Study On Selection Of Alfalfa Protein Suspension Cell With High Yield And Its Kinetics Of Metabolism

Induction Alfalfa suspension cell cultures in the kinetics of protein metabolism suspension cell line were studied. That are optimized from Alfalfa varieties, explants, culture conditions, callus and suspension cell lines, etc. The process of cell growth on the nutrition of Alfalfa material consumption and growth kinetics were discussed structural dynamics model for large-scale production of alfalfa protein in cell culture provide a basis for fundamental research.According to the paternity far five kinds of Alfalfa are determinate the protein protease alive and protein content that choice the Alfalfa (WL215). Callus of the Alfalfa induction and high production protein of Alfalfa lines suspended selection. Selected the optimized of suspension cell culture the explants size with epidemus was 0.3cm X 0.3cm, more loose callus could be induced from the WL215 in the medium of MB+ 3.0 mg/L 2,4-D+0.5mg/L 6-BA under condition of 600lux 12hours circular illumination. The maximum growth indices was 6.49 while the maximum total Protein activity was 10.14x 104U/L selected Alfalfa cell line suspension after 12 days' culture.The effects of major nutrients on Alfalfa cell growth and protein production were discussed. Sucrose was better for cell growth and protein production among different carbon sources. In the range of 1%⁶% sucrose concentration,highest biomass and protein production were achieved on 3%. Standard nitrogen concentration in MB medium was enough for Alfalfa cell growth and protein production. Mineral nitrate source and urea were suitable for cell growth. Phosphate was necessary for cell growth and protein production. Different initial phosphate concentration had remarkable effect on protein production. The standard phosphate concentration appeared in 1.25mM more suitable for cell growth and protein production.The suspension culture process of Alfalfa cell could be appeared"S"grow curve. Sucrose was growth limiting nutrient on low concentration condition. Increase of sucrose concentration stimulated cell growth, extended rapid growth phase and increased the final cell harvest. Substrate inhibition was observed when the concentration was above 3%. The influence of different NH₄⁺ / N03- ratio on cell growth was mainly reflected by the inhibition of high ammonium. Phosphate was also the growth limiting nutrient on low concentration condition. Increase of phosphate concentration increased cell growth rate and shortened the culture period. Under the standard culture condition, the specific cell growth rate was 0.31⁰.34 day-1, and the doubling time was 3.4³.8 days.The trends of protein production variation under different initial sucrose concentration were the same. Increase of sucrose concentration could also improve protein production to some extent, while higher sucrose concentration inhibited protein production. Protein production was the lowest when used ammonium as only nitrate source. The maximum total protein activity could be achieved while the ratio of NH₄⁺ / N03- was 20/40. Initial phosphate concentration was most effective on protein accumulation; the maximum protein yield could be obtained under the standard phosphate concentration in 1.25mM.Protein accumulation was partially-growth-associated, protein production by Alfalfa cell suspension was regard as synchronism with cell growth. Alfalfa cell rapidly hydrolyzed extra cellular sucrose before adsorb it. The hydrolyzing process could be described using substrate saturation kinetic equation. Glucose was absorbed more preferentially than fructose. The adsorbing rate, utilizing efficiency and intracellular accumulation of sugar were closely related to medium composition.Ammonium was rapidly adsorbed at the early stage. The adsorption of nitrate was much more slowly. The intracellular accumulation of ammonium and nitrate was not obvious.Phosphate was also rapidly adsorbed at the early stage. The higher the initial phosphate concentration was, the more rapid the utilization rate was. The intracellular phosphate accumulation level related to the initial phosphate concentration in the medium. Protein content in Alfalfa cell was decreased with the increasing of intracellular phosphate accumulation. The accumulation level showed strong influence on Alfalfa cell growth and protein production.A structural kinetic model was constructed in this thesis. The whole culture system was separated into biotic phase and abiotic phase. The former was the inside of Alfalfa cell, which was divided into 4 compartments: soluble sugar G, free phosphate P, Middle metabolites M and products S. The abiotic phase was extra cellular environment, which included sucrose Se, reductive sugar Ge, and phosphate Pe. Intracellular phosphate acted as a regulator on the cell growth and protein accumulation. Calculated result coincided well with experimental data. The model could be used for good description of culture process.