Identification of Cutinolytic Esterase from Microplastic-Associated Microbiota Using Functional Metagenomics and Its...

Plastic pollution has threatened biodiversity and human health by shrinking habitats, reducing food quality, and limiting the activities of organisms. Therefore, global interest in discovering novel enzymes capable of degrading plastics has increased considerably. Within this context, the functional metagenomic approach, which allows for unlocking the functional potential of uncultivable microbial biodiversity, was used to discover a plastic-degrading enzyme. First, metagenomic libraries derived from microplastic-associated microbiota were screened for esterases capable of degrading both tributyrin and polycaprolactone. Clone KAD01 produced esterase highly active against p-nitrophenyl esters (C2-C16). The gene corresponding to the enzyme activity showed moderate identity (≤ 55.94%) to any known esterases/cutinases. The gene was extracellularly expressed with a 6× histidine tag in E. coli BL21(DE3), extracellularly. Titer of the enzyme (CEstKAD01) was raised from 21.32 to 35.17 U/mL by the statistical optimization of expression conditions and media components. CEstKAD01 was most active at pH 7.0 and 30 °C. It was noteworthy stable over a wide pH (6.0-10.0) and temperature (20-50 °C). The enzyme was active and stable in elevated NaCl concentrations up to 12% (w/v). Pre-incubation of CEstKAD01 with Mg²⁺, Mn²⁺, and Ca²⁺ increased the enzyme activity. CEstKAD01 displayed an excellent tolerance against various chemicals and solvents. It was determined that 1 mg of the enzyme caused the release of 5.39 ± 0.18 mM fatty acids from 1 g apple cutin in 120 min. K_m and V_max values of CEstKAD01 against p-nitrophenyl butyrate were calculated to be 1.48 mM and 20.37 µmol/min, respectively. The enzyme caused 6.94 ± 0.55, 8.71 ± 0.56, 7.47 ± 0.47, and 9.22 ± 0.18% of weight loss in polystyrene, high-density polyethylene, low-density polyethylene, and polyvinyl chloride after 30-day incubation. The scanning electron microscopy (SEM) analysis indicated the formation of holes and pits on the plastic surfaces supporting the degradation. In addition, the change in chemical structure in plastics treated with the enzyme was determined by Fourier Transform Infrared Spectroscopy (FTIR) analysis. Finally, the degradation products were found to have no genotoxic potential. To our knowledge, no cutinolytic esterase with the potential to degrade polystyrene (PS), high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polyvinyl chloride (PVC) has been identified from metagenomes derived from microplastic-associated microbiota.