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.

Expression of a Cutinase of Moniliophthora roreri with Polyester and PET-Plastic Residues Degradation Activity

“…The maximum specific activity under the evaluated conditions was 34,036 U/mg after 3 h of induction. Interestingly, this value is higher than those of other fungal cutinases overexpressed in E. coli , such as the cutinase of Thermobifida fusca (167.4 U/mg) considering p -NPA as the substrate, even after improving T. fusca cutinase expression was done ( 21 , 28 , 29 ). Su and collaborators obtained 1,063.5 U/ml after cloning the truncated version of the extracellular T. fusca cutinase without the signal peptide, obtaining higher specific activity and the extracellular release of the enzyme ( 30 ).…”

Biotechnological Application of Cutinase: A Powerful Tool in Synthetic Biology

“…As presented in Figure 2, both fungal and bacterial cutinases present PETase activity. As higher temperatures (around 65) can promote the hydrolysis of PET, thermophilic cutinase is superior to mesophilic cutinases in practical applications [63][64][65], such as the LC cutinase (isolated from leaf compost) which can maintain activity under high temperatures [24,25]. In order to further improve the hydrolysis efficiency towards PET of natural cutinase, engineered cutinase have been developed.…”