Title: Balance-directed protein engineering of IsPETase enhances both PET hydrolysis activity and thermostability Lee SH, Seo H, Hong H, Park J, Ki D, Kim M, Kim HJ, Kim KJ Ref: Biorxiv, :, 2023 : PubMed
A mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low thermostability limits its industrial applications. We herein developed an engineering strategy for IsPETase to enhance PET hydrolysis activity, thermostability, and protein folding of the enzyme. Balance-directed Z1-PETase variant outperforms the stability-directed Z2-PETase variant under both mesophilic and thermophilic conditions, although Z2-PETase exhibits higher thermostability than Z1-PETase. The Z1-PETase is also superior to Fast-PETase, Dura-PETase, and LC-CICCG in terms of depolymerization rate regardless of temperature conditions we tested. Thus, maintaining a balance between PET hydrolysis activity and thermostability is essential for the developmentof high-performance PET hydrolases. In a pH-stat bioreactor, Z1-PETase depolymerized >90% of both transparent and colored post-consumer PET powders within 24 and 8 hours at 40C
and 55C, respectively, demonstrating that the balance-directed IsPETase variant produced herein may be applicable in the bio-recycling of PET.
        
Title: Three-directional engineering of IsPETase with enhanced protein yield, activity, and durability Lee SH, Seo H, Hong H, Park J, Ki D, Kim M, Kim HJ, Kim KJ Ref: J Hazard Mater, 459:132297, 2023 : PubMed
The mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low stability limits its industrial applications. Here, we developed a variant, Z1-PETase, with enhanced soluble protein yield and durability while maintaining or improving activity at lower temperatures. The selected Z1-PETase not only exhibited a 20-fold improvement in soluble protein yield compared to the previously engineered IsPETase(S121E/D186H/S242T/N246D) (4p) variant, but also demonstrated a 30% increase in low-temperature activity at 40 degreesC, along with an 11 degreesC increase in its Tm(D) value. The PET depolymerization test across a temperature range low to high (30-70 degreesC) confirmed that Z1-PETase exhibits high accessibility of mesophilic PET hydrolase and rapid depolymerizing rate at higher temperature in accordance with the thermal behaviors of polymer and enzyme. Additionally, structural interpretation indicated that the stabilization of specific active site loops in Z1-PETase contributes to enhanced thermostability without adversely impacting enzymatic activity. In a pH-stat bioreactor, Z1-PETase depolymerized > 90% of both transparent and colored post-consumer PET powders within 24 and 8 h at 40 degreesC and 55 degreesC, respectively, demonstrating that the utility of this IsPETase variant in the bio-recycling of PET.
        
Representative scheme of Polyesterase-lipase-cutinase structure and an image from PDBsum server
no Image
Databases
PDB-Sum
8H5O Previously Class, Architecture, Topology and Homologous superfamily - PDB-Sum server
FSSP
8H5OFold classification based on Structure-Structure alignment of Proteins - FSSP server