Selection Of NI²⁺ Binding Heavy Metal Peptides And The Microbial Functions Based On Zinc Finger Library

With the rapid development of social economy, on the one hand, it has greatly improved the people’s living standards, on the other hand, it has also brought great pressure and challenges to our living environment. The pollution accidents and hazards to the environment which is caused by heavy metal has given rise to high attention. As an important technology of environment heavy metals management, microbial remediation has been widely studied and developed in recent years and the genetically engineered microorganism (GMO) is the main direction of microbial remediation technology.The difficult point and the key points of it is to get high affinity heavy metal-binding peptide(molecules) and screen and obtain high enrichment and tolerance of environmental microorganisms. Zinc finger protein as an important transcription factor in vivo is involved in the intracellular DNA transcription of a variety of physiological and biochemical process, meanwhile,it provides important biological sources for obtaining metal-binding peptide of Zn2+、Ni2+. Meanwhile, heavy metal mining area provides a natural reservoir of heavy metal resistance and enrichment of microbial strains.This study was based on laboratory preliminary work-the constrained bacterial random peptide library. C2H2zinc finger protein was the scaffolds which had been used in the construction of the bacterial peptide library. In order to get the sequence of heavy metal-binding peptides, we used the percoll density gradient centrifugation to screen and isolate the heavy metal-binding peptides of Zn2+and Ni2+. Then, we analysed the heavy metal-binding peptides by multiple sequence alignment. The next step was to evaluate the characteristics of efficient or specific binding of these heavy metal-binding peptides to heavy metal ions by affinity reverse selection and determine the heavy metal adsorption by these pepdides displayed bacteria. Based on the conclusion above, we analysized the amino acid composition of the heavy metal-binding peptides in order to get the total nature and laws of these peptides. Also, we screened bacteria with high tolerance to nickel from soil in mine tailings and we identified those bacteria by molecular biology of16SrDNA and the physiological and biochemical characteristics.The purpose of our work was to obtain heavy metal-binding peptides and microorganisms with high tolerance to nickel heavy metals in order to provide the important basis for the microbial remediation of heavy metals pollution with further engineering bacteria.Main results: 1. We obtained19metal-binding peptides of Zn2+and Ni2+from bacterial random peptide library with the scaffold of C2H2zinc finger protein.2. The metal-binding peptide of Zn2+and Ni2+were enriched with the increased rounds of selection from bacterial random peptide library.The value of P/N can reach94.6and141.0.By SDS-PAGE, it can be determined that the molecular weight of the fusion protein is75KD. We detected the heavy metal adsorption by AAS of the display bacteria whose value of P/N were higher (P/N>10), it’s discovered that the adsorption capacityof display bacteria of6#and16#is preferably and the adsorption rate for heavy metals reached to about50%, however the adsorption capacity of display bacteria of5#and17#could not achieve the ideal effect.3. By bioinformatics analysis, we found that it is likely that the sequence of-H-X4-H-and-F-X-F-may be the motif sequence of metal-binding peptide of Zn2+and Ni2+. The content of some amino acids in the peptides such as:Ala、Thr、Trp、Val、Ser、Ile is high. It is probably that the two kinds of amino acids have some relationship with the function of metal-binding peptides.4. We obtained four strains of Ni2+resistant bacteria from nickel ore tailings,the resistant concentration of Ni2+resistant bacteria were over550mg/L and300mg/L in solid and liquid medium respectively.It was identified that the species of these bacteria were Bacillus spp., Sphingobacterium spp., Pseudomonas mendocina, Pseudomonas stutzeri.