Preparation And Characterzation Of Immobilized The Non-specific Serratia Nuclease

It is well known that the non-specific serratia nuclease is able to degrade all forms ofnucleic acids. It owns a highly efficient with a very small amount to hydrolyze nucleic acidand purify proteins. Previously, our laboratory has immobilized it; however, it turned out to beleaking. Therefore, my aim is to solve this problem. Firstly, site-directed mutagenesis wasconducted to change the184thHis into Cys in non-specific serratia nuclease gene, usingforegone recombinant expression vector pMAL-c4X-NU. Furthermore, we immobilized anddetected H184CNU and then optimized the condition. In addition, we further studiedcharacteristics of the immobilized enzyme. Contents would be presented precisely asfollowing.Based on constructed nuclease prokaryotic expression vector pMAL-C4X-NU designedprimer according to the mutation point, and contained mutant point by PCR ampliation. wehave obtained non-specific nuclease prokaryotic expression vector pMAL-C4X-NUH184C,Transferring it into E.coli BL21,78kD H184CNU expression did not decrease after mutationdetected by SDS-PAGE electrophoresis. We applied Amylose resin chromatography to get thetarget protein, and found no drop in its activity after mutation. Swiss-Pdbviewer3.7wasperformed to predict its structure,and it was founded that the distance between two subunits of184amino acids reduced from6.34nm to6.26nm and an increase in the charge and anenhancement in ionic bond energy.During the immobilization and optimization of H184CNU, covalent binding wasimplemented with a carrier of Sepharose CL6B. The optimal immobilized condition withhigh recovery is that the temperature is20℃, the enzyme amount0.15mg/mL,the length oftime is6h, pH8.0. When detecting the immobilized H184CNU, leakage did not appeared.With the comparison of characteristics of the immobilized H184CNU and thenon-immobilized enzyme, the optimal temperature for each of them is43℃and38℃,respectively. And the optimal pH of both enzymes is8.0. Compared with thenon-immobilized H184CNU, the immobilized one exhibited a higher stability for thermal, pH,storage and repeatability.In conclusion, we successfully site-directed mutated the non-specific serratia nuclease,immobilized and optimized H184CNU. It laid the foundation for the protein purification withenzyme at a low cost. Also, the study improved the utilization efficiency of non-specific nucleases.