Your browser doesn't support javascript.
loading
Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.
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.
Affiliation
  • Saunders JW; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Damry AM; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Vongsouthi V; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Spence MA; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Frkic RL; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Gomez C; ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Yates PA; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Matthews DS; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Tokuriki N; Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • McLeod MD; ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
  • Jackson CJ; Michael Smith Laboratories, The University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
Biochemistry ; 63(13): 1663-1673, 2024 Jul 02.
Article in En | MEDLINE | ID: mdl-38885634
ABSTRACT
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.
Subject(s)
Fulltext
Print
XML
PubMed Links
Search on Google

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Burkholderiales Language: En Journal: Biochemistry Year: 2024 Document type: Article
Fulltext
Print
XML
PubMed Links
Search on Google

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Burkholderiales Language: En Journal: Biochemistry Year: 2024 Document type: Article