Engineering and evaluation of thermostable IsPETase variants for PET degradation.
Engineering and evaluation of thermostable IsPETase variants for PET degradation.
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用于PET降解的耐热IsPETase变体的工程和评估。
DOI：
10.1002/elsc.202100105
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发表时间：
2022-03
影响因子：
2.7
通讯作者：
Bornscheuer UT
中科院分区：
文献类型：
--
作者：
Brott S;Pfaff L;Schuricht J;Schwarz JN;Böttcher D;Badenhorst CPS;Wei R;Bornscheuer UT
聚对苯二甲酸乙二醇酯（PET）是一种大规模生产的石油基合成聚合物。使用例如Ideonella sakaiensis PETase（IsPETase）的酶促PET降解可以是PET化学回收的更环保和节能的替代方案。然而，IsPETase是一种嗜温酶，其最佳反应温度低于PET的玻璃化转变温度（Tg），在该温度下，无定形聚合物可以容易地用于酶分解。在这项研究中，我们使用易错PCR来生成基于IsPETase的热稳定三重突变体（TM）的突变体文库。针对市售聚酯-聚氨酯Impranil DLN W 50筛选文库中更耐热的IsPETase变体，产生具有更高熔点的四种变体。最有前途的IsPETaseTMK 95 N/F201 I变体的熔点比IsPETaseTM高5.0°C。尽管该变体在较低的孵育温度下对PET显示出略低的活性，但其增加的热稳定性使其在高达60°C的较高反应温度下成为更有活性的PET水解酶。几种其他变体进行了比较，并与选定的先前发表的IsPETase突变体在热稳定性和对PET纳米颗粒和无定形PET膜的水解活性方面相结合。我们的研究结果表明，热稳定性是一个有效的PET水解酶的最重要的特征之一。
Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (T g) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error‐prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase.
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影响因子：
6
作者：
LANGEVIN, D;GRENET, J;SAITER, JM
通讯作者：
SAITER, JM
影响因子：
6.4
作者：
Moog, Daniel;Schmitt, Johanna;Maier, Uwe G.
通讯作者：
Maier, Uwe G.
DOI：
10.1098/rstb.2008.0205
发表时间：
2009-07-27
影响因子：
6.3
作者：
Barnes, David K. A.;Galgani, Francois;Barlaz, Morton
通讯作者：
Barlaz, Morton
影响因子：
5.5
作者：
Acero, Enrique Herrero;Ribitsch, Doris;Guebitz, Georg
通讯作者：
Guebitz, Georg
对聚乙二醇乙酸乙酯）降解的分子机制的结构洞察力。
影响因子：
16.6
作者：
Joo S;Cho IJ;Seo H;Son HF;Sagong HY;Shin TJ;Choi SY;Lee SY;Kim KJ
通讯作者：
Kim KJ