STUDY OF ACTIVITIES AND STABILITY RECOMBINANT MANGANESE SUPEROXIDE DISMUTASE Staphylococcus equorum S126C AND N73F

• Main Author: Sinthary, Venna
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/40447
• Institution: Institut Teknologi Bandung

Recombinant Manganese Superoxide Dismutase S. equorum (rMnSODSeq) has successfully been engineered in order to improve its stability by means of substituting asparagine amino acid at position 73 into phenylalanine (N73F) and substituting the serine at position 126 to cysteine (S126C). The present study was aimed to compare the stabiliies and activities of the recombinant rMnSODSeq and its N73F and S126C mutants, and further evaluate the relation to the structure. The study began with isolation and confirmation of the rMnSODSeq encoding DNA in the pJexpress414-sod. The nucelotide sequence analysis confirmed the substitutions of nucleotides coding for N73F and S126C. The enzymes were successfully overproduced in Escherichia coli BL21 (DE3), isolated and then purified. Overproduction of the native and mutant rMnSODSeq was carried out at 37 ° C 150 rpm with isopropyl ?-D-1-thiogalactopyranoside (IPTG) induction at a final concentration of 1 mM for 4 hours. Purification of the native and mutant rMnSODSeq was carried out using Ni 2 + -NTA affinity column chromatography. The results were pure protein as judged from the presence of a single band in the SDS- PAGE gel. The native and mutant rMnSODSeq activities were tested qualitatively and quantitatively with zymography and colorimetry, respectively. The activity assay showed that both substitution of N73F and S126C have decreased the enzyme activity. Specific activities of rMnSODSeq native, rMnSODSeq N73F, and S126C were 1958 Umg -1 , 417 Umg -1 , 666 Umg -1 respectively. However, the stability of the mutant protein against UVC is relatively better than the native protein. The residual activity of rMnSODSeq alami, rMnSODSeq N73F, and rMnSODSeq S126C after 45 minutes of UVC exposure was 71,3%, 97%, 103,3%. rMnSODSeq native, rMnSODSeq N73F, and rMnSODSeq S126C are relatively stable until 80 ºC and to the presence of denaturing agent urea but not with guanidine HCl.