RESUMO
The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.
Assuntos
Burkholderiales , Burkholderiales/enzimologia , Burkholderiales/genética , Burkholderiales/metabolismo , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/química , Hidrolases/metabolismo , Hidrolases/genética , Hidrolases/química , Solubilidade , Polietilenotereftalatos/metabolismo , Polietilenotereftalatos/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Engenharia de Proteínas/métodos , Dobramento de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Modelos MolecularesRESUMO
The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.
Assuntos
Burkholderiales , Hidrolases , Hidrolases/química , Burkholderiales/genética , Burkholderiales/metabolismo , Domínio Catalítico , Mutação , Polietilenotereftalatos/metabolismoRESUMO
Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.