Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellul...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Amar A Telke, Ningning Zhuang, Sunil S Ghatge, Sook-Hee Lee, Asad Ali Shah, Haji Khan, Youngsoon Um, Hyun-Dong Shin, Young Ryun Chung, Kon Ho Lee, Seon-Won Kim
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ae46e70a6a754314a428a97fe2a0f97a
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:ae46e70a6a754314a428a97fe2a0f97a
record_format	dspace
spelling	oai:doaj.org-article:ae46e70a6a754314a428a97fe2a0f97a2021-11-18T07:41:54ZEngineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.1932-620310.1371/journal.pone.0065727https://doaj.org/article/ae46e70a6a754314a428a97fe2a0f97a2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23785445/?tool=EBIhttps://doaj.org/toc/1932-6203Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.Amar A TelkeNingning ZhuangSunil S GhatgeSook-Hee LeeAsad Ali ShahHaji KhanYoungsoon UmHyun-Dong ShinYoung Ryun ChungKon Ho LeeSeon-Won KimPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e65727 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Amar A Telke Ningning Zhuang Sunil S Ghatge Sook-Hee Lee Asad Ali Shah Haji Khan Youngsoon Um Hyun-Dong Shin Young Ryun Chung Kon Ho Lee Seon-Won Kim Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
description	Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.
format	article
author	Amar A Telke Ningning Zhuang Sunil S Ghatge Sook-Hee Lee Asad Ali Shah Haji Khan Youngsoon Um Hyun-Dong Shin Young Ryun Chung Kon Ho Lee Seon-Won Kim
author_facet	Amar A Telke Ningning Zhuang Sunil S Ghatge Sook-Hee Lee Asad Ali Shah Haji Khan Youngsoon Um Hyun-Dong Shin Young Ryun Chung Kon Ho Lee Seon-Won Kim
author_sort	Amar A Telke
title	Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
title_short	Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
title_full	Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
title_fullStr	Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
title_full_unstemmed	Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
title_sort	engineering of family-5 glycoside hydrolase (cel5a) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/ae46e70a6a754314a428a97fe2a0f97a
work_keys_str_mv	AT amaratelke engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT ningningzhuang engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT sunilsghatge engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT sookheelee engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT asadalishah engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT hajikhan engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT youngsoonum engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT hyundongshin engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT youngryunchung engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT konholee engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates AT seonwonkim engineeringoffamily5glycosidehydrolasecel5afromanunculturedbacteriumforefficienthydrolysisofcellulosicsubstrates
_version_	1718423120184868864

Engineering of family-5 glycoside hydrolase (Cel5A) from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

Ejemplares similares