Production and Characterization of Cutinase from Polyethylene Terephthalate Degrading Bacterial Strain Isolated from Compost Soil

Abstract

Poly (ethylene terephthalate) (PET), a petroleum-derived synthetic plastic is considered chemically inactive and highly resistant to microbial attack. The accumulation of plastic waste in the environment not only is a source of pollution but also threatens the ecosystem through the release of microplastic. In the current study compost soil was screened out for isolation of polyethylene terephthalate degrading bacterial strain. A bacterium designated as CS7 possesses the ability to utilize PET as a sole carbon source at mesophilic temperature ~35°C. Strain CS7 showed biofilm formation ability over PET pieces and exhibited surface hydrophobicity. Gram staining, biochemical, and molecular characterization results indicated that the strain CS7 was similar to Bacillus subtilis. The degradation of PET was further confirmed through weight loss and Fourier transform infrared spectroscopy (FTIR). During PET biodegradation, the Bacillus subtilis CS7 exhibited cutinase activity. Various physicochemical conditions and nutritional factors were statistically analyzed and optimized for maximum cutinase production using the Plackett-Burman design and Central Composite Design software. Cutinase was purified to homogeneity by column chromatography technique using Sephadex G-100 gel resin. The specific activity of the purified cutinase from strain CS7 was found to be 7.58510 U/mg, achieving a 6.1062-fold purification and a yield of 64.01%. The kinetic parameters for this enzyme, Km and Vmax, were determined to be 2.87 mg/ml and 64.102 μmol/mg/min, respectively. The stability of purified enzyme was observed over a wide range of temperatures from 30 to 40°C and pH values from 8.0 to 10.0. The cutinase exhibited stability towards mesophilic temperature, and various pH ranges, and could further be tested for its resistance to solvents, metal ions, and detergents that will confirm its efficiency for various biotechnological applications under mesophilic conditions. This study suggests that Bacillus subtilis CS7 possesses the ability to biodegrade PET and could be further explored for enzymatic hydrolysis of plastic waste.