Rational mutagenesis of <i>Thermobifida fusca</i> cutinase to modulate the enzymatic degradation of polyethylene terephthalate

Mrigwani, Arpita; Pitaliya, Madhav; Kaur, Harman; Kasilingam, Bharathraj; Thakur, Bhishem; Guptasarma, Purnananda

doi:10.1002/bit.28305

Cited by 9 publications

(8 citation statements)

References 27 publications

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“…[9,10] In order to overcome the relatively slow catalytic rates of the enzymes, the structural and biophysical properties of PEThydrolases are taking center stage. [11][12][13][14][15] Enzyme engineering efforts on promising candidates [13,[16][17][18][19][20] have been intense, and several groups have succeeded in discovering and developing novel PET degrading enzymes that work at elevated rates [17,18,21] and/or have improved thermostability. [17,19] In contrast, fewer studies have focused on how the physical properties of the PET substrate influence the enzymatic degradation process.…”

Section: Introductionmentioning

confidence: 99%

Rate Response of Poly(Ethylene Terephthalate)‐Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase

et al. 2023

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The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (X C ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of X C on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with X C . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (X C � 10 %), but this enzyme was extremely sensitive to increased X C , whereas the enzymes LCC ICCG , LCC, and DuraPETase had higher tolerance to increases in X C and had activity on PET disks having X C of 24.4 %. Microscopy revealed that the X C -tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.

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Section: Introductionmentioning

confidence: 99%

Rate Response of Poly(Ethylene Terephthalate)‐Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase

et al. 2023

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“…TfCut2 G62A/F249R , TfCut2 S121P/D174S/D204P (Li et al, 2022;Mrigwani et al, 2023) Cutinase-like enzyme IsPETase Dura (IsPETase S214H−I168R−W159H−S188Q−R280A−A180I−G165A−Q119Y−L117F−T140D ) ( Y. Cui, Chen, et al, 2021b) Fast-PETase (IsPETase S121E/D186H/R224Q/N233K/R280A ) ( Lu et al, 2022) TS-PETase (IsPETase R280A S121E D186H N233C S282C ) ( Zhong-Johnson, Voigt, and Sinskey, 2021) HotPETase (IsPETase S121E/D186H/R280A/P181V/S207R/S214Y/Q119K/S213E/N233C/S282C/R90T/Q182M/N212K/R224L/S58A/S61V/K95N/M154G/N241C/K252M/T270Q ) (Bell, Smithson, and Kilbride, 2022) ThermoPETase (IsPETase S121E/D186H/R280A ) ( Joo et al, 2018) BhrPETase BhrPETase H184S/F93G/F209I/S213K (X.Q.…”

Section: Tfcut2mentioning

confidence: 99%

Current advances in the structural biology and molecular engineering of PETase

Liu,

Wang,

Yang

et al. 2023

Front. Bioeng. Biotechnol.

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Poly(ethylene terephthalate) (PET) is a highly useful synthetic polyester plastic that is widely used in daily life. However, the increase in postconsumer PET as plastic waste that is recalcitrant to biodegradation in landfills and the natural environment has raised worldwide concern. Currently, traditional PET recycling processes with thermomechanical or chemical methods also result in the deterioration of the mechanical properties of PET. Therefore, it is urgent to develop more efficient and green strategies to address this problem. Recently, a novel mesophilic PET-degrading enzyme (IsPETase) from Ideonella sakaiensis was found to streamline PET biodegradation at 30°C, albeit with a lower PET-degrading activity than chitinase or chitinase-like PET-degrading enzymes. Consequently, the molecular engineering of more efficient PETases is still required for further industrial applications. This review details current knowledge on IsPETase, MHETase, and IsPETase-like hydrolases, including the structures, ligand‒protein interactions, and rational protein engineering for improved PET-degrading performance. In particular, applications of the engineered catalysts are highlighted, including metabolic engineering of the cell factories, enzyme immobilization or cell surface display. The information is expected to provide novel insights for the biodegradation of complex polymers.

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“…It is believed that the optimal temperature of a desired PET hydrolase needs to be >60 or preferably >70°C. Based on this recognition, a few thermophilic enzymes, including the TfH variant (TfCut2) ( Mrigwani et al, 2023 ), LCC variant (LCC ICCG ) ( Tournier et al, 2020 ), Cut190 variant ( Kawai et al, 2022 ), HiC ( Carniel et al, 2021 ), and BhrPETase ( Xi et al, 2021 ), satisfy the requirements for thermostability above 60°C. In addition to thermophilic enzymes, the discovery of Is PETase from the mesophilic bacterium ( Ideonella sakaiensis ) caused mesophilic PET-hydrolysing enzymes to become a research hot spot.…”

Section: Promising Enzymes For Pet Degradationmentioning

confidence: 99%

“…The best enzymatic degradation reactions involving PET are carried out at (or near) PET’s glass transition temperature (~70°C), the temperature at which chains become mobile and accessible to the active site(s) of thermophilic hydrolases ( Kawai, 2020 ). The following enzymes are notable due to their ability to function at temperatures between 60°C and 70°C ( Sonnendecker et al, 2022 ): (i) metagenomically derived leaf branch compost cutinase (LCC) ( Zheng et al, 2022 ); (ii) Humicola insolens cutinase (HiC) ( Carniel et al, 2021 ); (iii) Thermobifida fusca cutinase (TfCut2) ( Mrigwani et al, 2023 ); (iv) PES-H1 (or polyester hydrolase 7, PHL7), a close homolog of LCC that is twice as active as LCC ( Richter et al, 2023 ); and (v) BhrPETase (from bacterium HR29), another close homolog of LCC that shows comparable activity.…”

Section: Promising Enzymes For Pet Degradationmentioning

confidence: 99%

“…MD simulations revealed that the higher flexibility of the substrate binding site of mutants L90A and I213A might lead to increased hydrolysing activities toward cutin. However, a subsequent study showed that the L90A mutant showed ~11.2-fold reduced degradation activity on commercially sourced PET film, but the L90F mutation displayed considerably increased degradation (~1.5-fold increased) activity ( Mrigwani et al, 2023 ). This might be caused by the more efficient binding of PET resulting from the replacement of the hydrophobic phenylalanine residue.…”

Section: Promising Enzymes For Pet Degradationmentioning

confidence: 99%

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Recent advances in the biodegradation of polyethylene terephthalate with cutinase-like enzymes

Sui,

Wang,

Fang

et al. 2023

Front. Microbiol.

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Polyethylene terephthalate (PET) is a synthetic polymer in the polyester family. It is widely found in objects used daily, including packaging materials (such as bottles and containers), textiles (such as fibers), and even in the automotive and electronics industries. PET is known for its excellent mechanical properties, chemical resistance, and transparency. However, these features (e.g., high hydrophobicity and high molecular weight) also make PET highly resistant to degradation by wild-type microorganisms or physicochemical methods in nature, contributing to the accumulation of plastic waste in the environment. Therefore, accelerated PET recycling is becoming increasingly urgent to address the global environmental problem caused by plastic wastes and prevent plastic pollution. In addition to traditional physical cycling (e.g., pyrolysis, gasification) and chemical cycling (e.g., chemical depolymerization), biodegradation can be used, which involves breaking down organic materials into simpler compounds by microorganisms or PET-degrading enzymes. Lipases and cutinases are the two classes of enzymes that have been studied extensively for this purpose. Biodegradation of PET is an attractive approach for managing PET waste, as it can help reduce environmental pollution and promote a circular economy. During the past few years, great advances have been accomplished in PET biodegradation. In this review, current knowledge on cutinase-like PET hydrolases (such as TfCut2, Cut190, HiC, and LCC) was described in detail, including the structures, ligand–protein interactions, and rational protein engineering for improved PET-degrading performance. In particular, applications of the engineered catalysts were highlighted, such as improving the PET hydrolytic activity by constructing fusion proteins. The review is expected to provide novel insights for the biodegradation of complex polymers.

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Rational mutagenesis of Thermobifida fusca cutinase to modulate the enzymatic degradation of polyethylene terephthalate

Cited by 9 publications

References 27 publications

Rate Response of Poly(Ethylene Terephthalate)‐Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase

Rate Response of Poly(Ethylene Terephthalate)‐Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase

Current advances in the structural biology and molecular engineering of PETase

Recent advances in the biodegradation of polyethylene terephthalate with cutinase-like enzymes

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