In order to improve manganese-SOD stability, three mutations were constructed via site-directed mutagenesis, and the root mean square fluctuation (RMSF) and root mean square deviation (RMSD) were used as stability assessment indexes. The amino acids of V140, E155 and E215 from wild-type mouse Mn-SOD was replaced to L140, W155 and W215, and a recombinant plasmid containing DNA segment coding wild-type and mutant Mn-SOD protein was transformed into Escherichia coli BL21 for expression. The highest enzyme activity of the mutations-MnSOD was 2050 U/mg. In addition, the recombinant protein, TM-MnSODV140L, E155W, E215W exhibited higher working temperature and improved stability compared with the wild-type Mn-SOD. Furthermore, CD spectrum analysis of the improved mutants and wild-type enzyme showed that there was no significant change in their secondary structures. This study not only expands the scope of the application of enzymes, but also helps us understand the relationship between protein structure and function.
Keywords: Manganese superoxide dismutase; Molecular dynamics simulation; Site-directed mutagenesis; Stability; ΔΔG(mut).
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