Construction, Expression Of Metallothionein Mutations And Heavy-metal Adsorption Capacity Of Engineered Bacteria

Based on PCR and isocaudamer techniques, a series of monkey metallothionein (mMT) mutations were constructed including MTα(αdomain of MT), MTα₂, MTα₃, MTα₅ (a cluster of 2, 3, 5 direct repeats of theαdomain). MTαwas expressed and purified and the characteristics of the protein were investigated. The resistance to Cd²⁺ and adsorption capacity for Cd²⁺ and Zn²⁺ by the engineered bacteria were examined. The main results are outlined as follows:Expression vector haboring MTαwas constructed. After expression and purification, highly purified MTαwas obtained. Its metal-adsorption ability is consistant with the theoretical result. According to secondary structure prediction, increased hydrophobic interaction was observed for MTα, MTα₂, MTα₃. Howeverα-helix was decreased in MTα₄,MTα₅, especially in MTα₅.Engineered bacteria expressing the cluster of MTαwere constructed as MTα/BL21,MTα₂/BL21, MTα₃/BL21 and MTα₅/BL21.Toxicity to control was found and no toxicity of Cd²⁺ to engineered bacteria was observed for MTα₂/BL21, MTα₃/BL21 and MTα₅/BL21 in the presence of 0.5 mmol/L Cd²⁺. As Cd²⁺ concentration increased to 1.0 mmol/L, MTα₅/BL21 showed higher resistance.Under low concentrations of Cd²⁺ (0.067 mmol/L), the engineered bacteria expressing mMT (MT/BL21) exhibited stronger adsorption capacity (0.0615 mmol/mg dry weight of cells) and the tendency became more obvious as Cd²⁺ concentration increased to 0.268 mmol/L. MT/BL21 adsorbed double amounts of Cd²⁺ (0.093 mmol/mg) as compared to the control when the concentration was 0.268 mmol/L. Among the series of engineered bacteria, MTα₃/BL21 removed the greastest percentage Cd²⁺ (76%), followed by MTα₂/BL21 (72%).The adsorption capacity for Zn²⁺ by MT/BL21 (0.118 mmol/mg dry weight of cells) was increased dramatically while no significant improvement was observed for the other engineered bacteria.