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].