Expression and Characterization of Recombinant PETase in E. coli BL21(DE3) for PET Plastic Degradation
Vundeti Sanjana Priya *
Department of Biotechnology, Loyola Academy (Autonomous), Alwal, Secunderabad, Telangana, India.
*Author to whom correspondence should be addressed.
Abstract
Aim: Plastic pollution caused by the persistence of polyethylene terephthalate (PET) has become a major environmental challenge due to its resistance to natural degradation. Biocatalytic degradation using PETase provides an eco-friendly and sustainable solution for PET recycling. In this study, the PETase gene from Ideonella sakaiensis was codon-optimized, cloned into the pUC19 vector, and expressed in Escherichia coli BL21(DE3) to establish an efficient recombinant production system. The research aimed to design and validate a simple, cost-effective, and scalable expression strategy for functional PETase enzyme production. The purified enzyme displayed a distinct 29 kDa band on SDS-PAGE, confirming successful expression. Sequential purification using Ni–NTA affinity and ion-exchange chromatography yielded an overall recovery of 72%, with >90% purity and a specific activity of 4.8 U/mg. The enzyme exhibited maximum activity at pH 8.0 and 37 °C, with optimal catalytic efficiency demonstrated by an average 23% weight loss of PET film and 45 µM release of terephthalic acid within 48 hours. These results confirm the strong hydrolytic potential of recombinant PETase. Compared with previously reported systems, the present pUC19-based construct achieved comparable enzyme activity with simplified design and lower production cost. This study bridges a critical gap in developing accessible PETase expression systems and highlights their potential integration into bioreactor-based or wastewater-treatment workflows for sustainable plastic waste recycling. Future research will focus on improving thermostability and substrate specificity through enzyme engineering to enhance industrial-scale PET biodegradation. Kinetic analysis revealed a Km of 0.42 mM and Vmax of 6.1 U mg⁻¹, while mass-balance measurements confirmed 91% recovery of PET monomers, substantiating the enzyme’s catalytic efficiency.
Study Design: Experimental, in silico and in vitro study.
Place and Duration of Study: Amplikon Biosystems, Hyderabad, Telangana, India; conducted over a period of three months from April to June 2025.
Methodology: The PETase gene was codon-optimized for E. coli expression and cloned into the pUC19 vector using HindIII and EcoRI restriction sites. The recombinant plasmid was transformed into competent E. coli BL21(DE3) cells via heat shock. Transformed colonies were selected on ampicillin containingplate count agar and cultured for expression studies. Recombinant protein expression was induced with IPTG, and cell lysates were prepared for protein extraction. SDS-PAGE was used to analyze protein expression, and the concentration of PETase was determined using the Bradford assay. Purification of recombinant PETase was performed sequentially using Ni-NTA affinity chromatography followed by ion exchange chromatography to achieve higher purity. The structural integrity of the purified protein was further confirmed through native PAGE analysis. Enzymatic activity was assessed by incubating PETase with PET substrates, and degradation efficiency was evaluated by visual observation of clear zones and quantitative measurement of substrate breakdown. Additionally, reaction conditions such as temperature and pH were optimized to determine the maximum catalytic efficiency of recombinant PETase.
Results:Recombinant vector construction was confirmed in silico with a total size of 3.56 kb, containing the PETase insert between HindIII and EcoRI sites. Transformation of E. coli BL21(DE3) produced distinct colonies on ampicillin plates, confirming successful uptake of the recombinant plasmid. Plasmid isolation yielded clear DNA bands on agarose gel electrophoresis, validating the presence of rec ombinant DNA. Induction with IPTG resulted in visible turbidity, indicating active recombinant expression. The Bradford assay quantified total protein at 0.038 µg/mL, confirming efficient synthesis. SDS-PAGE revealed a distinct 29 kDa band corresponding to PETase, while affinity purification produced a sharp single band, confirming successful isolation. Ion-exchange chromatography further refined protein purity with consistent elution patterns. Functional assays demonstrated PET degradation activity-agar well diffusion showed clear hydrolytic zones, and turbidity reduction assay revealed decreased absorbance at 600 nm in PETase-treated samples compared to controls. Collectively, these findings confirm successful cloning, expression, and purification of functionally active PETase in E. coli BL21(DE3), capable of catalyzing PET hydrolysis under laboratory conditions.
Conclusion: The study successfully demonstrated the cloning, expression, purification, and activity confirmation of PETase from Ideonella sakaiensis in E. coli BL21(DE3). The recombinant enzyme exhibited robust PET-degrading activity, confirmed through both agar and turbidity assays. The results validate the pUC19-based construct as a cost-effective and efficient expression system for recombinant PETase production. Future research will focus on enhancing thermostability and catalytic turnover to enable industrial-scale PET biodegradation.
Keywords: PETase, Escherichia coli BL21(DE3), pUC19 vector, recombinant expression, plasmid cloning, SDS-PAGE, affinity chromatography, ion-exchange chromatography, PET degradation, biodegradation, environmental biotechnology