Improving plastic degradation by increasing the thermostability of a whole cell biocatalyst with LC-cutinase activity

Abstract

Global consumption of polyethylene terephthalate (PET) increases each year, resulting in considerable buildup of plastic waste in the environment. A whole cell biocatalyst (WBC) with LC-cutinase bound to its outer membrane had been constructed to hydrolyze PET (optimum temperature 55°C). The aim of this study was to improve WBC viability at 55°C by inserting Ef-TU gene from sugarcane into WCB cells, with the hope of improving its hydrolytic activity. Escherichia coli BL21(DE3) was co-transformed with two plasmids, the first contained Lpp-OmpA-LC-cutinase fused gene and the second contained Ef-TU gene. Cells transformed with only the first plasmid were used as control. The cells were grown at 37°C and 55°C and viability was analyzed by total plate count. LC-cutinase activity was measured using pNPB as substrate and its capability to hydrolyze PET was observed by scanning electron microscopy. The presence of Ef-TU improved WCB viability at 55°C after 90 minutes incubation and LC-cutinase activity remained stable after 72 hours incubation at 55°C. LC-cutinase activity of WCB with Ef-TU was consistently higher than without EF-TU. Scanning electron micrograph of PET sheets incubated with WBC cultures with Ef-TU showed larger pockets than without EF-TU.