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*,
{"title":"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","authors":"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*, ","doi":"10.1021/acs.biochem.4c00165","DOIUrl":null,"url":null,"abstract":"<p >The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from <i>Ideonella sakaiensis</i> 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.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design\",\"authors\":\"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*, \",\"doi\":\"10.1021/acs.biochem.4c00165\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from <i>Ideonella sakaiensis</i> 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.</p>\",\"PeriodicalId\":28,\"journal\":{\"name\":\"Biochemistry Biochemistry\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.9000,\"publicationDate\":\"2024-06-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochemistry Biochemistry\",\"FirstCategoryId\":\"1\",\"ListUrlMain\":\"https://pubs.acs.org/doi/10.1021/acs.biochem.4c00165\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry Biochemistry","FirstCategoryId":"1","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.biochem.4c00165","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design
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.
期刊介绍:
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