OPTIMIZATION OF MUTANT PETASE EXPRESSION IN LURIA BERTANI (LB) AND TERRIFIC BROTH (TB) MEDIUM

• Main Author: Humaira Ratnapuri, Aisy
• Format: Final Project
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/74025
• Institution: Institut Teknologi Bandung

PET or polyethylene terephthalate is a type of plastic that is commonly used in various industrial fields due to its strong, durable, stable, airtight, as well as able to be reprocessed and recycled. In Indonesia, especially in Java, it is estimated that m ore than 37.000 tons of PET plastic waste produced per month. However, PET waste management is still not optimized and produces secondary pollution to the environment. Therefore, PET biodegradation research was carried out which could break down the PET po lymer into its monomer terephthalic acid and ethylene glycol using PETase enzyme from Ideonella sakaiensis 201 F6. Meanwhile, the wildtype PETase is thermolabile and has a low affinity for PET substrate, so mutations were carried out on multiple amino acid residues points such as L117F / Q119Y / S121E / G165A / D186H / R280A / S214H/ S238F to produce a mutant PETase which is thermostable and has a higher affinity toward PET substrate. However , in previous studies optimization of the expression of the recombinant enzyme had not been carried out. Therefore, in this study optimization of the culture medium and induction time will be carried out to express mutant PETase. Escherichia coli glycerol stock culture BL21(DE3) transformant from the transformation results of previous studies was confirmed using colony PCR. After the colonies were confirmed, growth curves were made at intervals of 30 minutes for 8 hours. The expression process was carried out by culturing activation on Luria Bertani medium and Terrific Broth and 1 mM IPTG induction in the lag, ½ log, and stationary phases. Results Recombinant protein expression was analyzed using the SDS-PAGE and ImageJ methods. The mutant PETase enzyme activity assay was carried out at 25°C and 40°C using pNPB substrate. In Escherichia coli BL21(DE3) transformants, confirmed the PET22b(+) plasmid band that has been inserted by the PET gene with a size of 1167 bp. The growth curve produced on TB medium shows a longer logarithmic phase compared to the logarithmic phase produced by culture on LB medium. From the analysis of protein expression, it was found that optimal results of Luria Bertani culture medium were induced in the ½-log phase. Meanwhile, from the enzyme activity test results, the highest activity was found in the mutant PETase enzyme cultured on Terrific Broth medium and induced in the ½ log phase with an absorbance value of 64.1 units/ml of enzyme. Based on this study, the optimal culture medium for expressing the extracellular fraction of the mutant PETase enzyme was Luria Bertani medium which was induced in the ½ log growth phase. Meanwhile, the highest mutant PETase enzyme activity test results were obtained in Terrific Broth medium. Further research can be carried out to optimize the PETase enzyme in Terrific Broth medium with IPTG concentrations and longer incubation times so as to minimize the formation of inclusion bodies.