#TITLE_ALTERNATIVE#

• Main Author: DZAKY RAZANI NIM : 10714020, MUTHIA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29416
• Institution: Institut Teknologi Bandung

Previous study on recombinant manganeese superoxide dismutase of Staphylococcus equorum <br /> <br /> (rMnSODSeq) showed that the enzyme has good stability in a wide range of pH and temperature, <br /> <br /> but this enzyme is unstable to long-term UVC exposure. This research aimed to increase <br /> <br /> rMnSODSeq stability towards UVC exposure by amino acid substitution at domain that allow dimer <br /> <br /> interface interaction. Interface interaction will increase dimer stability and UVC stability. This <br /> <br /> research started by determining site of substitution, in silico, using COOT program. Substitution <br /> <br /> sites from asparagin73 to phenylalanin (N73F) and serin126 to cystein (S126C) were obtained from <br /> <br /> screening process. Amino acid substitution was performed to rMnSODSeq gene in pJexpress414 <br /> <br /> (pJexpress414_sod) by site directed mutagenesis with Polymerase Chain Reaction with Two Single <br /> <br /> Reaction IN Parallel (SPRINP PCR) technique. Product of mutation was confirmed with sequece <br /> <br /> analysis, mutation of N73F and S126C were obtained using annealing temperature of 48°C and <br /> <br /> 50°C. Overproduction of wild-type and mutant rMnSODSeq was performed in Escherichia coli <br /> <br /> BL21(DE3) under condition of temperature 37°C, agitation 150 rpm, and isopropyl &#946;-D-1- <br /> <br /> thiogalactopyranoside (IPTG) induction at final concentration of 1 mM for 5 hours. Protein was <br /> <br /> purified with Ni2+ -NTA column and its activity determined qualitatively with zymography and <br /> <br /> quantitatively with colorimetry. Activity test on mutant protein showed that substitution <br /> <br /> asparagin73 to phenylalanin and serin126 to cystein are decrease protein activity. Pecent inhibition <br /> <br /> of 10 µg rMnSODSeq wild-type, rMnSODSeq_N73F, and rMnSODSeq_S126C are 76,1%, 62,8%, and <br /> <br /> 56,2%. Stability of rMnSODSeq_N73F and rMnSODSeq_S126C toward UVC exposure are increase. <br /> <br /> Residual activity of rMnSODSeq wild-type, rMnSODSeq_N73F, and rMnSODSeq_S126C after UVC <br /> <br /> exposure for 50 minutes are adalah 80,2%, 95,04%, 84,93%. <br />