Journal
PROCESS BIOCHEMISTRY
Volume 51, Issue 1, Pages 39-47Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2015.11.001
Keywords
Manganese superoxide dismutase; Site-directed mutagenesis; Multiple sequence alignment; Megaprimer-PCR; Metal-binding specificity; Thermostable Fe-SOD
Categories
Funding
- National High Technology Research and Development Program (863 Program) of China [2015AA020925]
- Chinese Postdoc Fund [2014M551981]
- Natural Science Foundation of China [41276135, 31172010]
- Program for New Century Excellent Talents in University [NCET-13-1031]
- Shandong Provincial Natural Science Foundation [ZR2015CL003]
- Doctoral Foundation of Shandong Province [2008BS02018]
- Fundamental Research Funds for the Central Universities [14CX02124A, 15CX05015A]
- UK Engineering and Physical Sciences Research Council [EPSRC EP/F062966/1]
- EPSRC [EP/N007174/1, EP/N023579/1] Funding Source: UKRI
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Three residues His29, His84 and His171 were found to be the ligands of metal ions of one hyperthermostable Mn-SOD produced by Thermus thermophilus HB27 after multiple sequence alignment. Conversion of these residues might cause a change of metal-binding specificity. Thus, three mutants His29Ala, His84Ala, and His171Ala were prepared. The mutant types changed, and the metal-binding specificities of His29Ala and His171Ala altered from Mn to Fe. The alpha-helix contents of His29Ala and His171Ala were 66% and 59% respectively, suggesting that the mutants folded with a reasonable secondary structure. His29Ala showed an activity comparable to that of the wild type, and His171Ala exhibited a 39.6% higher activity than the wild type. These two mutants were thermostable from 30 to 50 degrees C after incubation for 30 min. The activity of His171Ala retained 51% at 70 degrees C compared with 36.7% for His29Ala. Both mutants retained >80% residual activity in the pH range 5.0-10.0 (except their pI of 8.0) after incubation at 4 degrees C for 30min. This study provides a good foundation for accelerating the development of thermostable Fe-SOD for application in the fields of medicine and cosmetics. (C) 2015 Elsevier Ltd. All rights reserved.
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