SUBSTITUTION, OVERPRODUCTION, CHARACTERIZATION AND CRYSTALIZATION OF RECOMBINANT MANGANESE SUPEROXIDE DISMUTASE K38R-A121E AND K38R-A121Y FROM STAPHYLOCOCCUS EQUORUM
Manganese superoxide dismutase from Staphylococcus equorum recombinant (rMnSODSeq) is an enzyme that catalyzes the dismutation reaction of superoxide radical into a more stable oxygen species. rMnSODSeq is stable over a wide range of temperature and pH but its activity decreases drastically af...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/56671 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Manganese superoxide dismutase from Staphylococcus equorum recombinant
(rMnSODSeq) is an enzyme that catalyzes the dismutation reaction of superoxide radical
into a more stable oxygen species. rMnSODSeq is stable over a wide range of temperature
and pH but its activity decreases drastically after 45 minutes of exposure to the UVC.
Various modifications to increase protein stability have been succesfully performed but the
proteins have lower activity than Native rMnSODSeq. This study aims to improve thermal
stability of rMnSODSeq while maintaining its catalytic activity. Stability enhancement was
designed through additional interaction at the dimer interface of the protein which is not part
of the catalytic and conserved region. This study was initiated with a in silico design of
protein to promote new interaction at the protein dimer interface that do not interfere its
conserve region. Protein modifications through site directed mutagenesis have been
successfully done. The pJExpress414_sod mutant plasmids were transformed into
Escherichia coli TOP10 and BL21 (DE3) as host celland overproduction, characterization,
and crystallization of the rMnSODSeq K38R-A121E and K38R-A121Y was carried out.
Based on stability testing using Thermal Shift Assay (TSA), rMnSODSeq K38R-A121E
showed no dimer melting point that was hypothesized as better structural stability, while
rMnSODSeq K38R-A121Y showed lower monomer stability. Activity assessment using
zymography and colorimetric methods showed a similarity between wildtype and mutant of
rMnSODSeq. Further works will focus on elucidation of the three-dimensional (3D)
structure of the rMnSODSeq K38R-A121E and K38R-A121Y and investigation of the
structure and stability correlation.
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