other issues by traditional biosynthesis, at the same time it will achieve high efficiency, high-quality, low consumption and pollution-free production to meet the growing market demand for CLA, providing evidence about developing the agricultural waste-rapeseed oil sediment.Firstly, through adding different concentrations of the inducer LA into MRS medium, We found that Lactobacillus acidophilus CGMCC1.1854, Lactobacillus plantarum CGMCC1.557 and Lactobacillus plantarum 3-9 which was obtained from the pickle has the different sensitivity to the degree of the inducer. when the concentration of linoleic acid emulsion was 0.1mg/m L, the linoleic acid isomerase induced from Lactobacillus plantarum CGMCC1.557 and Lactobacillus plantarum 3-9 had the highest activity; when the concentration of linoleic acid emulsion was 0.3mg/m L, the linoleic acid isomerase induced from Lactobacillus acidophilus CGMCC1.1854 had the highest activity, thus we determined the best amount of inducer.Secondly, we identified the best ammonium sulphate saturation range through ammonium sulphate precipitation. The linoleic acid isomerase from Lactobacillus acidophilus CGMCC1.1854 was lower limit of 30% and up to 80%, the linoleic acid isomerase from Lactobacillus plantatrum CGMCC1.557 and Lactobacillus plantatrum 3-9 was lower limit of 40% and up to 80%. And then we achieved the pure linoleic acid isomerase from these three strains through dialysis, concentration by polyethylene glycol and gel filtration chromatography.Thirdly, we compared these three isomerase's optimum temperature, optimum p H, thermal stability, p H stability, and kinetic parameters. The optimum temperature of LAI from Lactobacillus acidophilus CGMCC1.1854 was 30?, and the LAI from Lactobacillus plantatrum CGMCC1.557 and Lactobacillus plantatrum 3-9 was 45?; the optimum p H of these three isomerase was 6.5; The LAI from Lactobacillus acidophilus CGMCC1.1854 was affected by temperature considerably, this enzyme was not heat resistant, Lactobacillus plantarum CGMCC1.557 and Lactobacillus plantarum 3-9 enzyme activity remained relatively stable at low temperatures, at high temperature stability of the former than the latter; The LAI from Lactobacillus acidophilus CGMCC1.1854 was affected by p H significantly, its acid-base stability was the worst, when p H was between p H3.0~6.0, LAI from Lactobacillus plantarum CGMCC1.557 was stable, thus the p H was above 6.0, the stability was not good, LAI from Lactobacillus plantarum 3-9 had the optimal p H stability which can adapt to a wide p H range; then we determined the kinetic parameters of these three kinds of linoleic acid isomerase, the apparent Km and Vmax of the enzyme from Lactobacillus acidophilus CGMCC1.1854 was 18.99 mmo L·L-1 and 1.58ug·m L-1·min-1; the apparent Km and Vmax of the enzyme from Lactobacillus plantarum CGMCC1.557 was 14.83 mmo L·L-1 and 1.75ug·m L-1·min-1; the apparent Km and Vmax of the enzyme from Lactobacillus plantarum 3-9 was 14.26 mmo L·L-1 and 1.94ug·m L-1·min-1.By comparing the capacity of LAI differently from Lactobacillus acidophilus CGMCC1.1854, Lactobacillus plantarum CGMCC1.557 and Lactobacillus plantarum 3-9 to convert LA into CLA under the condition of their respective optimal p H and optimal reaction temperature, we determined that Lactobacillus plantarum 3-9 had the strongest ability, the conversion of CLA was 1.17 times that of Lactobacillus plantarum CGMCC1.557 and was Lactobacillus acidophilus CGMCC1.1854 1.72 times; then we compared the capacity of LAI differently from these three strains to convert rapeseed oil sediment into CLA under the condition of their respective optimal p H and optimal reaction temperature, we determined that Lactobacillus plantarum 3-9 had the strongest ability, the conversion of CLA was 1.03 times that of Lactobacillus plantarum CGMCC1.557 and was Lactobacillus acidophilus CGMCC1.1854 1.19 times. Above all, the LAI from Lactobacillus plantarum 3-9 was selected to convert rapeseed oil sediment into CLA.Through a series of single factor experiments, we optimized the substrate dosage, buffer type, oscillation speed and conversion time, and then the optimum reaction conditions were determined through orthogonal experiments: the substrate was 3.0 m L, the buffer was potassium phosphate buffer,the oscillation speed was 100 r/min, transformation time was 18 h, under this condition,the yield of CLA was 244.85±4.91 ug/m L, which was 1.31 times than that before.Finally through the cofactors, metal ions and chelating agent for the influence of the enzymatic reaction, we determined the cofactor ATP, ADP, NADH and metal ions Ca2+, Mg2+ had a certain role in promoting. Then we designed the combination experiment and found that Ca2+ and ATP binding energy was very good to promote the reaction, and the optimized CLA generation was 1.27 times than that before. |
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