Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.

Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarr...

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Autores principales:	Marie Couturier, Julia Féliu, Sophie Bozonnet, Alain Roussel, Jean-Guy Berrin
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0201bf2255274d2f88432d2a516cb6ab
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spelling	oai:doaj.org-article:0201bf2255274d2f88432d2a516cb6ab2021-11-18T08:45:04ZMolecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.1932-620310.1371/journal.pone.0079800https://doaj.org/article/0201bf2255274d2f88432d2a516cb6ab2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24278180/?tool=EBIhttps://doaj.org/toc/1932-6203Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat /KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat /KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).Marie CouturierJulia FéliuSophie BozonnetAlain RousselJean-Guy BerrinPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 11, p e79800 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marie Couturier Julia Féliu Sophie Bozonnet Alain Roussel Jean-Guy Berrin Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.
description	Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in kcat /KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in kcat /KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).
format	article
author	Marie Couturier Julia Féliu Sophie Bozonnet Alain Roussel Jean-Guy Berrin
author_facet	Marie Couturier Julia Féliu Sophie Bozonnet Alain Roussel Jean-Guy Berrin
author_sort	Marie Couturier
title	Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.
title_short	Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.
title_full	Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.
title_fullStr	Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.
title_full_unstemmed	Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.
title_sort	molecular engineering of fungal gh5 and gh26 beta-(1,4)-mannanases toward improvement of enzyme activity.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/0201bf2255274d2f88432d2a516cb6ab
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Molecular engineering of fungal GH5 and GH26 beta-(1,4)-mannanases toward improvement of enzyme activity.

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