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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 mgPETasegCDW−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.
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Characterization and effect of metal ions on the formation of the Thermus thermophilus Sco mixed disulfide intermediate
Abstract The Sco protein fromThermus thermophilushas previously been shown to perform a disulfide bond reduction in the CuAprotein fromT. thermophilus, which is a soluble protein engineered from subunit II of cytochromeba3oxidase that lacks the transmembrane helix. The native cysteines onTtSco andTtCuAwere mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine inTtCuAand subsequent release upon incubation with the complementaryTtSco protein demonstrated the formation of the mixed disulfide intermediate. The cysteine ofTtSco that attacks the disulfide bond in the targetTtCuAprotein was determined to beTtSco Cysteine 49. This cysteine is likely more reactive than Cysteine 53 due to a higher degree of solvent exposure. Removal of the metal binding histidine, His 139, does not change MDI formation. However, altering the arginine adjacent to the reactive cysteine in Sco (Arginine 48) does alter the formation of the MDI. Binding of Cu2+or Cu+toTtSco prior to reaction withTtCuAwas found to preclude formation of the mixed disulfide intermediate. These results shed light on a mechanism of disulfide bond reduction by theTtSco protein and may point to a possible role of metal binding in regulating the activity. ImportanceThe function of Sco is at the center of many studies. The disulfide bond reduction in CuAby Sco is investigated herein and the effect of metal ions on the ability to reduce and form a mixed disulfide intermediate are also probed.
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- Award ID(s):
- 1726441
- PAR ID:
- 10076846
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 27
- Issue:
- 11
- ISSN:
- 0961-8368
- Page Range / eLocation ID:
- p. 1942-1954
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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