2. Materials and methods:
2.1 Bioinformatics studies : Structural comparisons of Tf C with LCC and PETase were done using PYMOL (Schrödinger). Molecular docking and Molecular Dynamics simulation studies, respectively, were performed GLIDE (Schrödinger) and Desmond (Schrödinger), employing parameters described previously [Mrigwani et al., 2022]. Docking was performed using Tf C (PDB ID: 4CG1) and 2HE-(MHET)3, containing three terephthalate moieties. Structural alignments were done using the Tm align web server [Zhang and Skolnick, 2005].
2.2 Cloning, expression and protein purification of wild type Tf Cand its mutants : The gene encoding Tf C was amplified from the genomic DNA of Thermobifida fusca (MTCC No. 1754T) and cloned between Bam HI and Hind III restriction sites in pQE-30 (Qiagen) for expression in fusion with an N-terminal 6xHis tag. Tf C variants/mutants were created through site directed mutagenesis of this gene through splicing by overlap extension PCR (SOE-PCR), prior to cloning into the same vector, between the same sites. All clones were transformed into XL1-Blue for constitutive expression. Transformed XL1-Blue cells (grown at 37 °C for 9 h at 220 rpm) were harvested and disrupted. Cell debris was separated through centrifugation at 12,000 rpm for 1 h, and lysates subjected to Ni-NTA (IMAC) chromatography. The second step of purification involved size exclusion chromatography (SEC) on a GE Superdex-75 Increase 10/300 GL column, run on a GE AKTA Purifier-10 workstation, using 25 mM sodium dihydrogen phosphate buffer of pH 8.0.
2.3 Circular dichroism (CD) studies : An MOS-500 CD spectrometer (BioLogic, France) was used to examine secondary structure in enzymes, using concentrations of 0.2 mg/ml, and a quartz cuvette of 2 mm path length. Mean Residue Ellipticity was calculated as MRE= (θ×mean residue weight×100)/(1000×concentration in mg/ml×pathlength in cm); where, θ is the raw ellipticity. For chemical and thermal kinetics, enzyme was incubated at various guanidium hydrochloride (Gdm.HCl) concentrations and temperatures for 2 h, with monitoring of CD spectral intensity at 222 nm. Folded fractions were determined and used to determine rates of unfolding and thermodynamic parameters of stability.
2.4 Differential Scanning Calorimetry (DSC) studies : Using VP-DSC (Microcal), thermal histories were created through repeated heating/cooling (10-15 up/down scans) at 90 °C/hr (upscan; 20-90°C) and 60 °C/hr (downscan; 90-20 °C). Then, buffer in the sample cell was replaced by 0.5 mg/ml. One cycle of up/down scans was performed to determine enzyme melting temperature, refolding ability and enthalpy associated with unfolding, with raw data fitted using a non-2-state cursor init model (Microcal).
2.5 PET-binding and activity against PET/BHET : Prior to binding/activity assays, circular discs of commercial PET film (Goodfellow, Product code: GF25214475) were washed with 1% SDS, water and ethanol for 30 min, and air dried. For binding assays, 2 µM enzyme was incubated with PET for 40 h at 60 °C. Films were washed with buffer. Enzyme fractions in solution, or bound to PET (extracted through boiling with SDS-PAGE sample loading buffer) were determined through electrophoresis, and densitometry using Image lab software (Bio-Rad). For activity assays, films were incubated with 2 µM enzyme(s) at 60 °C for 50 h in phosphate buffer, pH 8.0. Ability to hydrolyse BHET (Sigma Aldrich, Product Code 465151) was determined by incubating 250 µM BHET with 1 µM enzyme at 60 °C, for either 4 h, or 12 h. Reverse-phase HPLC was used to quantify degraded species on a Shimadzu HPLC workstation using previously described methods [Yoshida et al., 2016].