Structural Analysis And Site-directed Mutagenesis Of A Non-specific Nuclease From Yersinia Enterocolitica Subsp. Palearctica

Nuclease from Y. enterocolitica subsp. Palearctica is a non-specific nuclease which can degrade various DNA and RNA, whether it is single-stranded, doublestranded, linear, cyclic, or supercoiled form. Previously, gene of Y.e.p non-specific nuclease(nuc) was obtained by PCR in our group. Expression vector pET24a-nuc was constructed by the ligation of plasmid pET24 a with the gene of nuc. Recombinant nuclease was induced by IPTG. Characteristics analysis of recombinant nuclease showed that Y.e.p non-specific nuclease not only has high nuclease activity, but also has super heat resistance, acid and alkali resistance, and chemicals resistance.Therefore, in order to provide the basis for the function evolutionary study of Y.e.p non-specific nuclease, effect of the factors on enzyme activity and thermal stability of Y.e.p non-specific nuclease by bioinformatics methods and site-directed mutagenesis was studied in this work. The main studies and results were as follows:(1) Using bioinformatics software, we analyzed primary structure, secondary structure, the domain structure and the tertiary structure of Y.e.p non-specific nuclease.This work provided the theoretical basis for site-directed mutagennesis study of Y.e.p non-specific nuclease.(2) The results of active site, salt bridge and disulfide bond of Y.e.p non-specific nuclease by site-directed mutagenesis method, showed that when changing amino acids of the active site, or breaking salt bridge and disulfide bond, enzyme activity and thermal stability of Y.e.p non-specific nuclease decreased significantly, indicating active site played an important role in enzyme activity of Y.e.p non-specific nuclease and salt bridge and disulfide bond played an important role in thermal stability of Y.e.p non-specific nuclease.(3) The amino acid sequences of Y.e.e and Y.e.p non-specific nuclease were analyzed, respectively. 15 amino acids were designed for site-directed mutagenesis.The results showed the mutations of E202 A, I203 F and D264 E significantly affected the enzyme activity and thermal stability of Y.e.p non-specific nuclease, indicating Glu202, Ile203 and Asp264 might be key residues of Y.e.p non-specific nuclease.