Study On The Preparation Of Citrulluine

As one of the nonprotein amino acids, citrulline plays important role in the urea cycle. Recent years many foreign researches show citrulline has a lot of important physiological functions such as radical-scavenging hydroxyl, indicator of the foreign exclusive effect, vasodilatation, stabilizing blood pressure, diagnosing rheumatoidal arthritis, antioxidation and so on. The thesis investigated the analysis approach of citrulline and the producing process of citrulline by extract process and fermentation.The thesis first established a HPLC method for analyzing citrulline by 2,4-DNFB pre-column derivation and optimized the HPLC analysis conditions. The optimum HPLC conditions included ultraviolet wavelength 360nm, column temperature 30℃, flow rate of mobile phase 1mL/min and proportion of mobile phase A. Learner relationship of this analysis method was very good (r=0.9990) within the range of 2μg－10μg and the precision was 1.7%.Using Trichosanthin as raw material, the optimum extraction conditions were obtained by investigating the dissolvent, time and temperature of extraction and so on. Deionized water as dissolvent, extraction processed once for 2hrs at room temperature with the proportion of liquid and solid 10:1.The thesis studied on the separation and purification of citrulline and the process and the result were listed as follow. First the extracting solution was preprocessed by ultrafiltration in order to get rid of macromolecular proteins and polysaccharide. Then adjusted to pH 2.0 with HCl, the solution passed through a column packed with 732 cationic-exchange resin(H+ form) at 1.2BV/h, room temperature. After eluting with 1.0% NH4OH, the eluant was decolored by polarized macroreticular resin NKA-II and evaporated to crude crystals of citrulline by dissolved in alcohol liquor. The elution rate was 98%, the purity of the product was 47.8% and the yield of citrulline in the whole process was 40%.The thesis also researched on the process for producing citrulline by fermentation. By culturing arginine-requiring Corynebacterium hydrocarboclastus ATCC 21242, the optimum culturing conditions were determined based on the optimized composition and proportion of medium. The optimum culturing conditions included inoculation 20%, medium volumes 30mL/250mL, 30℃, pH8.5, 180rpm, culturing time 120hrs. Under these conditions, the wet weight of the cell was 29g/L and the concentration of citrulline was 0.84g/L.