Structural and functional characterization of an auxiliary domain-containing PET hydrolase from Burkholderiales bacterium

Sagong, Hye-Young; Kim, Seong–Min; Lee, Dong‐Hoon; Hong, Hwaseok; Lee, Seul Hoo; Seo, Hogyun; Kim, Kyungjin

doi:10.1016/j.jhazmat.2022.128267

Cited by 27 publications

(12 citation statements)

References 47 publications

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“…Still, more mutations would be encouraged for the MD simulation to help understand other PETase variants and consolidate the simulation protocol. Furthermore, not limited to PETase, our protocol could act as a preliminary guide to identify potential mutation sites of other PET-degrading enzymes, such as BbPETase from bacterium Burkholderiales [ 56 ], and MoPE from bacterium Moraxella sp. [ 57 ].…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…21,50 Pb -PETase and Bb -PETase have been used as purified enzymes for the degradation of PET. 5,51,52 Sequence analysis showed that the protein sequence of Bb -PETase was longer on the N-terminus than other PETases (Fig. S1†) and a truncated enzyme has been reported.…”

Section: Resultsmentioning

confidence: 95%

“…This likely reflects the propensity of Ad -PETase to degrade aliphatic polymers and Bb -PETase has been described to primarily degrade PET to MHET, rather than TPA. 52,53 While the background hydrolysis of BHET increased between pH 6.0 and 9.0, generally no significant effect of pH on enzyme activity was observed. At 50 °C, Ad -PETase and Bb -PETase showed little if any activity above background levels, while for Pb -PETase and short Bb -PETase yields (by HPLC) dropped to 38% and 67% respectively.…”

Section: Resultsmentioning

confidence: 97%

“…S1 † ) and a truncated enzyme has been reported. 52 Here, 141 amino-acid residues were removed from the N-terminus, and the resulting subcloned gene named short Bb -PETase. Ad -PETase has been highlighted as a potential PETase, and is known to degrade poly(tetramethylene succinate), but to the best of our knowledge it has not been described to degrade PET and was therefore included.…”

Section: Resultsmentioning

confidence: 99%

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Mechanoenzymatic reactions for the hydrolysis of PET

et al. 2023

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“…IsPETase produced by the bacteria can hydrolyze PET into small molecules at moderate temperature. In addition, several PETase-like enzymes were determined through phylogenetic tree analysis, such as the RgPETase from Rhizobacter gummiphilus [16], the PbPETase from Polyangium brachysporum, and the BbPETase from Burkholderiales bacterium [17]. Of these, BbPETase differs from other type IIb enzymes in having an extra 143 amino acids (AAs) at the N-terminus.…”

Section: Introductionmentioning

confidence: 99%

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation

2023

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Polyethylene terephthalate (PET) is one of the most prevalent transparent thermoplastics. It is commonly utilized due to its low cost and high durability. With the massive accumulation of waste PET, however, serious environmental pollution has become a global problem. Compared to traditional chemical degradation, biodegradation of PET catalyzed by PET hydrolase (PETase) is more environmentally friendly and energy-efficient. BbPETaseCD from the Burkholderiales bacterium is a PETase that shows favorable properties for application in the biodegradation of PET. To enhance the enzymatic performance of this enzyme, this work focuses on the rational design of disulfide bridges in BbPETaseCD. We utilized two computational algorithms to predict the probable disulfide-bridge mutations in BbPETaseCD, and five variants were acquired from the computations. Among these, the N364C/D418C variant with one additional disulfide bond showed higher expression than the wild-type enzyme (WT) and the best enzymatic performance. The melting temperature (Tm) of the N364C/D418C variant presented an increase of 14.8 °C over that of WT (56.5 °C), indicating that the additional disulfide bond significantly raised the thermodynamic stability of the enzyme. Kinetic experiments at different temperatures also demonstrated the thermal stability increase of the variant. The variant also showed significantly increased activity over WT when using bis(hydroxyethyl) terephthalate (BHET) as the substrate. More remarkably, the N364C/D418C variant exhibited approximately an 11-fold increase over the WT enzyme in the long-term (14 days) degradation of PET films. The results prove that the rationally designed disulfide bond significantly improved the enzymatic performance of the enzyme for PET degradation.

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Structural and functional characterization of an auxiliary domain-containing PET hydrolase from Burkholderiales bacterium

Cited by 27 publications

References 47 publications

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

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

Mechanoenzymatic reactions for the hydrolysis of PET

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation

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