Enhanced Biosorption Of Heavy Metal By Displaying Metallothionein On Cell Surface Of Saccharomyces Cerevisiae

In this study, two engineering strains, S. cerevisiae HACg, were achieved by vector which contained cell surface display cassette and cup1 was integrated into chromosome by HO locus. The origin strains were-S1. cerevisiae 4126. The tolerated and adsorption capacity of engineering strains were evaluated and the bioaroption of metal by S. cerevisiae HACg was evaluated.The metallothionein encoding DNA fragment (cup1) and pgk promoter gene PPGK1 were amplified from S. cerevisiae 288c by PCR amplification. The cupl fragment was inserted into the yeast episomal vector pYDl carrying the a-agglutinin receptor, which was named pYD1/cup1. The AGA2 -cup1 sequence with a-agglutinin encoding gene and Xpress epitope tag, was amplified from the plasmid pYD1/cup1 by PCR, the amplified product was ligated to the integration plasmid HO-2, generating the recombinant plasmid HAC. The PPGK1 from S. cerevisiae 288c was inserted into the HAC, which is called HACg. And the control plasmid without cup1 gene was named as HAg. The yeast strain S. cerevisiae 4126 was transformed with the HACg and HAg by electroporation, the verified transformants were designated as S. cerevisiae HACg, S. cerevisiae HAg.The metal tolerance and biosorption ability of S. cerevisiae HACg were evaluated. The results showed that the metal tolerance of S. cerevisiae HACg was obviously enhanced compared with control strains. When 50 mg/L Cd2+ was supplemented into the water, the Cd2+ removal rate of S. cerevisiae HACg enhanced 19.39% compared with control strains. S. cerevisiae HACg for Cu2+ biosorption ability has increased significantly compared with control strains, Cu2+(50 mg/L) removal rate of S. cerevisiae HACg increased by 13.35%. The reduction ability of S. cerevisiae HACg also increased, when initial Cr6+ concentration was 50 mg/L, the completed reduction time less 6 hours than control strains and save a third of the time, and total Cr6+ removal rate of S. cerevisiae HACg increased by 10%.The results of SEM (Scanning Electron Microscopy) dislayed that after metal biosorption, some changes occurred in the cell, such as disrupt of cell in some degree and the hollow of the surface, but the changes of cell surface are small in S. cerevisiae HACg after metal biosorption compared with control strains after metal biosorption, this suggested that the cell surface display of metallothionein was useful to decrease the hurt of metal to the cell. The Fourier Transform Infrared Spectroscopy (FTIR) showed that the main function groups of biosorption, which were hydroxyl, amino, phosphoric acid and amine groups, the main function groups of biosorption did not have impact after metallothionein display in cell surface. Voltammetric studies of yeast before and after biosorption metal showed that the functional groups of cell surface have changed. These results indicated that the display of metallothionein on the surface of the yeast plays an important role in enhancing the metal tolerance and biosorption ability of yeasts.The results of isothermal adsorption showed that the S. cerevisiae HACg adsorption Cd2+, Cu2+ and total Cr were consistent with the Langmuir, Freundlich and Freundlich adsorption isotherm model.