| L-Citrulline has many important physiological functions, such as improving immune system, helping absorb the harmful free radicals to improve antioxidant capacity a nd assisting in the treatment of arthritis deformans. Therefore, citrulline is widely used in cosmetics, health food, food additives and nutritional supplements. Biosynthsis of citrulline refers to the L-arginine transforming into L-citrulline in the act of Arginine Deiminase(ADI). The advantage of the enzymatic synthesis includes the milder production conditions, the higher concentration of the product and the less purification steps. However, the present studies on the biosynthsis of L-citrulline are still at the primary stage. This study focused on the optimal medium and fermentation conditions of Enterococcus faecalis SK32.001, the purification and characterization of ADI from Enterococcus faecalis SK32.001 and the optimal conditions for purifying L-citrulline by solventing-out crystal ization.Firstly, based on the optimum flask culture conditions, fermentation process of ADI was carried out in 3 L fermentor. The influence of DO, p H, glucose feeding time, the fed strategy of glucose, the induction time of arginine and the arginine concentration was investigated in order to obtain higher enzyme activity. The optimal culture conditions were determined as follows: DO 20%, p H 5.8(1~10 h),p H 6.3(10~18 h), glucose feeding time was 12 h after inoculation when the glucose concentration was 4 g/L, the fed strategy for glucose was adding 100 g/L glucose in 4 h with the constant flow rate at 0.42 m L/min, the induction time of arginine was 8 hours after fermention and the arginine concentration was 12 g/L, the fermentation time was 18 h. Under the optimized conditions, OD600 of thalli increased from 2.30 to 6.99, and the arginine deiminase activity increased from 1.43 U/m L to 3.76 U/m L.Secondly, the crude enzyme of the arginine deiminase was purified and the enzymatic characterization was studied. The result indicated that the arginine deiminase was successfully purified to homogeneity level after ammonium sulfate precipitation, Hi Prep Q FF 16/10 following with Sephadex G-75 filtration chromatography. The molecular weight of the purified arginine deiminase was about 42 k Da. The optimum condition for temperature and p H of the purified arginine deiminase were 50 oC and 6.5, respectively. The temperature had a significant effect on the arginine deiminase activity, the enzyme activity showed good stability in the temperature range of 30~40 oC. And p H had modest influence on the arginine deiminase activity, the enzyme activity kept stability at p H 5.5~7.5. 1.0 mmo I/L Zn2+ was found to be the most valid promoter while 100 mmo I/L Cu2+ showed the most obvious inhibitory effect on arginine deiminase activity. The Michaelis constant Km was 1.33 mmol/L and Vmax was 2.41 μmol/min.Thirdly, the separation and crystallization of L-citrulline and superficial characteristics analysis of crystal were investigated. The method used to purify L-citrulline was solventing-out crystallization. The results manifested that the optimal solvent/anti-solvent was water/ethanol, the crystallization temperature was 40 oC, the amount of crystal seed addition was 3%, the stir speed was 50 r/min, the addition rate of anti-solvent was 0.33 m L/min, the ratio of water and ethanol was 1:4.Finally, the crystal of L-citrulline was analyzed by high performance liquid chromatography(HPLC), infrared spectroscopy(IR), magnetic resonance imaging(MRI) and scanning electron microscope(SEM). The results domenstrated that the product of solventing-out crystallization was L-citrulline and the purity was over 99%. The picture of crystal showed that the crystal of citrulline appeared to be rectangular in shape with smooth surface and homogeneous morphology. |
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