Purification Of A Linoleic Acid Isomerase From Lactobacillus Plantarum And It's Application

Production of a linoleic acid isomerase by fermentation with a Lactobacillus plantarum (Lplantarum LT2-6) was studied. The enzyme was purified and its characteristic was determined. Besides, the improvement of CLA content in soybean dregs by microbe fermentation and enzyme reaction was investigated.Lplantarum LT2-6 can synthesize linoleic acid siomerase in its cell with the addition of linoleic acid (LA) as an inducer, so the bacteria has the ability to transform LA into conjugated linoleic acid (CLA) by fermentation. Results of experiment show that the appropriate conditions under which Lplantarum LT2-6 can produce more linoleic acid isomerase are: temperature 37°C, initial pH6.5, anaerobic environment, shaker rotate speedl20r/min. LA 0.075%, fermentation time 22~24h. The produced isomers were cis,trans-9,ll-CLA. Penicillin(28.8~115.2xl0~3U/ml) can change the cell wall, helping to crack the bacteria, without decreasing the enzyme activity considerably.After collecting cells by centrifugation, the linoleic acid isomerase was purified from LT2-6 by ammonium sulfate precipitation . DEAE Sepharose fast flow and Sephadex G-100 column chromatography. The molecular weight of the enzyme was about 43kDa. The enzyme exhibited higher activity when temperature and pH were 30℃ .. 6.0 respectively. Co~2+ Fe~2+ improved the activity of the enzyme, and Cu~2+ Zn~2+ instead decreased the activity of the enzyme. Mcihaelis constants of the enzyme were Km=2.53xl0-5 mol/L and Vmax=2.57x10-8 mol/(min-mg) when substrate was linoleic acid.The right fermentation conditions of enhancing CLA content in soybean dregs with Lplantarum LT2-6 are: temperature 30℃, water content 80%, adding CoCl2(l %, w/w), fermentation time 3days more. The highest CLA production was 450g CLA/g dry soybean dregs. The preferably enzyme reaction conditions in enhancing CLA content in soybean dregs are: temperature 30℃, water content 80%, fermentation time 7.5h more, pH of buffer system 5.5~7.5. After treatment with enzyme, CLA content increased about 330g CLA/g dry soybean dregs.