EXPRESSION OF MUTANT PETASE IN E. COLI ORIGAMI B (DE3) BY OPTIMIZING IPTG INDUCTION TIME

• Main Author: Loho, Elizabeth
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/81844
• Institution: Institut Teknologi Bandung

PET (Polyethylene terephthalate) is one of the most widely used types of plastic in everyday life. However, PET is difficult to decompose, resulting in the accumulation of plastic waste in the environment. In 2016, PETase was isolated from Ideonella sakaiensis bacteria in nature. However, this enzyme is thermolabile, so in previous studies, in silico mutations have been carried out and thermostable mutant PETase was produced. Optimization of mutant PETase expression has been carried out in E. coli BL21 (DE3), but the highest protein expression is still found in the insoluble intracellular fraction. Therefore, in this study, mutant PETase expression will be carried out in E. coli Origami B (DE3) with optimization of induction time which is expected to increase protein expression in the extracellular fraction. Plasmid pET 22b(+) containing the mutant PETase gene was isolated, transformed into E. coli Origami B (DE3), and confirmed by colony PCR and DNA sequencing. Furthermore, a growth curve of the recombinant bacteria for 16 hours was constructed. Mutant PETase was then expressed at mid log phase, induction temperature of 24oC, and IPTG concentration of 0.5 mM with variations in induction time of 12, 18, and 24 hours. Tests were carried out in three repetitions on three protein fractions, namely extracellular, intracellular soluble, and intracellular insoluble. After induction, protein analysis was carried out by SDS-PAGE method and colorimetric activity test using pNPB substrate. The transformation results were confirmed through colony PCR and DNA sequencing. From the SDS-PAGE results, the protein band with the highest color intensity was produced by E. coli Origami B (DE3) in the insoluble intracellular fraction with an induction time of 18 hours. The mutant PETase activity was significantly different (p<0.05) in E. coli BL21 (DE3) and E. coli Origami B (DE3) with an induction time of 12 hours in the soluble fraction, 18 hours in the extracellular fraction, and 24 hours in all fractions. In E. coli Origami B (DE3), induction time variation had a significant effect (p<0.05) on mutant PETase activity for all protein fractions. The highest activity was obtained in the intracellular non soluble fraction at 18 hours induction time. In conclusion, E. coli Origami B (DE3) can express the mutant PETase recombinant enzyme and increase the expression of extracellular fraction proteins, with the highest level of expression and activity obtained in the insoluble intracellular fraction. Further research is needed to characterize the recombinant PETase enzyme that has been produced.