Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design

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...

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Bibliographic Details
Published in:Biochemistry (Easton) 2024-07, Vol.63 (13), p.1663-1673
Main Authors: Saunders, Jake W., Damry, Adam M., Vongsouthi, Vanessa, Spence, Matthew A., Frkic, Rebecca L., Gomez, Chloe, Yates, Patrick A., Matthews, Dana S., Tokuriki, Nobuhiko, McLeod, Malcolm D., Jackson, Colin J.
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Burkholderiales - enzymology
Burkholderiales - genetics
Burkholderiales - metabolism
depolymerization
Escherichia coli - genetics
Escherichia coli - metabolism
ethylene glycol
hydrolases
Hydrolases - chemistry
Hydrolases - genetics
Hydrolases - metabolism
Ideonella
industrial applications
Models, Molecular
Phthalic Acids - chemistry
Phthalic Acids - metabolism
Polyethylene Terephthalates - chemistry
Polyethylene Terephthalates - metabolism
Protein Engineering - methods
Protein Folding
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Solubility
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Summary: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.
ISSN:0006-2960
1520-4995
1520-4995
DOI:10.1021/acs.biochem.4c00165