Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.

ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have n...

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Autores principales:	Su Ma, Gaofei Duan, Wengang Chai, Cunliang Geng, Yulong Tan, Lushan Wang, Frédéric Le Sourd, Gurvan Michel, Wengong Yu, Feng Han
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/75420a985862409ba16a36af1a5c3f53
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spelling	oai:doaj.org-article:75420a985862409ba16a36af1a5c3f532021-11-18T07:43:36ZPurification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.1932-620310.1371/journal.pone.0064666https://doaj.org/article/75420a985862409ba16a36af1a5c3f532013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23741363/?tool=EBIhttps://doaj.org/toc/1932-6203ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0-10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites -1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites -1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites -1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.Su MaGaofei DuanWengang ChaiCunliang GengYulong TanLushan WangFrédéric Le SourdGurvan MichelWengong YuFeng HanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 5, p e64666 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Su Ma Gaofei Duan Wengang Chai Cunliang Geng Yulong Tan Lushan Wang Frédéric Le Sourd Gurvan Michel Wengong Yu Feng Han Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.
description	ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0-10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites -1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites -1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites -1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.
format	article
author	Su Ma Gaofei Duan Wengang Chai Cunliang Geng Yulong Tan Lushan Wang Frédéric Le Sourd Gurvan Michel Wengong Yu Feng Han
author_facet	Su Ma Gaofei Duan Wengang Chai Cunliang Geng Yulong Tan Lushan Wang Frédéric Le Sourd Gurvan Michel Wengong Yu Feng Han
author_sort	Su Ma
title	Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.
title_short	Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.
title_full	Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.
title_fullStr	Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.
title_full_unstemmed	Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.
title_sort	purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from cellulophaga sp. qy3.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/75420a985862409ba16a36af1a5c3f53
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Purification, cloning, characterization and essential amino acid residues analysis of a new ι-carrageenase from Cellulophaga sp. QY3.

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