Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability

Abstract Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme’s commercial utilization. To obtain endoglucanases with substantially imp...

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Autores principales:	Xiutao Chen, Weiguang Li, Peng Ji, Yang Zhao, Chengyao Hua, Chao Han
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1fbef55c5f7848819f325ba76a9c1345
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spelling	oai:doaj.org-article:1fbef55c5f7848819f325ba76a9c13452021-12-02T15:07:55ZEngineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability10.1038/s41598-018-21246-82045-2322https://doaj.org/article/1fbef55c5f7848819f325ba76a9c13452018-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-21246-8https://doaj.org/toc/2045-2322Abstract Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme’s commercial utilization. To obtain endoglucanases with substantially improved activity and thermostability, structure-based rational design was carried out based on the Chaetomium thermophilum β-1,4-endoglucanase CTendo45. In this study, five mutant enzymes were constructed by substitution of conserved and noncatalytic residues using site-directed mutagenesis. Mutants were constitutively expressed in Pichia pastoris, purified, and ultimately tested for enzymatic characteristics. Two single mutants, Y30F and Y173F, increased the enzyme’s specific activity 1.35- and 1.87-fold using carboxymethylcellulose sodium (CMC-Na) as a substrate, respectively. Furthermore, CTendo45 and mutants exhibited higher activity towards β-D-glucan than that of CMC-Na, and activities of Y173F and Y30F were also increased obviously against β-D-glucan. In addition, Y173F significantly improved the enzyme’s heat resistance at 80 °C and 90 °C. More interestingly, the double mutant Y30F/Y173F obtained considerably higher stability at elevated temperatures but failed to inherit the increased catalytic efficiency of its single mutant counterparts. This work gives an initial insight into the biological function of conserved and noncatalytic residues of thermostable endoglucanases and proposes a feasible path for the improvement of enzyme redesign proposals.Xiutao ChenWeiguang LiPeng JiYang ZhaoChengyao HuaChao HanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-10 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xiutao Chen Weiguang Li Peng Ji Yang Zhao Chengyao Hua Chao Han Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability
description	Abstract Endoglucanases are increasingly applied in agricultural and industrial applications as a key biocatalyst for cellulose biodegradation. However, the low performance in extreme conditions seriously challenges the enzyme’s commercial utilization. To obtain endoglucanases with substantially improved activity and thermostability, structure-based rational design was carried out based on the Chaetomium thermophilum β-1,4-endoglucanase CTendo45. In this study, five mutant enzymes were constructed by substitution of conserved and noncatalytic residues using site-directed mutagenesis. Mutants were constitutively expressed in Pichia pastoris, purified, and ultimately tested for enzymatic characteristics. Two single mutants, Y30F and Y173F, increased the enzyme’s specific activity 1.35- and 1.87-fold using carboxymethylcellulose sodium (CMC-Na) as a substrate, respectively. Furthermore, CTendo45 and mutants exhibited higher activity towards β-D-glucan than that of CMC-Na, and activities of Y173F and Y30F were also increased obviously against β-D-glucan. In addition, Y173F significantly improved the enzyme’s heat resistance at 80 °C and 90 °C. More interestingly, the double mutant Y30F/Y173F obtained considerably higher stability at elevated temperatures but failed to inherit the increased catalytic efficiency of its single mutant counterparts. This work gives an initial insight into the biological function of conserved and noncatalytic residues of thermostable endoglucanases and proposes a feasible path for the improvement of enzyme redesign proposals.
format	article
author	Xiutao Chen Weiguang Li Peng Ji Yang Zhao Chengyao Hua Chao Han
author_facet	Xiutao Chen Weiguang Li Peng Ji Yang Zhao Chengyao Hua Chao Han
author_sort	Xiutao Chen
title	Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability
title_short	Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability
title_full	Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability
title_fullStr	Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability
title_full_unstemmed	Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability
title_sort	engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/1fbef55c5f7848819f325ba76a9c1345
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Engineering the conserved and noncatalytic residues of a thermostable β-1,4-endoglucanase to improve specific activity and thermostability

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