open access publication

Preprint, 2023

De novo Design of a Polycarbonate Hydrolase

In: bioRxiv, Page 2023.03.10.532063, 10.1101/2023.03.10.532063

Contributors (7)

Holst, Laura H. [1] Madsen, Niklas G. [1] Toftgård, Freja T. [1] Rønne, Freja [1] Moise, Ioana-Malina (0000-0003-2513-3638) [1] Petersen, Evamaria I (0009-0001-5599-6269) (Corresponding author) [1] Fojan, Peter (0000-0002-0626-4766) (Corresponding author) [1]


  1. [1] Aalborg University
  2. [NORA names: AAU Aalborg University; University; Denmark; Europe, EU; Nordic; OECD]


Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural α/β-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the α/β-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. This has been used to design an enzyme that can catalyze the hydrolysis of polycarbonate, which no known natural enzymes can degrade. Rosetta enzyme design is used to introduce a catalytic triad into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in E. coli. CD spectroscopy suggests that the design has a melting temperature of >95°C. Activity towards a commercially used polycarbonate (Makrolon 2808) was confirmed using AFM, which showed that a PCase had been designed successfully.


AFM, CD spectroscopy, E. coli, Hydrolysis of polycarbonate, PCase, activity, approach, catalytic activity, catalytic triad, coli, computational evaluation, de novo design, degradation, degrading enzymes, design, different proteins, engineering approach, enzymatic degradation, enzyme, enzyme design, evaluation, exists, hydrolase, hydrolysis, interest, likely exists, melting temperature, method, mutations, natural enzymes, novo design, plastic, plastic degrading enzymes, polycarbonate, protein, scaffolds, sequence space, set, space, spectroscopy, temperature, thermostability, thermostable enzymes, triad, use, β fold