Immobilization of PETase enzymes on magnetic iron oxide nanoparticles for the decomposition of microplastic PET

Polyethylene terephthalate (PET) is responsible for a large amount of environmental contamination with microplastics. Based on its high affinity, the PET degrading enzyme PETase can be immobilized on superparamagnetic iron oxide nanoparticles through a His-tag. The His-tag increases enzyme stability, and allows magnetic separation for recovery. Multiple recycling steps are possible and microplastic particles can be decomposed depending on the PET's crystallinity. The separation or decomposition of PET allows for a sustainable way to remove microplastic from water.

The culture was transferred to shake flasks the following day and incubated at 37 °C for approximate 3 h at 200 rpm until an optical density (OD 600 ) between 0.6-0.8 was reached. Next, the cultures were induced with 0.1 mM IPTG and incubated at 16 °C for 20 h at 200 rpm. Cells were harvested by centrifuging at 3200xg for 20 min and subsequently the supernatant was discarded. The remaining pellets were stored at -20 °C. For cell disruption the previously obtained pellets were dissolved in 5 mL equilibration buffer (20 mM Na 3 PO 4 , 500 mM NaCl) by vortexing. Protease inhibitor was added to each suspension. The tubes with the dissolved cells were put on ice and disrupted mechanically by a French press (Julabo GmbH, Seelbach) at 1.8 kbar and 8 °C. The lysate was centrifuged at 17000xg to get rid of cell debris. From the obtained lysate PETase was purified by an Immobilized Metal Ion Chromatography (IMAC) system (Äkta Explorer, GE Healthcare, Chicago). The analytical detection of PETase was carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a 5 % collection and a 12 % separation gel (160 V, 300 mA and 30 W for 60 min). The E. coli lysate and the purified PETase enzyme after IMAC elution were compared on the gel. The success of the PETase purification was determined by comparing the contrast intensity between the band of interest (~30 kDa) and other bands on the gel. Therefore, the Coomassie-stained SDS-PAGE (0.1% Coomassie, 10% acetic acid, 40% ethanol) was analysed in a biomolecular imager (Amersham Typhoon, GE Healthcare, Chicago).
Before quantifying the corresponding PETase concentrations, the purified lysate was rebuffered in Tris(hydroxymethyl)aminomethane(Tris)-HCl buffer at pH 7.5. The lysate was filled in tubes with a pore size of 10 kDa and centrifuged at 4 °C and 3200 g for 20 min. The filtered liquid was disposed while the remaining liquid containing the target protein was refilled again with buffer. This procedure was repeated three times. Afterwards, each remaining liquid containing PETase was aliquoted in 200 µL portions and dried at ~80 °C.
Colorimetric protein quantification was determined by a bicinchoninic acid (BCA) assay (Merck KGaA, Darmstadt, Germany) according to the manufacturer's user guide. Analysis was performed at 562 nm with an Infinite M200 Microplate reader (Tecan Deutschland, Germany). The calibration curve was calculated by averaging the corresponding triplicates and subtracting the buffer blank. Protein concentrations were calculated from the corresponding linear relationship of absorption and concentration of the standard. The experiments were performed in triplicates. Green fluorescent protein (GFP) without (woT) and with an (RH)4-tag (containing four histidines and four arginines) has been synthesized and purified as described by Zanker et al. 3 Histidine hexapeptides have been obtained from ? and mixed with magnetic nanoparticles in a range of 1:1 (w/w) with 0.5 g L -1 nanoparticles in a 1 mL suspension for 30 min at 25 °C under vigorous shaking.
Magnetite was synthesized following the co-precipitation of Fe 2+ and Fe 3+ in alkaline environment as described by The crystallinity of the (GF)-PET samples was determined by differential scanning calorimetry (DSC 204, Netzsch, Waldkraiburg). MNP-or NBC-treated (GF-)PET samples were separated with a magnetic holder and the supernatant was analyzed. For DSC measurements, the PET samples were heated from room temperature to 300 °C at a rate of 10 K min -1 and held for 60 s at this temperature. The melting heat enthalpy ∆H was obtained by the integral of the melting peak around 250 °C. The degree of crystallinity (%DOC) is calculated with the following equation 7 : Raman spectra for further crystallinity analysis were recorded by a Raman spectrometer (SENTERRA, Bruker Optics GmbH, Ettlingen). Therefore, samples containing MNPs or NBCs were separated by a magnetic holder.
The spectra were measured with a laser at a wavelength of 488 nm and laser power of 4 mW with a 50x objective.
The integration time for each measurement was 60 s. Each spectrum was measured twice and averaged. The