BIORESOURCE TECHNOLOGY
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2023
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IF 11.4
Lizhu Aer,Huiling Qin,Peng Wo,Juan Feng,Lixia Tang
The use of polyethylene terephthalate (PET) results in a significant amount of plastic waste, which poses a threat to the environment and human health. Dual-enzyme system is promising candidate for PET depolymerization. However, its production in Escherichia coli is challenging, especially for secretory expression. Herein, a novel bifunctional dual-enzyme, TfH-FPE, was constructed through fusion of FAST-PETase and TfH. TfH modifies cell membrane permeability via phospholipid degradation, thus facilitating the secretion of TfH-FPE into the medium. After systematic optimization, purified secreted TfH-FPE reached 104 ± 5.2 mg/L, which is 32.5-fold higher than that of the secreted enzyme using a signal peptide. TfH-FPE exhibits remarkable PET depolymerization capacity compared to FAST-PETase, releasing 6-fold more product than FAST-PETase and 2-fold more product than an equimolar enzyme mixture. Collectively, this study explores a novel secretory approach for efficient production of TfH-FPE and provides a valuable tool to promote PET bio-depolymerization via multi-enzyme cascades.
研究领域:
环境科学
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生物技术
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生物化学
·
微生物学
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高影响因子
JOURNAL OF HAZARDOUS MATERIALS
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2026
·
IF 10.6
Zhi Qu,Zongyang Tian,Zehui Guo,Yan Sun
Extensive accumulation of polyethylene terephthalate (PET) plastic waste causes serious pollution to the global environment, and developing superior PET hydrolases is vital for the enzymatic degradation and biorecycling of PET. Here, we propose a “locking” strategy for the rational redesign of FAST-PETase, one of the best-performing PETase variants reported so far, to further improve its performance. The best variant, FAST-PETase DC (A171C/S193C), exhibits 1.9-fold enhanced degradation efficiency compared with FAST-PETase at 50 °C, and a 4.1 °C increase in melting temperature ( T m ). FAST-PETase DC can almost completely depolymerize untreated post-consumer PET film within 3 d at 50 °C with periodic enzyme replenishment, two-fold faster than FAST-PETase. Molecular dynamics simulations reveal that the mutation locks Helix 5 and Loop 10 through a stable disulfide bond, and reduces the flexibility of the mutation sites and their connected regions. The structural changes consequently promote the substrate binding to the enzyme, facilitate the interaction within the catalytic triad, and rigidify the overall structure of the enzyme, leading to the improved degradation efficiency and thermostability. The study underscores the “locking” strategy as an effective way to enzyme redesign, and the engineered FAST-PETase variant is a promising hydrolase for the treatment and recycling of low-to-medium crystallinity PET plastic waste.
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高影响因子
Biomolecules
·
2025
·Vol. 15
·期 6
·888
·
IF 4.8
Jun Yang,Binyang Deng,Pingan Liao,Siyu Lin,Liqi Zheng,Xing Yang,Fei Wang,Chao Zhai,Lixin Ma
Previous reports indicated that self-assembling amphipathic peptide S1v1 (AEAEAHAH)2significantly enhances the soluble expression, thermostability, and activity of the target proteins when fused to them. In order to obtain high-efficiency enzymes for the large-scale degradation of polyethylene terephthalate (PET), this multifunctional peptide was fused to the N- and C-terminus of FAST-PETase, a variant ofIdeonella sakaiensisPETase (IsPETase), with a PT-linker (TTVTTPQTS) harbored between the target protein and the multifunctional peptide. Consistent with previous reports, S1v1 increased the solubility of FAST-PETase slightly. Moreover, it increased the activity of FAST-PETase dramatically. The amount of terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) released from PET substrate after 24 h of digestion at 50°C by fusion enzymes bearing N- and C-terminal S1v1 tag was approximately 2.9- and 4.6-fold that of FAST-PETase, respectively. Furthermore, the optimal temperature and thermostability of the fusion proteins increased in comparison with FAST-PETase. The present study provides a novel strategy to improve the depolymerization efficiency of FAST-PETase.
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高影响因子
ACS Applied Materials & Interfaces
·
2024
·
IF 8.3
Zhi Qu,Yan Sun
The enormous accumulation of poly(ethylene terephthalate) (PET) waste has posed a serious threat to the environment and human health, and biodegradation with PET hydrolase (PETase) can be a possible solution. Herein, we propose site-specifically modifying PETase with amphiphilic polymers to improve the enzyme performance at ambient temperature. For this purpose, we devise a computer-aided strategy to prioritize the conjugation site, and polyethylene glycol (PEG) preparations of 0.55 to 10 kDa are site-specifically conjugated to PETase. The most active conjugate PETase–PEG 5k (PETase-5K) shows an increase of melting temperature (3.88 °C) and significantly improves PET degradation performance (3.5- and 3.1-fold increases at 30 and 40 °C, respectively). Experimental investigation and molecular dynamics simulations reveal that the site-specific PEGylation increases the hydrophobic solvent-accessible surface area and the binding capability to the PET surface, thickens the hydration layer, increases the intramolecular hydrogen bonding, reduces the interactions between water and the conjugated enzyme surface, and rigidifies the enzyme structure via hydrogen bonding and hydrophobic interactions between the polymer and the enzyme, thus leading to improved enzymatic performance of PETase-5K. We further validate the versatility of the site-specific PEGylation in one of the most evolved variants of PETase, FAST-PETase, by 1.8-fold improvement in PET degradation at 30 °C. The presented computer-aided site-specific conjugation strategy has opened a new avenue to enhancing PETase performance at ambient temperature, and the contribution of PEGylation to PETase unraveled in this work laid a foundation for the rational engineering of PET hydrolases.
研究领域:
环境科学
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生物化学
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生物技术
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分子生物学
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材料科学
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高影响因子
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