Ke Ding , Zarina Levitskaya , Barindra Sana , Rupali Reddy Pasula , Srinivasaraghavan Kannan , Abdurrahman Adam , Vishnu Vadanan Sundaravadanam , Chandra Verma , Sierin Lim , John F. Ghadessy
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Activity of both enzymes on PET film was inhibited by the K95A mutation, highlighting complex interplay between mutation context and substrate morphology. Further installing the distal R132N and R280A surface charge mutations potentiated activity of V3 on all substrates tested. This variant afforded 100 % degradation of pre-processed bottle-grade PET powder in 3 days at 40<sup>°</sup>C reaction temperature, a 3-fold improvement over <em>Is</em>PETase. Whilst reduction of positive charge on the PETase surface is known to reduce interaction with PET, molecular dynamics simulations suggest this can be offset by context-dependent modulation of active site flexibility, which differentially impacts both hydrolysis of morphologically distinct PET substrates and the concentration-dependent inhibition phenomenon observed for PETase.</p></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Modulation of PETase active site flexibility and activity on morphologically distinct polyethylene terephthalate substrates by surface charge engineering\",\"authors\":\"Ke Ding , Zarina Levitskaya , Barindra Sana , Rupali Reddy Pasula , Srinivasaraghavan Kannan , Abdurrahman Adam , Vishnu Vadanan Sundaravadanam , Chandra Verma , Sierin Lim , John F. 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引用次数: 0
摘要
酶水解聚对苯二甲酸乙二醇酯(PET)废料是对一种主要污染物进行环境友好型回收利用的一项引人注目的策略。在这里,我们研究了嗜中性 PET 降解酶 IsPETase 活性位点近端和远端的表面电荷点突变的影响,以及具有更高活性的恒温 V3 变体。近端 K95A 突变显著抑制了机械加工 PET 粉末上 IsPET 酶的活性。相反,这种突变明显增加了 V3 PET 酶对 PET 粉的水解作用。K95A 突变抑制了两种酶在 PET 薄膜上的活性,突显了突变背景和底物形态之间复杂的相互作用。进一步安装远端 R132N 和 R280A 表面电荷突变可增强 V3 在所有测试底物上的活性。在 40°C 的反应温度下,该变体可在 3 天内 100% 降解预处理的瓶级 PET 粉末,比 IsPETase 提高了 3 倍。虽然已知 PET 酶表面正电荷的减少会降低与 PET 的相互作用,但分子动力学模拟表明,这可以被活性位点灵活性的上下文依赖性调节所抵消,这种调节会对形态各异的 PET 底物的水解以及 PET 酶观察到的浓度依赖性抑制现象产生不同的影响。
Modulation of PETase active site flexibility and activity on morphologically distinct polyethylene terephthalate substrates by surface charge engineering
Enzymatic hydrolysis of polyethylene terephthalate (PET) waste is a compelling strategy for environmentally friendly recycling of a major pollutant. Here, we investigate the effects of surface charge point mutations both proximal and distal to the active site of the mesophilic PET-degrading enzyme IsPETase and the thermostable V3 variant with superior activity. The vicinal K95A mutation significantly inhibited IsPETase activity on mechanically processed PET powder. Conversely, this mutation significantly increased hydrolysis of PET powder in the V3 PETase. Activity of both enzymes on PET film was inhibited by the K95A mutation, highlighting complex interplay between mutation context and substrate morphology. Further installing the distal R132N and R280A surface charge mutations potentiated activity of V3 on all substrates tested. This variant afforded 100 % degradation of pre-processed bottle-grade PET powder in 3 days at 40°C reaction temperature, a 3-fold improvement over IsPETase. Whilst reduction of positive charge on the PETase surface is known to reduce interaction with PET, molecular dynamics simulations suggest this can be offset by context-dependent modulation of active site flexibility, which differentially impacts both hydrolysis of morphologically distinct PET substrates and the concentration-dependent inhibition phenomenon observed for PETase.
期刊介绍:
The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology.
The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields:
Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics
Biosensors and Biodevices including biofabrication and novel fuel cell development
Bioseparations including scale-up and protein refolding/renaturation
Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells
Bioreactor Systems including characterization, optimization and scale-up
Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization
Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals
Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release
Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites
Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation
Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis
Protein Engineering including enzyme engineering and directed evolution.