IsPETase Is a Novel Biocatalyst for Poly(ethylene terephthalate) (PET) Hydrolysis.

Poly(ethylene terephthalate) (PET) is one of the most widely used synthetic polyesters, but also a major cause of plastic pollution. Because the chemical degradation of PET would be uneconomical and rather burdensome, considerable efforts have been devoted to exploring enzymatic processes for the disposal of PET waste. Many PET-hydrolyzing enzymes have been reported in recent decades, some of which demonstrate excellent potential for industrial applications. This review sets out to summarize the state of investigation into IsPETase, a cutinase-like enzyme from Ideonella sakaiensis possessing ability to degrade crystalline PET, and to gain further insight into the structure-function relationship of IsPETase. Benefiting from the continuing identification of novel cutinase-like proteins and growing availability of the engineered IsPETase, we may anticipate future developments in this type of enzyme would generate suitable biocatalyst for industrial use.

Kan Y ，He L ，Luo Y ，Bao R ... -  《-》

Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis.

Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70 °C. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETaseS242T and IsPETaseN246D, with higher enzymatic activity at both 25 and 37 °C compared with IsPETaseWT. We also developed the IsPETaseS121E/D186H/S242T/N246D variant by integrating the S242 T and N246D mutations into the previously reported IsPETaseS121E/D186H/R208A variant. At the 37 °C incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETaseWT that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETaseWT.

Son HF ，Joo S ，Seo H ，Sagong HY ，Lee SH ，Hong H ，Kim KJ ... -  《-》

Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis.

Polyethylene terephthalate (PET) hydrolase from Ideonella sakaiensis (IsPETase) can be used to degrade PET. In order to use IsPETase in industry, we studied the enzymatic activity of IsPETase in different conditions containing environmental and physicochemical factors commonly found in nature. We observed that salts and glycerol enhanced the enzymatic activity, while detergents and organic solvents reduced the enzymatic activity. IsPETase hydrolyzed p-nitrophenyl (p-NP) esters instead of naphthyl esters. To make IsPETase an enzyme capable of hydrolyzing naphthyl esters, site-directed mutagenesis was carried out based on the structural information provided by the crystal structure. We found that the IsPETaseS93M, IsPETaseW159F, and IsPETaseN241F mutants can hydrolyze naphthyl esters. IsPETase engineering can direct researchers to use this α/β-hydrolase protein scaffold to design enzymes that can hydrolyze a variety of polyesters.

Liu C ，Shi C ，Zhu S ，Wei R ，Yin CC ... -  《-》

Protein engineering of stable IsPETase for PET plastic degradation by Premuse.

Poly(ethylene terephthalate) (PET) is used widely by human beings, but is very difficult to degrade. Up to now, the PET degradation effect of PETase from Ideonella sakaiensis 201-F6 (IsPETase) variants with low stability and activity was not ideal. In this study, a mutation design tool, Premuse, was developed to integrate the sequence alignment and quantitative selection of the preferred mutations based on natural sequence evolution. Ten single point mutants were selected from 1486 homologous sequences using Premuse, and then two mutations (W159H and F229Y) with improved stability were screened from them. The derived double point mutant, W159H/F229Y, exhibited a strikingly enhanced enzymatic performance. Its Tm and catalytic efficiency values (kcat/Km) respectively increased by 10.4 °C and 2.0-fold using p-NPP as the substrate compared with wild type. The degradation activity for amorphous PET was increased by almost 40-fold in comparison with wild type at 40 °C in 24 h. Additionally, the variant could catalyze biodegradation of PET bottle preform at a mean rate of 23.4 mgPET/h/mgenzyme. This study allowed us to design the mutation more efficiently, and provides a tool for achieving biodegradation of PET pollution under mild natural environments.

Meng X ，Yang L ，Liu H ，Li Q ，Xu G ，Zhang Y ，Guan F ，Zhang Y ，Zhang W ，Wu N ，Tian J ... -  《-》

Assessment of the PETase conformational changes induced by poly(ethylene terephthalate) binding.

Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the β1-β2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the β7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems.

da Costa CHS ，Dos Santos AM ，Alves CN ，Martí S ，Moliner V ，Santana K ，Lameira J ... -  《-》