OLIGOMERIZATION TO INCREASE THERMAL STABILITY OF RECOMBINANT MANGANESE DISMUTASE SUPEROXIDE STAPHYLOCOCCUS EQUORUM
Superoxide dismutase (SOD) is a natural antioxidant that prevents the accumulation of free radicals. This enzyme is active in dimeric form and its stability increases when oligomerization. Recombinant MnSOD from Staphylococcus equorum (rMnSODSeq) is dimeric and its stability might be improved upo...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/66352 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Superoxide dismutase (SOD) is a natural antioxidant that prevents the
accumulation of free radicals. This enzyme is active in dimeric form and its stability
increases when oligomerization. Recombinant MnSOD from Staphylococcus
equorum (rMnSODSeq) is dimeric and its stability might be improved upon
oligomerisation. This study aims to increase thermal stability of rMnSODSeq
through introduction of disulfide or hydrogen bonds between dimers that may lead
the formation of its oligomer. The research is initiated by determining amino acid
candidate to substituted with the criteria of not part of the conserved region, away
from the active site, and does not in direct contact with the manganese. This initial
study was conducted in silico using COOT software. Based on the preliminary
studies, the recommended substitution was aspartic acid 47 to cysteine /serine
(D47C/D47S) or glutamic acid 115 to cysteine/serine (E115C/E115C). D47C-
E115C and D47S-E115S substitutions were performed with directed site
mutagenesis on DNA encoding rMnSODSeq. The confirmed pJExpress414_sod
mutants were transformed into Escherichia coli BL21(DE3) for production mutants
protein. Characterization of the mutants showed that 2 mutants formed oligomers:
they are D47C and D47C-E115C. The activity assay of oligomeric rMnSODSeq
mutants showed better thermal stability at 90 °C than dimeric form. The structural
stability test using Thermal Shift Assay (TSA) showed the melting point of
rMnSODSeq D47C was 70 °C and rMnSODSeq D47C-E115C was 73, higher than
natural rMnSODSeq. Thus, efforts to improve thermal stability by forming
rMnSODSeq oligomers were successful. Further research can focus onto calculate
the activity units of rMnSODSeq D47C and D47C-E115C and their complete
characterization.
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