Thermal stabilization of the deglycating enzyme Amadoriase I by rational design

Abstract Amadoriases are a class of FAD-dependent enzymes that are found in fungi, yeast and bacteria and that are able to hydrolyze glycated amino acids, cleaving the sugar moiety from the amino acidic portion. So far, engineered Amadoriases have mostly found practical application in the measuremen...

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Autores principales:	Federica Rigoldi, Stefano Donini, Francesca Giacomina, Federico Sorana, Alberto Redaelli, Tiziano Bandiera, Emilio Parisini, Alfonso Gautieri
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6eefa2ae873945d49454695efac1e953
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spelling	oai:doaj.org-article:6eefa2ae873945d49454695efac1e9532021-12-02T15:08:28ZThermal stabilization of the deglycating enzyme Amadoriase I by rational design10.1038/s41598-018-19991-x2045-2322https://doaj.org/article/6eefa2ae873945d49454695efac1e9532018-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-19991-xhttps://doaj.org/toc/2045-2322Abstract Amadoriases are a class of FAD-dependent enzymes that are found in fungi, yeast and bacteria and that are able to hydrolyze glycated amino acids, cleaving the sugar moiety from the amino acidic portion. So far, engineered Amadoriases have mostly found practical application in the measurement of the concentration of glycated albumin in blood samples. However, these engineered forms of Amadoriases show relatively low absolute activity and stability levels, which affect their conditions of use. Therefore, enzyme stabilization is desirable prior to function-altering molecular engineering. In this work, we describe a rational design strategy based on a computational screening method to evaluate a library of potentially stabilizing disulfide bonds. Our approach allowed the identification of two thermostable Amadoriase I mutants (SS03 and SS17) featuring a significantly higher T 50 (55.3 °C and 60.6 °C, respectively) compared to the wild-type enzyme (52.4 °C). Moreover, SS17 shows clear hyperstabilization, with residual activity up to 95 °C, whereas the wild-type enzyme is fully inactive at 55 °C. Our computational screening method can therefore be considered as a promising approach to expedite the design of thermostable enzymes.Federica RigoldiStefano DoniniFrancesca GiacominaFederico SoranaAlberto RedaelliTiziano BandieraEmilio ParisiniAlfonso GautieriNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-14 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Federica Rigoldi Stefano Donini Francesca Giacomina Federico Sorana Alberto Redaelli Tiziano Bandiera Emilio Parisini Alfonso Gautieri Thermal stabilization of the deglycating enzyme Amadoriase I by rational design
description	Abstract Amadoriases are a class of FAD-dependent enzymes that are found in fungi, yeast and bacteria and that are able to hydrolyze glycated amino acids, cleaving the sugar moiety from the amino acidic portion. So far, engineered Amadoriases have mostly found practical application in the measurement of the concentration of glycated albumin in blood samples. However, these engineered forms of Amadoriases show relatively low absolute activity and stability levels, which affect their conditions of use. Therefore, enzyme stabilization is desirable prior to function-altering molecular engineering. In this work, we describe a rational design strategy based on a computational screening method to evaluate a library of potentially stabilizing disulfide bonds. Our approach allowed the identification of two thermostable Amadoriase I mutants (SS03 and SS17) featuring a significantly higher T 50 (55.3 °C and 60.6 °C, respectively) compared to the wild-type enzyme (52.4 °C). Moreover, SS17 shows clear hyperstabilization, with residual activity up to 95 °C, whereas the wild-type enzyme is fully inactive at 55 °C. Our computational screening method can therefore be considered as a promising approach to expedite the design of thermostable enzymes.
format	article
author	Federica Rigoldi Stefano Donini Francesca Giacomina Federico Sorana Alberto Redaelli Tiziano Bandiera Emilio Parisini Alfonso Gautieri
author_facet	Federica Rigoldi Stefano Donini Francesca Giacomina Federico Sorana Alberto Redaelli Tiziano Bandiera Emilio Parisini Alfonso Gautieri
author_sort	Federica Rigoldi
title	Thermal stabilization of the deglycating enzyme Amadoriase I by rational design
title_short	Thermal stabilization of the deglycating enzyme Amadoriase I by rational design
title_full	Thermal stabilization of the deglycating enzyme Amadoriase I by rational design
title_fullStr	Thermal stabilization of the deglycating enzyme Amadoriase I by rational design
title_full_unstemmed	Thermal stabilization of the deglycating enzyme Amadoriase I by rational design
title_sort	thermal stabilization of the deglycating enzyme amadoriase i by rational design
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/6eefa2ae873945d49454695efac1e953
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Thermal stabilization of the deglycating enzyme Amadoriase I by rational design

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