| Silica,as the main component of silicate,has been widely used in areas including infrastructure construction and real estate.With the high development of China’s modernization,the demand for silica is increasing.But serious environment pollution and shortage of silica has been occurred due to unreasonable excavation and utilization.An efficient and envirmentally friendly method is of vital importance for silica enrichment.Under this context,one kind of engineering yeast was constructed to enrich silica by surface displaying silica-binding proteins-Si-tag.Additionally,its biological function and microstructure of Si-tag displayed on the surface Yarrowia lipolytica were investigated.The main contents of this study were summarized as follows:(1)The biological function and most suitable conditions of Si-tag about absorbing silica was verified in this study.Si-tag fused with enhanced green fluorescent protein EGFP was expressed in Escherichia coli BL21(DE).After incubated with silica,The purified fusion protein was washed and then detected with green fluorescence around cell surface via fluorescence microscope which indicated the biological function of Si-tag.The optmal conditions of Si-tag were further explored.As the results showed the optimal time,temperature and pH were respectly 50 min,30℃ and 8.0.(2)The Si-tag fused with C-anchored protein Ylcwp3 was successfully displayed on Yarrowia lipolytica.It is an efficient strategy to enrich silica by yeast surface display.Firstly,the fusion sequence of Si-tag and Ylcwp3 was respectively cloned into pINA1296 and pINA1297,then recombinant plasmids were respectively transformed into Po1 g and Po1 h.Finally confocal microscope indicated that the green fluorescence around the cell wall was detected from reconmbinants Po1g/pINA1296 F #3 and Po1h/pINA1297F’ #8incubated with secondary antibody linked with FITC,and the latter was brighter.This demonstrated that Si-tag has been displayed on the cell wall and positively related to the protein’s expression.(3)The biological function and structure of the engineering Y.lipolytica were further investigated.The results of silica absorbed experiment showed that the amount of cells Po1h/pINA1297F’ #8 remained on the silica-glass piece was more than Po1g/pINA1296F#3,both of them remained more cells than control groups,which indicating that protein Si-tag displayed on the cell wall performed the right silica-absorbed founction and related to the amount of the displayed protein.More importantly,the scanning electron microscope(SEM)indicated that the mutual contact microstructure between Po1h/pINA1297F’ #8 and silica obviously had Mosaic and Capture two typcle forms,which indicated that Si-tag had changed the ficial structure about the cell wall.This study had proved the silica-absorbed function of Si-tag,which offered an environmentally friendly method to enrich silica efficiently. |
