Efficient polyethylene terephthalate biodegradation by an engineered Ideonella sakaiensis PETase with a fixed substrate-binding W156 residue

Abstract

The S92P/D157A variant of Ideonella sakaiensis PETase (IsPETase) showed significantly enhanced thermostability and PET degradation activity. The W156 residue of the variant was fixed in the substrate-binding conformation.

Enhancing PET Degrading Enzymes: A Combinatory Approach

“…Because the crystal structure of the S121P/D186A double mutant could not resolve various conformations of W185 side chain, the authors explained the increased activity was caused by a reduction in the flexibility. [38] However, the authors did not note that this is also common in other crystal structures of PsPETase variants, and that the different conformations of the so-called wobbly tryptophan W185 is not always resolved in multiple conformations. Nailing down all the possible conformations is difficult-seeing that density is missing is easy.…”

Enhancing the biodegradation of bis(2‐hydroxyethyl) terephthalate by an IsPETasePA and MHETase dual‐enzyme system

“…Escherichia coli BL21 (DE3) (TransGen Biotech, Beijing, China) was used as the host for over-expression of IsPETase, leaf-branch compost cutinase (LCC), T. fusca carboxylesterase (TfCa), LCC-ICCG, IsPETase PA and MHETase. 24 The strains in this study were cultivated in Luria broth (LB) medium (10 g L −1 tryptone, 5 g L −1 yeast extract and 10 g L −1 sodium chloride) containing ampicillin (Amp, 100 mg L −1 ) at 37 °C and 220 rpm. Cliscent Technology Co. Ltd (Beijing, China) kindly provided liquid preparations of Lipozyme CALB L.…”

“…The 3D structure is obtained from the protein database. IsPETase PA is an A-chain crystal structure with 1.97 Å resolution (PDB: 8J17) 24 and MHETase is an A-chain crystal structure with 1.8 Å resolution (PDB: 6QZ2). 28 Free water was removed.…”

“…The catalytic pathways of IsPE-Tase PA and MHETase operate through similar mechanisms. 24 They involve Ser-His proton transfer, leading to a nucleophilic attack on the carbonyl group of the substrate, and follow the classical mechanism of action of ⊍/⊎hydrolases. [34][35][36] In addition, it can be seen from Fig.…”

“…23 Recently, our group conducted semi-saturation mutation toward the key residues of IsPETase and obtained a S92P/D157A (IsPETase PA ) variant with 9.9-fold higher activity over IsPETase WT at 40 °C. 24 Besides, the complicated operation and high cost of protein purification limit the industrial application of enzymes; thus, whole cells or crude enzyme extracts without prior purification are popular in practical use. 25,26 Therefore, in this study, we applied a IsPETase PA /MHETase dualenzyme synergistic system, focusing on the optimal modulator conditions for BHET degradation, and the crude enzyme extracts were adopted for convenience.…”