Continuous Production Of L-Citrulline By Immobilized Streptococcus Faecalis Cells

L-citrulline is a non-protein amino acids, playing an important role in urea cycle. In recent years, much foreign research shows that L-citrulline has many important physiological functions, such as scavenging free radicals, serving as an allograft rejection indicator, helping in vasorelaxation, stabilizing blood pressure, diagnosing of rheumatoid arthritis and functioning in anti-oxidation. Thus it has drawn much attention from nutritional and medical industries. In this paper, the continuous process of transforming L-citrulline through immobilized microbial cells in packed bed reactor was studied, aiming at establishing a set of low-cost, easy-operation, high-quality L-citrulline production systems in laboratory and laying the foundation for industrial production of L-citrulline in the future.Firstly, the use of sodium alginate as the main carrier for immobilization was identified in this paper. Then 5% diatomite and 3% silica polymer were added into the mixture to form immobilized beads, which contain biomass 10%, in the immobilizing process. The operation of immobilization was of high stability, which could be repeatedly conducted eight times with the activity of enzyme remaining unchanged.In addition, the optimal conditions for preparation of converting L-citrulline through immobilized cells in the shaker were identified: pH6.5, temperature 35℃, acetate buffer 0.2mol/L, shaker speed 100r/min, reaction time 20h, L-Arginine concentration 100g/L, then the highest output of L-Citrulline was received--85g/L. After that, continuous production of L-citrulline in the packed bed reactor was further studied. The experimental results show that when the effective volume of the packed bed was 589mL, filled with immobilized particles 623g containing 10% of the bacterial cells, with the reaction temperature of 35℃, reaction liquid containing 100g/L L-Arginine, with the pH value of 6.5 fed by peristaltic pump in a flow rate of 30mL/h, the converting reaction could continue 73d. After the long-time reaction, the immobilized cell particles turned to be soft. In this process, the average daily output of L-citrulline was 95.6g/L, with a molar yield of 95.1%.Separation and purification of L-citrullinen were also studied to determine the optimum conditions:The extract was adjusted to a pH value of about 10, at room temperature, with 1.2BV/h flow rate and the cation exchange resin D201 was used in the adsorption process. Then the resin was washed with deionized water and 0.5mol/L of ammonia elution and the eluent was treated by vacuum distillation and washed by ethanol to form crystalline--the product of L-citrulline. After the whole process, we could get a product purity of 98.4% and the overall yield products of 86.4%.The production cost of both in shaking batch and packed bed reactor were separately estimated in this paper. The conclusion is that the former is, the latter is, so it was highly recommended to use the packed bed reactor to operate continuous production of L-citrulline.