IJEP 41(9): 989-996 : Vol. 41 Issue. 9 (September 2021)
Yashaswini M. and Priya R. Iyer*
Women’s Christian College, PG and Research Department of Biotechnology, Chennai – 600 006, India
Abstract
Plastics have been a huge nuisance to the world. In order to degrade the most widely used polyethylene terephthalate (PET), this research has been conducted. Organisms were isolated using minimal media alongwith shredded PET plastic provided as the sole carbon source. Eight individual colonies were isolated and were named PET1 to PET8. Organisms were identified by Gram staining and biochemical assays. Thin layer chromatography was performed alongwith standard to check the presence of terephthalic acid produced as a result of PET degradation. High pressure liquid chromatography was performed to confirm the production of terephthalic acid. A colourimetric assay was standardized using bromocresol green to quantitatively measure the terephthalic acid produced following the degradation of the plastic. Lowry’s method was performed to check the presence of protein. Since the enzyme is the only protein available in the media it is a confirmatory test used to check the presence of the enzyme. PETase theoretically being an esterase should utilize tributyrin as a substrate. Hence esterase activity was viewed by well diffusion method on tributyrin agar. A colourimetric assay was standardized using p-nitrophenyl acetate to analyze the amount of PETase produced by the organisms. The temperature, pH, shaking and static conditions were standardized and optimum characteristics for the production of the enzyme were determined. The highest producers were given for 16s rRNA sequencing. The weight of the plastic was determined before and after the degradation. The surface of the plastic was studied using scanning electron microscopy. The enzyme was purified and sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed. Plasmids were isolated and then transformation was carried out into E. coli DH5a. This analysis is the need of the hour and further the enzyme activity can be studied and the enzymes can be used to treat the plastic filled dump yards.
Keywords
Polyethylenase, Plastic biodegradation, Polyethylene terephthalate, Microbial degradation, Esterase
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