Perpustakaan Digital ITB

PETase is an enzyme derived from Ideonella sakaiensis, known for its ability to degrade polyethylene terephthalate (PET) plastic waste in an environmentally friendly manner. Although previous studies have achieved improvements in the activity and thermostability of PETase, its application at the industrial scale remains challenging, particularly due to fluctuating environmental conditions and the need for large-scale, sustainable enzyme production. This study aims to evaluate an immobilization strategy based on the formation of hybrid nanoflowers using CuSO? and Co(NO?)? to enhance enzyme stability and enable reusability. Mutant PETase was produced using recombinant Escherichia coli BL21(DE3) induced with IPTG and subsequently purified from the culture supernatant. Immobilization was performed by mixing 0.5 mg/mL mutant PETase with 10 mM CuSO? or Co(NO?)? solution, followed by incubation at 25°C for 72 hours. The catalytic performance of free and immobilized mutant PETase was evaluated through measurements of initial activity, thermostability (25–70°C), storage stability at room temperature over 10 days, and activity at a pH range of 4–8. Reusability was also assessed over 5 cycles for the immobilized enzymes. The PET-degrading activity of both free and immobilized PETase was tested against PET film for 10 days at 25°C, and morphological characterization of the immobilized enzymes was conducted using SEM and EDS analyses. Mutant PETase was successfully expressed, as confirmed by the presence of a 1,210 bp gene fragment and a corresponding protein of approximately 23–30 kDa. The immobilization resulted in an activity recovery of Co(NO?)?–PETase (102.52%) and CuSO?–PETase (59.16%). Thermostability tests showed that all three enzyme types exhibited optimum activity at 40°C. In terms of storage stability at room temperature over 10 days, Co(NO?)?–PETase showed the highest stability, retaining 61.04% of its initial activity, compared to 51.22% for free mutant PETase and 43.45% for CuSO?–PETase. Both immobilized enzymes also demonstrated reusability, with optimum activity observed at pH 8. By the fifth cycle, Co(NO?)?–PETase and CuSO?–PETase retained 46.47% and 69.07% of their initial activity, respectively. Both free and immobilized mutant PETase exhibited the ability to degrade PET film, as evidenced by the formation of surface holes after 10 days of incubation. Morphological characterization revealed that CuSO?–PETase formed a nanoflower-like structure, while Co(NO?)?–PETase exhibited irregular aggregates. This study concludes that CuSO?- and Co(NO?)?-immobilized mutant PETase successfully enhanced enzyme stability and reusability, indicating their potential for sustainable plastic biodegradation applications.