Primary Study Of Aerobic Ocean Bacteriaum YSC-1 And Purification Of Its Ferric Reductase

Our lab got an aerobic ocean bacterium YSC-1, donated by Pro.XIAO from Institute of Oceanology, Chinese Academy of Sciences. YSC-1 is able to synthesize electron dense particles in vivo.Collaborating with Pro.XIAO's lab, after a series of morphology and physiology researches, we identified YSC-1 a distinct phylogenetic group of α-Proteobacteria, closely related to Arctic seawater bacterium and Roseobacter sp. Analyzed with atomic absorption spectrometry, the content of iron in dried YSC-1 cell was measured to be 26848.5mg/kg, which is about ten times that of E.coli.Through single factor and orthogonal experiments, the culture conditions of YSC-1 were determined. The results indicates that the proper content of culture medium is yeast extract 1.0g/l, peptone 5.0g/l, and ferric quinate 10μM. And the proper culture condition is pH 7.5, incubation time 24℃, and rotation speed 200rpm. Under these optimal conditions, the strain density of YSC-1 in liquid medium reached 171% of the original..With ion-exchange chromatography of CM-Sephorase, DEAE-Sephorase and Q-Sephorase, a ferric reductase named Ysc was separated from YSC-1. Its molecular weight was identified to be 36,458Da based on SDS-PAGE. And its pI was calculated to be 6.3 by IEF. Enzyme response researches showed that its heat-resistant ability is poor, and the enzyme activity decreases sharply when the environment temperature is over 30*C. The enzyme activity is high in pH6.0 and stable in pH7.0. The influence of metal ion on the enzyme activity was studied. Mn~2+, Zn~2+, Mg~2+,K~+,Ca~2+, Na~+ each activates the enzyme activity of Ysc, but Ag~+, Cu~2+, Co~2+ and EDTA suppress it.We explored fermentation condition of both YSC-1 and the production of its in vivo electron dense particles. Finally we found an approving technique forfermentation, which is the air flow about 4L/min, the agitating rate 250rpm, and the fermentation time 60h. Thus, we established the foundation for late studies of the mathematical models on fermentation and the possible large scale production of electronic dense particles.