OLIGOMERIZATION TO INCREASE THERMAL STABILITY OF RECOMBINANT MANGANESE DISMUTASE SUPEROXIDE STAPHYLOCOCCUS EQUORUM

• Main Author: Dika Fadilah, Muhammad
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66352
• Institution: Institut Teknologi Bandung

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.