INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Ting Wu,Huashan Sun,Wenyao Wang,Bin Xie,Zhengjie Wang,Jianqi Lu,Anming Xu,Weiliang Dong,Jie Zhou,Min Jiang
Enzymatic PET recycling has emerged as a promising green solution in addition to mechanical recycling, but low soluble expression levels of the inherently hydrophobic PET hydrolases hinder large-scale applications. Here, we propose a novel strategy for enhanced production of FastPETase in Escherichia coli using co-expression of molecular chaperones from Ideonella sakaiensis . Co-expression of cognate DnaK and DnaJ chaperones significantly increased soluble FastPETase expression (up to 2.5-fold), surpassing commercial chaperone plasmids. Furthermore, a combinatorial approach employing co-expression of DnaK/DnaJ chaperones and fusion of FastPETase with the VNp6-tag significantly boosted FastPETase secretion, yielding over 2 g/L of target protein in a 5-l bioreactor. Notably, the crude FastPETase in fermentation broth displayed comparable PET hydrolysis effects to the purified enzyme. This work not only provides new insights into the process of chaperones in protein folding but also suggests a novel and efficient strategy for producing recombinant proteins.
研究领域:
生物化学
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环境科学
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蛋白质工程
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PubMed
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
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2024
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IF 12.2
Lizhu Aer,Qifa Jiang,Linling Zhong,Qiuyue Si,Xianghong Liu,Yan Pan,Juan Feng,Hongjuan Zeng,Lixia Tang
Although many efforts have been devoted to the modification of polyethylene terephthalate (PET) hydrolases for improving the efficiency of PET degradation, the catalytic performance of these enzymes at near-ambient temperatures remains a challenge. Herein, a multi-enzyme cascade system (PT-EC) was developed and validated by assembling three well-developed PETases, PETase EHA , Fast-PETase, and Z1-PETase, respectively, together with carboxylesterase TfCa, and hydrophobic binding module CBM3a using scaffold proteins . The resulting PT-EC EHA , PT-EC FPE , PT-EC ZPE all demonstrated outstanding PET degradation efficacy. Notably, PT-EC EHA exhibited a 16.5-fold increase in product release compared to PETase EHA , and PT-EC ZPE yielded the highest amount of product. Subsequently, PT-ECs were displayed on the surface of Escherichia coli , respectively, and their degradation efficiency toward three PET types was investigated. The displayed PT-EC EHA exhibited a 20-fold increase in degradation efficiency with PET film compared to the surface-displayed PETase EHA . Remarkably, an almost linear increase in product release was observed for the displayed PT-EC ZPE over a one-week degradation period, reaching 11.56 ± 0.64 mM after 7 days. TfCa I69W/L281Y evolved using a docking-based virtual screening strategy showed a further 2.5-fold increase in the product release of PET degradation. Collectively, these advantages of PT-EC demonstrated the potential of a multi-enzyme cascade system for PET bio-cycling.
研究领域:
生物化学
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环境科学
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合成生物学
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高影响因子
CHINESE JOURNAL OF CHEMICAL ENGINEERING
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2023
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IF 3.8
Renwen Tian,Yan Sun
Extensive use of polyethylene terephthalate (PET) has brought about global environmental problems. A recently reported PET hydrolase (PETase) discovered from Ideonella sakaiensis showed high potential for degrading PET at moderate temperatures, but its activity and stability need further improvement for practical applications. Herein, we proposed to use α-synuclein (αS) as a fusion chaperone and created six PETase-αS fusion enzymes with linkers of different types and lengths. All the fusion enzymes exhibited improved enzymatic performance, presenting 1.5 to 2.6-fold higher activity towards bis-2(hydroxyethyl) terephthalate than PETase, as well as significantly increased stabilities. Fluorescence spectroscopy indicated that the chaperone fusion tightened the overall conformation and resulted in the opening of the substrate binding pocket, which led to the improved thermal stability and catalytic activity of the fusion enzymes. Remarkably, one of the fusion proteins, PETase-[(GS)(EK)] 10 -αS, showed 3.2 to 5.1 times higher PET degradation capability than PETase. The significantly boosted PET degradation performance was not only attributed to the enhanced enzymatic activity and stability, but also possibly due to the binding affinity of the fused αS domain for PET. These findings demonstrated that αS was an effective fusion chaperone for significantly enhancing the enzymatic performance of PETase.
研究领域:
酶学
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生物技术
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环境科学
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蛋白质工程
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高影响因子
BIORESOURCE TECHNOLOGY
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2024
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IF 9.7
Xiao-Qian Chen,De-Ming Rao,Xing-Yu Zhou,Yang Li,Xiao-min Zhao,De-min Kong,Han Xu,Chu-Qi Feng,Lei Wang,Ling-Qia Su,Zheng-Fei Yan,Jing Wu
Biodegradation, particularly via enzymatic degradation, has emerged as an efficient and eco-friendly solution for Poly (ethylene terephthalate) (PET) pollution. The production of PET hydrolases plays a role in the large-scale enzymatic degradation. However, an effective variant, 4Mz, derived from Thermobifida fusca cutinase (Tfu_0883), was previously associated with a significant reduction in yield when compared to the wild-type enzyme. In this study, a novel cell membrane protection strategy was developed to enhance the yield of 4Mz. This approach increased the yield of 4Mz by 18.2-fold from shaken flasks to 3-L bioreactors, reaching a yield of 3.1 g·L −1 , the highest yield of a PET hydrolase described thus far. In addition, the raw culture broth from 4Mz was applied directly for the enzymatic degradation of PET bottles, achieving a 91.2 % degradation rate. These advancements render the large-scale enzymatic degradation of PET more feasible, thus contributing to the more sustainable management of plastic waste.
研究领域:
生物技术
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环境科学
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生物工程
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Guoqiang Zhang,Jieke Du,Chengsong Zhang,Zhiyi Zhao,Yuexing Chen,Mingyu Liu,Jianwei Chen,Guangyi Fan,Li Ma,Shengying Li,Kun Liu
Widespread use of polyethylene terephthalate (PET) plastics and their recycling challenges have led to substantial accumulation of PET wastes in global environments, with inevitable consequences for their entry into the food chains. Recent studies have increasingly documented the ingestion of microplastics by humans through food and beverages. However, the fate of these microplastics within the gastrointestinal tract, particularly the role of the human gut microbiota, remains inadequately understood. To address this knowledge gap, we employed a bioinformatics workflow integrated with functional verification to investigate the PET digestion/degradation capabilities of intestinal microorganisms. This approach identified a novel PET hydrolase—HGMP01 from the human gut metagenome, which exhibits the capacity to hydrolyze PET nanoparticles. Moreover, comprehensive exploration for HGMP01 homologues in the human gut metagenome and metatranscriptome unveil their distribution in diverse intestinal microorganisms. This study provides biochemical evidence for an unforeseen role of human gut microbiome in plastic digestion, thus holding substantial implications for human health.
研究领域:
环境科学
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微生物学
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生物化学
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人类健康
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高影响因子
MOLECULES
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2024
·Vol. 29
·期 6
·1338
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IF 4.2
Zhi Qu,Lin Zhang,Yan Sun
PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETaseD186Nimproved both the activity and thermostability with an increase inTmby 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWTat 30 and 40 °C, respectively. The most stable PETaseD186Vshowed an increase inTmof 12.91 °C over PETaseWT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase.
研究领域:
生物化学
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酶学
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塑料降解
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分子生物学
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高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2022
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IF 4.446
Zixuan Li,Kun Chen,Linling Yu,Qinghong Shi,Yan Sun
Cost-effective and eco-friendly treatment or recycled use of poly (ethylene terephthalate) (PET) is highly desired. Enzymatic PET degradation is considered a promising approach, but currently available PET hydrolases (PETases) have low activity at ambient temperature. Thus, elevating the reaction system temperature in a cost-effective way becomes a key to the economical enzymatic PET degradation. Herein, we proposed solar-driven enzymatic PET degradation with DuraPETase, a stable mutant of PETase, by using Fe 3 O 4 nanoparticles (NPs) as the solar-to-thermal agent. Simulated solar irradiation could elevate the suspension of Fe 3 O 4 NPs from 25 °C to 46 °C for the effective PET degradation. The enzyme immobilized onto Fe 3 O 4 NPs, [email protected] 3 O 4 , had higher stability than free DuraPETase, but a 10-d PET degradation experiments under simulated solar irradiation revealed that free DuraPETase in the presence of Fe 3 O 4 NPs was approximately three times more effective than the [email protected] 3 O 4 system because of the superiority of free DuraPETase in degrading PET. This implies that enzyme immobilization is unnecessary in such a single-use system. By contrast, the DuraPETase-only system showed only 15.5% efficiency of the Fe 3 O 4 -mediated solar-driven system with free DuraPETase. The research demonstrated the potential of the Fe 3 O 4 -mediated solar-driven enzymatic PET degradation strategy for further development in different practical scenarios
研究领域:
环境科学
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材料科学
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生物技术
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可持续化学
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高影响因子
ACS Applied Materials & Interfaces
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2024
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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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产品关联
DOI
PubMed
高影响因子
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