Search for: All records

Abstract BackgroundDepolymerizing polyethylene terephthalate (PET) plastics using enzymes, such as PETase, offers a sustainable chemical recycling route. To enhance degradation, many groups have sought to engineer PETase for faster catalysis on PET and elevated stability. Considerably less effort has been focused toward expressing large quantities of the enzyme, which is necessary for large-scale application and widespread use. In this work, we evaluated severalE. colistrains for their potential to produce soluble, folded, and activeIsPETase, and moved the production to a benchtop bioreactor. As PETase is known to require disulfide bonds to be functional, we screened several disulfide-bond promoting strains ofE. colito produceIsPETase, FAST-PETase and Hot-PETase. ResultsWe found expression in SHuffle T7 Express results in higher active expression ofIsPETase compared to standardE. coliproduction strains such as BL21(DE3), reaching a purified titer of 20 mg enzyme per L of culture from shake flasks using 2xLB medium. We characterized purifiedIsPETase on 4-nitrophenyl acetate and PET microplastics, showing the enzyme produced in the disulfide-bond promoting host has high activity. Using a complex medium with glycerol and a controlled bioreactor,IsPETase titer reached 104 mg per L for a 46-h culture. FAST-PETase was found to be produced at similar levels in BL21(DE3) or SHuffle T7 Express, with purified production reaching 65 mg per L culture when made in BL21(DE3). Hot-PETase titers were greatest in BL21(DE3) reaching 77 mg per L culture. ConclusionsWe provide protein expression methods to produce three important PETase variants. Importantly, forIsPETase, changing expression host, medium optimization and movement to a bioreactor resulted in a 50-fold improvement in production amount with a per cell dry weight productivity of 0.45 mg_PETaseg_CDW⁻¹ h⁻¹, which is tenfold greater than that forK. pastoris. We show that the benefit of using SHuffle T7 Express for expression only extends toIsPETase, with FAST-PETase and Hot-PETase better produced and purified from BL21(DE3), which is unexpected given the number of cysteines present. This work represents a systematic evaluation of protein expression and purification conditions for PETase variants to permit further study of these important enzymes. 

Abstract Designing complex synthetic materials for enzyme immobilization could unlock the utility of biocatalysis in extreme environments. Inspired by biology, we investigate the use of random copolymer brushes as dynamic immobilization supports that enable supra-biological catalytic performance of immobilized enzymes. This is demonstrated by immobilizingBacillus subtilisLipase A on brushes doped with aromatic moieties, which can interact with the lipase through multiple non-covalent interactions. Incorporation of aromatic groups leads to a 50 °C increase in the optimal temperature of lipase, as well as a 50-fold enhancement in enzyme activity. Single-molecule FRET studies reveal that these supports act as biomimetic chaperones by promoting enzyme refolding and stabilizing the enzyme’s folded and catalytically active state. This effect is diminished when aromatic residues are mutated out, suggesting the importance of π-stacking and π-cation interactions for stabilization. Our results underscore how unexplored enzyme-support interactions may enable uncharted opportunities for using enzymes in industrial biotransformations.