U 2910 spectrophotometer

All reagents were of commercially analytical grade and used directly.
Fluorescent spectra were recorded using a Hitachi F-4600 spectrofluorometer (Tokyo, Japan), and UV-Vis spectra were determined on a Hitachi U-2910 spectrophotometer. ¹H- and ¹³C-NMR spectra were carried out with a Brucker AV 400 nuclear magnetic resonance instrument (Faellanden, Switzerland), and the chemical shift is given in ppm from tetramethylsilane (TMS). Mass spectra were obtained using a thermo TSQ Quantum Access Agilent 1100 mass spectrometer (Santa Clara, CA, USA). Fluorescence imaging was performed with Olympus FluoView Fv3000 laser scanning microscope (Tokyo, Japan).

After that, the expression plasmids, pET-30a (+): HCSD, pET-30a (+): CSD and pET-30a (+): HCSD-HFBII were transformed into E. coli BL21(DE3) competent cells via heat shock. The successful transformation was confirmed via agarose gel electrophoresis and Sanger sequencing. 200 μl pre-cultures were inoculated from glycerol stocks and grown in a 10 ml LB medium overnight. After that, 1 ml pre-cultures were transferred to a 50 ml LB medium for expression. Cell growth of the cultures was measured using a HITACHI U-2910 spectrophotometer (Japan). After the cultures reached an OD₆₀₀ of 3, induction was conducted at 18°C and 150 rpm using IPTG at final concentration of 0.6 mM. For the separation of free PETase, 50 ml culture medium was centrifuged for 5 min at 4°C. The collected E. coli cells were resuspended in 5 ml sodium phosphate (10 mM, pH 7.4). After that, the resuspended cells were disrupted by an ultrasonic homogenizer at 4°C. And finally, the crude enzyme solution containing PETase was collected via centrifugation at 4°C for 30 min and applied to the degradation process of PET directly.

UV–Vis titration experiments were performed in CH₃CN solution at 298K using a Hitachi U-2910 spectrophotometer. To a 10 mm cuvette was added 2.5 mL of freshly prepared (B1 − c = 1.0 × 10⁻⁵ M; B2 − c = 1.1 × 10⁻⁵ M) solution of the studied receptor and, in the case of ion-pair-binding studies, 1 mol equivalent of cation (KPF₆ or NaClO₄) was added before titrations. Small aliquots of ca. 1.0 × 10⁻³ M TBAX solution containing the receptor at the same concentration as in the cuvette were then added, and a spectrum was acquired after each addition. The resulting titration data were analyzed using the BindFit (v0.5) package, available online at http://supramolecular.org (accessed on 10 November 2022). Each titration was carried out in duplicate. Reported values were calculated as a weighted arithmetic mean, where the weights were the errors obtained for each value separately. The given uncertainty of the association constants was the largest of the variance (external or internal).

Optical absorption spectra were recorded in the wavelength range of 400–1100 nm using the Hitachi U-2910 spectrophotometer. Due to the high concentration of SWCNTs in the water medium, the samples were diluted with demineralized water for analysis with demineralized water. All spectra were normalized to the global minimum between 600 and 900 nm to compare the samples, which is a standard procedure [43 (link)].

¹H, ¹³C and ¹⁹F NMR spectra of intermediates were recorded on a JEOL resonance ECZ 400S spectrometer (400 MHz) in CDCl₃. Tetramethylsilane (TMS) and CDCl₃ were used as internal standards for ¹H and ¹³C NMR, respectively; CF₃CO₂H was used as an external standard for ¹⁹F NMR. FT-IR spectra were recorded on a Nicolet AVATAR-360 FT-IR spectrophotometer with a resolution of 4 cm⁻¹. Number-average molecular weights (M_n) and molecular weight distributions (M_w/M_n) were obtained on a conventional gel permeation chromatography (GPC) system equipped with a Waters 515 Isocratic HPLC pump, a Waters 2414 refractive index detector, and a set of Waters Styragel columns (HR3 (500–30 000), HR4 (5000–600 000) and HR5 (50 000–4 000 000), 7.8 × 300 mm, particle size: 5 μm). GPC measurement was carried out at 35 °C using tetrahydrofuran (THF) as eluent with a flow rate of 1.0 mL min⁻¹. The system was calibrated with linear polystyrene standards. Differential scanning calorimetry (DSC) was performed on a TA Q200 DSC instrument in N₂ with a heating rate of 10 °C min⁻¹. Thermogravimetry analysis (TGA) was conducted on a TA Discovery TGA 55 thermal analysis system in N₂ with a heating rate of 10 °C min⁻¹. UV/vis spectra were acquired on a Hitachi U-2910 spectrophotometer.