Directional-path modification strategy enhances PET hydrolase catalysis of plastic degradation

Chen, Xiaoqian; Guo, Zhiyong; Wang, Lei; Yan, Zheng‐Fei; Jin, Chang-Xu; Huang, Qingsong; Kong, Demin; Rao, Deming; Wu, Jing

doi:10.1016/j.jhazmat.2022.128816

Cited by 46 publications

(24 citation statements)

References 37 publications

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“…The value and significance of this method has been demonstrated by several studies that successfully generated improved variants of PET degrading enzymes using MD simulations. 14 , 58 , 59 , 60 These studies not only confirm a mode of interaction comparable to the one we observe, with a strong proportion of aromatic interactions and simultaneous participation of polar interactions, but also illustrate for example the diverse binding of the substrate by means of several binding modes. 16 …”

Section: Resultssupporting

confidence: 85%

“…Recently a number of exciting articles have been published reporting improved catalytic activities of either IS, PET2, or LCC. 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 A variety of strategies targeting sequence‐based knowledge, substrate‐binding or the catalytic mechanism have been used including machine‐learning or directed evolution that resulted in enhanced degradation capabilities thereby emphasizing the high potential of engineered PET‐hydrolases. In fact, Tournier et al already presented a proof of concept for enzymatic PET recycling employing engineered variants of LCC.…”

Section: Introductionmentioning

confidence: 99%

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Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

et al. 2022

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The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three‐fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environments.

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Section: Resultssupporting

confidence: 85%

Section: Introductionmentioning

confidence: 99%

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

et al. 2022

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“…Tournier et al ( 2020 ) reported that mutating Y127 in LCC (corresponding to Q138 of Cut190) to glycine (smaller than alanine) improved the thermostability of the enzyme. Chen et al ( 2022 ) compared Q92 mutants of T. fusca cutinase (corresponding to Q138 of Cut190), resulting in the higher activity of Q92G than Q92A. Cui et al ( 2021 ) reported the efficiency of L136F/Q138Y (expressed as corresponding positions in Cut190) introduced in PETase from Ideonella sakaiensis ( Is PETase) (DuraPETase).…”

Section: Resultsmentioning

confidence: 99%

“…Tournier et al ( 2020 ) replaced the tyrosine of LCC with glycine, not alanine, and no comparison of glycine and alanine was made. Q92 of T. fusca cutinase was replaced with serine, alanine and glycine, the best performance being Q92G (Chen et al 2022 ). The Q138 of Cut190 is positioned in the direction of a polymer chain extension from the active site cleft (Kawabata et al 2017 ), and the replacement of Q138 with a small amino acid, such as alanine or glycine, may improve the extension of the polymer chain.…”

Section: Discussionmentioning

confidence: 99%

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

et al. 2022

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The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day. Graphical Abstract

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“…In addition, these enzymes have been further optimized and modified by protein engineering [ 13 ]. For example, introducing a disulfide bond (D204C/E253C) at the calcium binding site [ 14 ] or mutations to remodel the binding groove of TfCut2 [ 15 , 16 ], introducing mutations (S226P, S226P/R228S) to increase Ca 2+ -binding sites on the protein surface of Cut190 [ 11 , 17 ], and introducing a disulfide bond (D238C/S283C) of LCC [ 18 ], showed an improvement of the enzyme catalytic efficiency and stability. However, the uses of these enzymes are limited because of their low enzymatic activities [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

In Silico Identification of Potential Sites for a Plastic-Degrading Enzyme by a Reverse Screening through the Protein Sequence Space and Molecular Dynamics Simulations

et al. 2022

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The accumulation of polyethylene terephthalate (PET) seriously harms the environment because of its high resistance to degradation. The recent discovery of the bacteria-secreted biodegradation enzyme, PETase, sheds light on PET recycling; however, the degradation efficiency is far from practical use. Here, in silico alanine scanning mutagenesis (ASM) and site-saturation mutagenesis (SSM) were employed to construct the protein sequence space from binding energy of the PETase–PET interaction to identify the number and position of mutation sites and their appropriate side-chain properties that could improve the PETase–PET interaction. The binding mechanisms of the potential PETase variant were investigated through atomistic molecular dynamics simulations. The results show that up to two mutation sites of PETase are preferable for use in protein engineering to enhance the PETase activity, and the proper side chain property depends on the mutation sites. The predicted variants agree well with prior experimental studies. Particularly, the PETase variants with S238C or Q119F could be a potential candidate for improving PETase. Our combination of in silico ASM and SSM could serve as an alternative protocol for protein engineering because of its simplicity and reliability. In addition, our findings could lead to PETase improvement, offering an important contribution towards a sustainable future.

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Directional-path modification strategy enhances PET hydrolase catalysis of plastic degradation

Cited by 46 publications

References 37 publications

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes

Efficient depolymerization of polyethylene terephthalate (PET) and polyethylene furanoate by engineered PET hydrolase Cut190

In Silico Identification of Potential Sites for a Plastic-Degrading Enzyme by a Reverse Screening through the Protein Sequence Space and Molecular Dynamics Simulations

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