4. Conclusions:
We created 17 mutants of Tf C, with a view to (a) creating or removing space in the active site, to modulate catalysis, or (b) increasing or decreasing hydrophobicity in-and-around the binding site, or at distal locations, to modulate Tf C’s binding of PET. In every instance attempting to alter catalytic efficiency through changes made to residues at the active site, we only observed a reduction or complete abolition of activity upon solid PET, sometimes without a reduction in activity against BHET, indicating the sensitivity ofTf C’s active site to binding to crystalline PET chains. In stark contrast, we achieved significant success with mutations designed to reduce non-specific (hydrophobic) interactions of Tf C with PET, at locations distal to the active site, where our intention was to facilitate either (i) improved dissociation of enzyme from PET, following catalysis, to free up enzyme for further rounds of binding and catalysis, or (ii) the improved presence of enzyme in solution, to concomitantly convert OETs, BHET or MHET into TPA. Mutants G62A/F209I, G62A/F249R, and G62A/L90F displayed between ~2.3-fold and ~3.0-fold enhancement in PET degradation overTf C, and between ~1.3-fold and ~2.0-fold enhancement over G62A Tf C, which is known to be the currently best-performing mutant/variant of Tf C.
Acknowledgements: AM thanks DBT, India, and HK and BT thank CSIR, India, for doctoral fellowships. MP was a Masters’ thesis student. PG thanks DBT, India, for grant BT/PR/31706/PBD/26/705/2019. The work was mainly conceived and executed by AM under the supervision of PG, and the first draft was written up by AM and then edited by AM and PG. MP and HK worked under the immediate supervision of AM and the overall supervision of PG. MP produced and studied four of the seventeen mutants. HK worked with AM in determining the activities of mutants against BHET and PET. BT participated in discussions about the work, suggested the making of certain mutants, and helped with trouble-shooting in the production of genes/proteins in respect of several mutants.
Declaration of conflicting interests: The authors have no conflicting interests.
Supplementary Data: The seven figures in the supplementary data for this article can be viewed online.