Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation
- 15 Downloads
Thermophilic or hyperthermophilic SODs (superoxide dismutase) usually offer substantial biotechnological advantages over mesophilic SODs. Previously a 244-amino acid N-terminal domain (NTD) from a heatresistant SOD of Geobacillus thermodenitrificans NG80-2 was discovered and demonstrated to be able to confer thermostability to homologous mesophilic SODs, which revealed a new type of heat resistance mechanism. To further improve the heat resistance and stress tolerance of thermophilic cambialistic superoxide dismutase (Fe/Mn- SOD Ap ) from Aeropyrum pernix K1 through metal incorporation and fusion with the newly found peptide NTD for broadening its industrial application, the wildtype SOD Ap and NTD-fused ntdSOD Ap were expressed in E. coli BL21 and incorporated with metal cofactors by two ways. Recombinant fusion SOD obtained by in vitro reconstitution (Mn-rec ntdSOD Ap ) exhibited improved optimum temperature at 70°C and dramatically enhanced thermostability especially at 110°C with enhanced pH stability from 4 to 10 and higher tolerance for denaturants and organic media than Mn-rec SOD Ap . To the best of our knowledge, Mn-rec ntdSOD Ap could be the most heat resistant SOD. In addition, metal incorporation of SOD Ap and ntdSOD Ap via in vivo modification have been developed and proved to be more practical for industrial use. These results indicate that fusion with NTD along with metal incorporation can generate superimposed effect and be applied to enhance the stability of cambialistic thermophilic SODs, thus providing a universal and convenient bioengineering method for generating extremely stable SODs.
KeywordsAeropyrum pernix K1 Geobacillus thermodenitrificans NG80-2 metal incorporation superoxide dismutase stress tolerance thermostability
Unable to display preview. Download preview PDF.
- 1.Fridovich, I. (1978) Superoxide dismutases: Defence against endogenous superoxide radical. Ciba Found. Symp. 77–93.Google Scholar
- 6.Pinto, V. H., D. Carvalhoda-Silva, J. L. Santos, T. Weitner, M. G. Fonseca, M. I. Yoshida, Y. M. Idemori, I. Batinic-Haberle, and J. S. Reboucas (2013) Thermal stability of the prototypical Mn porphyrin-based superoxide dismutase mimic and potent oxidative-stress redox modulator Mn(III) meso-tetrakis(Nethylpyridinium-2-yl)porphyrin chloride, MnTE-2-PyP(5+). J. Pharm. Biomed. Anal. 73: 29–34.CrossRefGoogle Scholar
- 7.Zhang, H. W., F. S. Wang, W. Shao, X. L. Zheng, J. Z. Qi, J. C. Cao, and T. M. Zhang (2006) Characterization and stability investigation of Cu,Zn-superoxide dismutase covalently modified by low molecular weight heparin. Biochem. 71: S96–100, 105.Google Scholar
- 15.Nam, J. S., J. H. Yoon, H. I. Lee, S. W. Kim, and Y. T. Ro (2011) Molecular cloning, purification, and characterization of a superoxide dismutase from a fast-growing Mycobacterium sp. Strain JC1 DSM 3803. J. Microbiol. 49: 399–406.Google Scholar
- 19.Whittaker, J. R. (1994) Principles of enzymology for the food sciences. 2nd ed. CRC Press.Google Scholar
- 25.Lim, J. H., K. Y. Hwang, J. Choi, D. Y. Lee, B. Y. Ahn, Y. Cho, K. S. Kim, and Y. S. Han (2001) Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability. Biochem. Biophys. Res. Commun. 288: 263–268.CrossRefGoogle Scholar
- 26.Wintjens, R., C. Noël, A. C. May, D. Gerbod, F. Dufernez, M. Capron, E. Viscogliosi, and M. Rooman (2004) Specificity and phenetic relationships of iron- and manganese-containing superoxide dismutases on the basis of structure and sequence comparisons. J. Biol. Chem. 279: 9248–9254.CrossRefGoogle Scholar