Studies On Breeding Of The Strain Of High-producing Linoleic Acid Isomerase And Its Enzymatic Properties

Conjugated linoleic acid (CLA) had the effect of immune regulation,anti-atherosclerosis, anti-cancer to the body, weight loss, antioxidant, etc. Atpresent, CLA was mainly synthesized by chemical method and biological method.Biosynthesis method showed good applied potentialities due to its mild reactionconditions and single product.Pickled cucumber, pickled radish, cowpea kimchi were chosen as the rawmaterial, from which a strain was screened with relatively high ability to produceconjugated linoleic acid (CLA) by gas chromatograph-mass spectrum technology.With conversion rate of CLA as index, optimize the conditons for producing CLAby response surface analysis statistical method. Purification of linoleic acidisomerase and enzymatic properties were studied. The main conclusions were asfollows:(1) A strain named PC-3was screened from the pickle environment withrelatively high ability to produce c9, t11-CLA and t10, c12-CLA which were allwith biological activity. Combined with GC-MS machine, strain PC-3wasidentified as Lactobacillus plantarum subsp. Plantarum strain: PC-3by the analysis of thecell and colony morphology, Physio-biochemical characteristics, and16S rDNA sequence.The16S rDNA gene sequence was submitted to GenBank and the accession numberwas JX270774.1. The length of the sequence was1460bp.(2) The main fermenting factors of Lactobacillus plantarum subsp. Plantarum strain:PC-3were incubation time, inoculation, and the concentration of LA by single factorexperiment and response surface experimental design method. The optimalconditons were as follows: MRS media, incubation time43.0h, inoculation3.0%(v/v), the concentration of LA1.30‰(v/v), lactose: ammonium sulfate=3:2,the dosage of Tween-802‰(w/v).(3) we took the ultrasonic and enzymatic method to break cells. The cells weredisrupted by sonication for80times at power400w working3s and resting4s in0℃,lysozyme concentration was0.8%, biomass concentration was15%g/mL, Fragmentationvolume was4mL, the concentration of NaCl was0.1mol/L. The optimum saturation ofAmmonium sulfate for linoleate isomerase was20%~80%. Sephadex G-100gelfiltration chromatography was used after ultrafiltration， achieving an overall purification of4.60-fold. The relative molecular weight of about42.88kDa wasestimated by SDS-PAGE electrophoresis. The optimum temperature of linoleateisomerase was35℃, the optimum pH of linoleate isomerase was4. The thermalstability of linoleate isomerase was poor. The linoleate isomerase was easy to store atthe condition of low temperature and weak acid. The Km of Linoleic isomerase was13.95mmol/L, the Vmax was9.03mmol/L·h.