Construction and Cloning of Plastic-degrading Recombinant Enzymes (MHETase)

Abstract

Background: Polyethylene terephthalate (PET) is the most widely produced polyester plastic in the world. PET is very difficult to catalyze or biological depolymerization due to the limited access to ester bonds. Consequently, plastic will be stockpiled or flowed into the environment which is projected until hundreds of years. The most effective and environmental friendly plastic degradation method is biodegradation with microorganisms. Two specific enzyme for PET hydrolase, PETase and MHETase have been identified from Ideonella sakaiensis 201-F6. Recombinant genes are made to increase the effectiveness of enzymes in degrading PET. Previous studies of the PETase gene have been carried out, but to produce the final degradation PET product, the enzyme MHETase is needed. Thus, in this study the MHETase gene construction was carried out.

Methods: The goal of this study is to construct MHETase gene in pUCIDT plasmid with native signal peptide from I. sakaensis 201-F6 and constitutive promoter J23106 was expressed in Escherichia coli BL21 (DE3) by heats shock. Expression analysis using SDS-PAGE and activity of enzyme is analyzed by spectrophotometry method and SEM.

Results: MHETase gene protein was successfully constructed in pUCIDT +Amp plasmid with native signal peptide from Ideonella sakaensis 201-F6, T7 terminator and constitutive promoter J23106. PCR analysis showed that the gene successfully contained in the cells by band size (1813 bp) in electrophoresis gel. Analysis using Snap Gene, pairwise alignment using MEGA X, and NCBI was demonstrated that MHETase sequence the gene was in-frame in pUCIDT plasmid.

Conclusion: MHETase gene was successfully constructed in plasmids by in silico method. Synthetic plasmids transformed in E. coli BL21 (DE3) contain MHETase gene sequences which were in frame. Hence, the E. coli BL21 (DE3) cells have the potential to produce MHETase proteins for the plastic degradation testing process. We will patent the construct of MHETase gene using constitutive promoter and signal peptide from native which expressed in E. coli BL21 (DE3). This patent refers to a more applicable plastic degradation system with a whole cell without the need for purification and environmental conditioning of pure enzymes.

Keywords: MHETase; PET; biodegradable; enzyme; plastic; recombinant.