OPTIMIZATION OF RECOMBINANT MUTANT PETASE ENZYME EXPRESSION AT 37°C BY IPTG INDUCTION VARIATIONS

• Main Author: Rachelisa Rudjito, Resmila
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/67741
• Institution: Institut Teknologi Bandung

Polyethylene terephthalate (PET) is one of the most used type of plastic due to it’s excellent stability and mechanical properties. However, the overuse of PET has resulted in the accumulation of PET waste that is extremely difficult to degrade and eventually polluting the environment. In 2016, a bacterial species Ideonella sakaiensis was discovered that had the ability to express PETase and MHETase enzymes that could degrade PET. PETase had higher enzymatic activity compared to other PET degrading enzymes, but it was still not sufficient enough for production on an industrial scale. In a previous study, a mutant PETase in the form of PETase L117F/Q119Y/S121E/G165A/D186H/R280A/S214H/S238F was designed in silico and had higher stability and stronger interaction with PET compared to wildtype PETase. However, the results of the in silico design has never been tested in vitro. Therefore, in this study, optimization of IPTG concentration & incubation time after induction was carried out to produce the mutant PETase enzymes in Escherichia coli BL21 (DE3) at 37°C. The gene encoding the mutant PETase protein was cloned into pET-22b(+) plasmid carrying a pelB signal peptide and transformed into E. coli BL21 (DE3) cells. Expression of mutant PETase protein in E. coli BL21 (DE3) was carried out at 37°C with variations in induction duration (4, 8 and 16 hours) and IPTG concentrations (0; 0.05; 0.1; 0.25; and 0.5 mM) in the extracellular and intracellular fractions (soluble and insoluble). Then, the bands suspected as target proteins in the SDS PAGE gel visualization results were analyzed using ImageJ and SPSS to examine the relative density and the significance of the variations. The activity of the mutant PETase was then tested using a colorimetric method using p-nitrophenyl butyrate (pNPB). The transformation results were confirmed by PCR and DNA sequencing. The highest expression was found in the insoluble fraction, with an IPTG concentration of 1 mM for 4 hours of induction time. The enzymatic activity assay results did not show any enzyme activity in the extracellular fraction but revealed activity in the intracellular fraction where the sample with the variation of 4 hours and 1 mM IPTG had the highest activity with an absorbance delta of 0,15557. Although the proteins produced in the insoluble fraction are generally inclusion bodies, activity was still found, indicating that the formation of inclusion bodies does not always have to be a disadvantage. Thus, it can be concluded that in this study, mutant PETase could not be expressed extracellularly at 37°C but could be expressed intracellularly with a duration of 4 hours and a concentration of 1 mM IPTG as the most optimal variation. Further studies using lower temperatures can be carried out to produce mutant PETase proteins extracellularly.