SUBSTITUSI K38R-A121E DAN K38R-A121Y PADA SUPEROKSIDA DISMUTASE MANGAN STAPHYLOCOCCUS EQUORUM REKOMBINAN

• Main Author: Pajatiwi, Ismiana
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48633
• Institution: Institut Teknologi Bandung

Manganese superoxide dismutase Staphylococcus equorum recombinant (rMnSODSeq) is an enzyme to catalyze the changing of reactive oxygen species (ROS) into more stable oxygen species. rMnSODSeq has good catalytic activity and wide range stability of pH and temperature but poor against UVC exposure. Dimer formation through interactions on the surface of rMnSODSeq monomer is predicted to increase SOD stability against UVC exposure. This study aims to improve the stability of rMnSODSeq against UVC exposure by forming interaction between monomers in the dimer interface area. The study started with determining the amino acid candidates based on sequence alignment and followed by designing the interactions on the dimer interface using in silico approach. The COOT program was used to substitute the amino acid candidates. Not included in the conserved region, far from the active cite, located on the interface of the dimer, and close distance between the amino acid side chains are the characteristics of substituted amino acids that should be present. Based on preliminary studies, lysine as the 38th amino acid sequence was substituted into arginine (K38R), while alanine as the 121st amino acid sequence was substituted into glutamate (A121E). Alternatively, alanine was also substituted into tyrosine (A121Y). Mutations are performed at the DNA level using site directed mutagenesis by polymerase chain reaction (PCR). Plasmid product from AAA-38-AGA (K38R) substitution on pJExpress414_sod was used as a template for GCA-121-GAA (A121E) and GCA-121-TAT (A121Y) substitution. Substituted PCR products were transformed into Eschericia coli TOP10 then later confirmed through transformant screening, restriction analysis, and DNA sequencing. K38R-A121E substitution has been successfully carried out while the K38R-A121Y substitution has not succeeded presumably due to cross hybridization during annealing process using PCR.