Optimization And Identification Of Violacein By Janthinobacterium Lividum And Its Effect On The Colon Cancer Cell HT-29

Violacein is a kind of secondary metabolites produced by microorganism, which belonged to the indole derivatives. It was composed of two tryptophan molecules after oxidative condensation with various biological activities like antiprotozoon, antiviral,antibacterial and antitumor. In this experiment, as the original strain,Janthinobacterium lividum ZSJ was cultivated in fermenter to extract violacein so as to study its effect on the growth and proliferation of colon cancer cell HT-29. The main results of this paper are as follows.1. Fermentation media and culture conditions for J. Lividum B01 were optimized. The optimized medium ingredient is: maltose 1.25g/L, peptone 9.5g/L,beef extract 2.5g/L, and tryptophan 1.25g/L. Violacein yield reach 2.87g/L when the strain was cultured with a initial pH value 6, a 7% of inoculation quantity, and a 50 m L of culture volume in 250 m L conical flask, which increased by 34.7% compared with the original yield 2.13g/L.2. By identifying the strain through 16 S rDNA analysis, the homology of the strain was 99% with that of a classic J. Lividum BR01 strain. By undertaking HPLC,ultraviolet visible spectrum and nuclear magnetic resonance, the extracted blue-purple pigment was verified to be composed of deoxidation violacein and violacein with the ratio of about 1:9, and with a maximum absorption wavelength of visible light 565 nm and 584 nm respectively.3. The pigment's stability was influenced by light, heat, acid and alkali, solubility,color and metal ions. The results showed that the pigment had good thermal stability.It also had a good pH stability, hence, the obvious color and light absorption value changes were observed only when pH value exceeds 12. But it was very sensitive to light, and the color was easy to fade with direct light.4. The effects of violacein on colon cancer cell HT-29 were studied. The results showed that the inhibitory effect of the violacein on cell was more obvious than that of 5-Fluorouracil as the dose and time increasing. Its maximum inhibition rate exceeded 90%. The vacuolar structure in cells was observed after the treatment with violacein, and the structure increased with dose-effect relationship. The apoptosis occured in late period, and was positively correlated with the concentration and time.5. The differential protein expression in HT-29 cells treated with violacein was conducted using proteomics technology. 492 differential proteins were identified incells treated with high dose of violacein, 112 and 336 in cells treated with low dose of violacein and 5-Fu, respectively. Differential proteins were mainly enriched in the process of signal transduction and translation, enriched in the location of cytoplasm,plasma membrane, nucleic acid, and enriched in the function of protein binding.Differential proteins were involved in 50 pathways, the main pathway were the transcription, translation and central metabolism.This experiment established a violacein seperation techniques from J. Lividum,and certified its obvious inhibitory effect on colon cancer cell lines. Its inhibitory mechanism was studied by proteomics technology. The study provides a basis for the further study of the cancer treatment using violacein.