Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 50, Issue 25, Pages 7264-7270Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jf0207070
Keywords
lemon; Citrus limon; SOD; expression; PCR; pET-20b(+)
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A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/ZnSOD) from lemon (Citrus limon) was cloned by PGR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21 (DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni2+-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30%. dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K-d value calculated for the dimer at 90 degreesC was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.
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