Isolation Of Serratia Marcescens And The Preliminary Study On Mechanism Of Prodigiosin Production

Prodigiosin (PG) is one of a red-pigment own prodigisin, it is an important natrual red-pigment which can be used in industries like medicine, chemistry, dye, etc.. In this study, a serratia marcecens strain producing red-pigment at 30℃was isolated from soil samples, and the red-pigment was identified as prodigiosin after purifying. The yield was enhanced via fermentation medium optimization and fermentation condition optimization. Due to its sensitive characteristic towards temperature, we get tentative exploration of its mechanism---the differential expression intracellular proteins under different temperatures(37℃&28℃) were investigated via two-dimensional gel electrophoresis. The main points and results are as follows:(1) Four strains can producing red-pigment at 30℃were isolated from nature samples. B5-1, which could produce higher levels of strains, was selected for research after initial fermentation test. It was identified by general morphological and biochemical characteristics, then identified by 16S rDNA sequence and systematic analysis. The results showed B5-1 was a serratia marcecens, and was named as Serratia marcecens JNB5-1.(2) The red-pigment was dissolved and extracted by ethanol and chloroform. Through the full wavelength scanning it acquired the absorption peak at 535nm under acid conditions and the molecular weight(Mr)was 323.2 by the preliminary identification of LC-MS. After repeated isolation and purification of red-pigment, I finally obtained the prodigiosin sample with the purity of 98% by TLC.(3) To determine fermentation medium, first of all we get research about the influences different carbon and nitrogen sources, inorganic salts and amino acids on bleeding red pigment yield (refer to the metal ion initial medium), single-factor experiment results show that sucrose, beef extract, calcium chloride and proline can effectively improve bleeding red pigment production. By four factors and three-level orthogonal optimization experiments to determine the best formulation for the fermentation medium: 2% sucrose, 1.5% beef extract, 1% CaCl2, proline0.75%, MgSO4·7H2O 0.02%, FeSO4·7H2O 0.006%.(4) Mainly on the fermentation conditions, inoculum size, temperature, pH, shaking speed and liquid volume yield of prodigiosin factors influence the optimum fermentation temperature is 28℃, by three factors (inoculum size, pH, liquid volume) three-level orthogonal experiments, and ultimately determine the optimum fermentation conditions were: 6% inoculum, liquid volume 30ml, pH 7.0, in this condition prodigiosin prime fermentation production reached 6.9g / L, which has seen a substantial increase compared with the initial production.(5) Through the experiment of 2-DE, we made research on situations of JNB5-1 did produce prodigiosin at 28℃instead of at 37℃. Successfully identified by TOF-MS, including temperature regulation by heat shock protein GroEL (37℃up-regulate), and prodigiosin synthesis pathway methoxy-transferase (37℃down-regulate), etc., these 13different proteins may be one of the main factors to determine JNB5-1`s prodigiosin synthesis when it comes to different temperatures.