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Rational mutagenesis of Thermobifida fusca cutinase to modulate the enzymatic degradation of polyethylene terephthalate
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  • Arpita Mrigwani,
  • Madhav Pitaliya,
  • Harman Kaur,
  • Bhishem Thakur,
  • Purnananda Guptasarma
Arpita Mrigwani
Indian Institute of Science Education and Research Mohali

Corresponding Author:mrigwaniarpita@gmail.com

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Madhav Pitaliya
Indian Institute of Science Education and Research Mohali
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Harman Kaur
Indian Institute of Science Education and Research Mohali
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Bhishem Thakur
Indian Institute of Science Education and Research Mohali
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Purnananda Guptasarma
Indian Institute of Science Education and Research Mohali
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Abstract

Thermobifida fusca cutinase ( TfC ) is a carboxylesterase (CE) that degrades the environmental pollutant, polyethylene terephthalate (PET). TfC also acts upon PET’s degradation intermediates (DIs), such as oligoethylene terephthalate (OET), and bis-/mono-hydroxyethyl terephthalate (BHET/MHET), to convert these into terephthalic acid (TPA), the terminal product of PET degradation. We examined TfC’s surface, compared it to that of other CEs, and performed molecular docking and MD simulations with an OET, 2HE-(MHET) 3, to understand interactions between TfC’s surface and the OET, at TfC’s active site as well as vicinal regions. We mutated 17 residues on TfC’s surface, mostly individually, but sometimes using pairs of mutations, to see how these modulate TfC’s activity. Most mutants/variants showed a decrease in activity against solid PET. Some killed activity completely. However, three mutations (L90F, F209I and F249R), made using a background mutation (G62A) already reported to improve activity by almost ~2.0-fold, yielded increases in activity that were between ~1.3- and ~2.0-fold higher than that of G62A TfC (which we found to display a ~1.7-fold increase in activity over TfC, in our own experiments). TfC variants, G62A/F249R, and G62A/F209I, exhibit the highest activities yet observed in any TfC mutants/variants, against PET, and BHET, respectively.
07 Jul 2022Submitted to Biotechnology and Bioengineering
08 Jul 2022Submission Checks Completed
08 Jul 2022Assigned to Editor
17 Jul 2022Reviewer(s) Assigned
14 Aug 2022Review(s) Completed, Editorial Evaluation Pending
14 Aug 2022Editorial Decision: Revise Major
07 Nov 20221st Revision Received
07 Nov 2022Assigned to Editor
07 Nov 2022Submission Checks Completed
07 Nov 2022Review(s) Completed, Editorial Evaluation Pending
13 Nov 2022Reviewer(s) Assigned
04 Dec 2022Editorial Decision: Revise Minor
09 Dec 20222nd Revision Received
09 Dec 2022Assigned to Editor
09 Dec 2022Submission Checks Completed
09 Dec 2022Review(s) Completed, Editorial Evaluation Pending
11 Dec 2022Editorial Decision: Accept
22 Dec 2022Published in Biotechnology and Bioengineering. 10.1002/bit.28305