An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films

Abstract Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measureme...

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Autores principales:	En Ze Linda Zhong-Johnson, Christopher A. Voigt, Anthony J. Sinskey
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/77c84e244cec4422b5875ad9b32fed97
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spelling	oai:doaj.org-article:77c84e244cec4422b5875ad9b32fed972021-12-02T14:01:23ZAn absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films10.1038/s41598-020-79031-52045-2322https://doaj.org/article/77c84e244cec4422b5875ad9b32fed972021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-79031-5https://doaj.org/toc/2045-2322Abstract Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C. This was used to assess the method’s ability to determine relative enzyme kinetics across variants and reveal structure–function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.En Ze Linda Zhong-JohnsonChristopher A. VoigtAnthony J. SinskeyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q En Ze Linda Zhong-Johnson Christopher A. Voigt Anthony J. Sinskey An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
description	Abstract Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C. This was used to assess the method’s ability to determine relative enzyme kinetics across variants and reveal structure–function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.
format	article
author	En Ze Linda Zhong-Johnson Christopher A. Voigt Anthony J. Sinskey
author_facet	En Ze Linda Zhong-Johnson Christopher A. Voigt Anthony J. Sinskey
author_sort	En Ze Linda Zhong-Johnson
title	An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
title_short	An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
title_full	An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
title_fullStr	An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
title_full_unstemmed	An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
title_sort	absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/77c84e244cec4422b5875ad9b32fed97
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An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films

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