Cloning, Expression And Characterization Of A Manganese-containing Superoxide Dismutase From Bacillus Subtilis ATCC 9372

In order to achieve the soluble expression of SOD gene in E. coli, the SOD gene was amplified from the genomic DNA of Bacillus subtilis ATCC 9372 according to the sodA gene sequence of Bacillus subtilis 168. Amino acids' sequences deducing by this gene had the highest similarity with Bacillus subtilis natto BEST 195 and Bacillus subtilis 168. The cloned SOD gene was inserted into the expression vector pET28a to construct recombinant plasmid pET28a-SOD and was then transformed for expression in E. coli BL21(DE3). The SOD protein with molecular weight of about 26 kD was expressed in E. coli after induction with IPTG, which accounted for approximately 45.6% of total bacterial protein. After purification, the protein was determined as rMn-SOD by riboflavin-nitroblue tetrazolium method. The enzyme activity of rMn-SOD was assayed with improved pyrogallic acid autoxidation method. The results showed that the specific activity of the rSOD was 2553.211 U/mg, the activity of this rSOD was relatively stable from pH 6.0 to 11.0, and the enzyme showed maximum activity at about pH 8.0. It still had 75% activities at 55℃after 90 min. The conformational changes of rMn-SOD in the presence of different pH buffer and different concentrations of GuHCl or Urea were investigated by using fluorescence spectra (FR), and compared with the activity recovery of denatured rMn-SOD. The results showed that the changes between enzyme activity and protein conformation which was shown by fluorescence spectroscopy was almost identical. The mutant rSOD[E167A] was made for clarification of the physical role of Glu167 in rSOD, using site-directed mutagenesis technology to change the codon of Glu167. The maximum fluorescent emission peak of the mutant showed a red shift and the fluorescence intensity at about 335 nm increased compared with the wild-type SOD, which is possible to explain that the space structure of rSOD [E167A] has changed. The enzymatic activity of rSOD[E167A] was only 74.989 U/mg,2.94% compared to that of the wild-type SOD, the protein thermal stability and the resistance ability to guanidine hydrochloride became poor. It was concluded that Glu167 has important contribution on the activity and maintaining the stability of protein structure. These results will provide theoretical and experimental foundation for further studies on the structure and function of the Mn-SOD and on establishing a convenient and efficient way to produce Mn-SOD. These will be helpful for the development and application of SOD.