A cell-free microtiter plate screen for improved [FeFe] hydrogenases.

<h4>Background</h4>[FeFe] hydrogenase enzymes catalyze the production and dissociation of H(2), a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including the...

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Autores principales:	James A Stapleton, James R Swartz
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/21ee64d798ee42d3a1366daff9215b25
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spelling	oai:doaj.org-article:21ee64d798ee42d3a1366daff9215b252021-12-02T20:21:48ZA cell-free microtiter plate screen for improved [FeFe] hydrogenases.1932-620310.1371/journal.pone.0010554https://doaj.org/article/21ee64d798ee42d3a1366daff9215b252010-05-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20479937/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>[FeFe] hydrogenase enzymes catalyze the production and dissociation of H(2), a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms.<h4>Methodology/principal findings</h4>Here we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity approximately 4 times that of the wild-type enzyme.<h4>Conclusions/significance</h4>Our results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment.James A StapletonJames R SwartzPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 5, p e10554 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q James A Stapleton James R Swartz A cell-free microtiter plate screen for improved [FeFe] hydrogenases.
description	<h4>Background</h4>[FeFe] hydrogenase enzymes catalyze the production and dissociation of H(2), a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms.<h4>Methodology/principal findings</h4>Here we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity approximately 4 times that of the wild-type enzyme.<h4>Conclusions/significance</h4>Our results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment.
format	article
author	James A Stapleton James R Swartz
author_facet	James A Stapleton James R Swartz
author_sort	James A Stapleton
title	A cell-free microtiter plate screen for improved [FeFe] hydrogenases.
title_short	A cell-free microtiter plate screen for improved [FeFe] hydrogenases.
title_full	A cell-free microtiter plate screen for improved [FeFe] hydrogenases.
title_fullStr	A cell-free microtiter plate screen for improved [FeFe] hydrogenases.
title_full_unstemmed	A cell-free microtiter plate screen for improved [FeFe] hydrogenases.
title_sort	cell-free microtiter plate screen for improved [fefe] hydrogenases.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/21ee64d798ee42d3a1366daff9215b25
work_keys_str_mv	AT jamesastapleton acellfreemicrotiterplatescreenforimprovedfefehydrogenases AT jamesrswartz acellfreemicrotiterplatescreenforimprovedfefehydrogenases AT jamesastapleton cellfreemicrotiterplatescreenforimprovedfefehydrogenases AT jamesrswartz cellfreemicrotiterplatescreenforimprovedfefehydrogenases
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A cell-free microtiter plate screen for improved [FeFe] hydrogenases.

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