Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design

IF 2.9 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemistry Biochemistry Pub Date : 2024-06-17 DOI:10.1021/acs.biochem.4c00165

Jake W. Saunders, Adam M. Damry, Vanessa Vongsouthi, Matthew A. Spence, Rebecca L. Frkic, Chloe Gomez, Patrick A. Yates, Dana S. Matthews, Nobuhiko Tokuriki, Malcolm D. McLeod and Colin J. Jackson*,

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Abstract

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

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通过共识设计提高塑料降解酶 MHETase 的可溶性表达和全细胞活性

来自堺伊甸菌（Ideonella sakaiensis）的对苯二甲酸单（2-羟乙基）水解酶（MHETase）在聚对苯二甲酸乙二醇酯（PET）塑料经酶解聚成单体对苯二甲酸（TPA）和乙二醇（EG）的过程中执行第二步操作。尽管 MHETase 具有潜在的工业和环境应用价值，但其重组表达能力较差一直是其工业应用的障碍。为了克服这一障碍，我们开发了一种测定方法，可对细胞裂解液和全细胞悬浮液中的 MHETase 活性进行中等通量定量，从而筛选出工程变体库。通过共识设计，我们生成了几种改进的变体，它们的全细胞活性比野生型（WT）MHETase 高出 10 倍以上。生化和结构分析表明，这主要是由于蛋白质折叠得到改善，从而增加了可溶性表达。

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来源期刊

Biochemistry Biochemistry 生物-生化与分子生物学

CiteScore

5.50

自引率

3.40%

发文量

336

审稿时长

1-2 weeks

期刊介绍： Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.