THE ROLE OF INTRAMOLECULAR DISULPHIDE BOND ADDITION ON MONOMERIC THERMAL STABILITY OF RECOMBINANT MANGANESE SUPEROXIDE DISMUTASE STAPHYLOCOCCUS EQUORUM

• Main Author: Aryani Putri, Nanik
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/56614
• Institution: Institut Teknologi Bandung

Recombinant manganese superoxide dismutase from Staphylococcus equorum (rMnSODSeq) is an enzyme that functions to protect against cell damage as effects of reactive oxygen species. rMnSODSeq has good stability over temperature and pH but various modifications are still performed to increase its stability. This study aims to improve thermal stability of the monomer by introducing intramolecular disulphide bond without affecting the protein activity. In this study, rMnSODSeq was introduced with an intra-molecular disulfide bond. The Threonine-89, asparagine-187, valine-132, and lysine-154 were substituted to cysteine independently. Substitution was performed by site-directed mutagenesis with each of mutagenic primer annealing temperature on 55 and 58?. The confirmed mutation products were then transformed into Escherichia coli BL21 (DE3) for overproduction followed by purification, characterization, and crystallization. The zimography test showed a decrease in the activity of rMnSODSeq T89C- N187C. The colorimetric test showed that rMnSODSeq T89C-N187C activity was 41.8% lower than rMnSODSeq, with 78.5% residual activity at 75?. The structural stability test of rMnSODSEq T89C-N187C using thermal shift assay showed an increase in the monomer melting temperature from 62 to 66 ? in the absense of additives, and from 61 to 63 ? in the presence of ?-mercaptoethanol. The T89C-N187C substitution affects the crystallization conditions that were different from wild type rMnSODSeq crystalization condition. On the other hands, the crude V132C-K154C mutant displayed very low activity, difficult to express in E. coli BL21 (DE3) and difficult to be purified from its contaminant.