Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites

Abstract Background Endoglucanase has been extensively employed in industrial processes as a key biocatalyst for lignocellulosic biomass degradation. Thermostable endoglucanases with high catalytic activity at elevated temperatures are preferred in industrial use. To improve the activity and thermos...

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Main Authors: Chao Han, Qunqing Wang, Yanxu Sun, Ruirui Yang, Mengyu Liu, Siqi Wang, Yifan Liu, Lifan Zhou, Duochuan Li
Format: Article
Language:English
Published: BMC 2020-02-01
Series:Biotechnology for Biofuels
Subjects:
Online Access:http://link.springer.com/article/10.1186/s13068-020-1668-4
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author Chao Han
Qunqing Wang
Yanxu Sun
Ruirui Yang
Mengyu Liu
Siqi Wang
Yifan Liu
Lifan Zhou
Duochuan Li
author_facet Chao Han
Qunqing Wang
Yanxu Sun
Ruirui Yang
Mengyu Liu
Siqi Wang
Yifan Liu
Lifan Zhou
Duochuan Li
author_sort Chao Han
collection DOAJ
description Abstract Background Endoglucanase has been extensively employed in industrial processes as a key biocatalyst for lignocellulosic biomass degradation. Thermostable endoglucanases with high catalytic activity at elevated temperatures are preferred in industrial use. To improve the activity and thermostability, site-directed mutagenesis was conducted to modify the N-glycosylation sites of the thermostable β-1,4-endoglucanase CTendo45 from Chaetomium thermophilum. Results In this study, structure-based rational design was performed based on the modification of N-glycosylation sites in CTendo45. Eight single mutants and one double mutant were constructed and successfully expressed in Pichia pastoris. When the unique N-glycosylation site of N88 was eliminated, a T90A variant was active, and its specific activity towards CMC-Na and β-d-glucan was increased 1.85- and 1.64-fold, respectively. The mutant R67S with an additional N-glycosylation site of N65 showed a distinct enhancement in catalytic efficiency. Moreover, T90A and R67S were endowed with extraordinary heat endurance after 200 min of incubation at different temperatures ranging from 30 to 90 °C. Likewise, the half-lives (t 1/2) indicated that T90A and R67S exhibited improved enzyme thermostability at 80 °C and 90 °C. Notably, the double-mutant T90A/R67S possessed better hydrolysis activity and thermal stability than its single-mutant counterparts and the wild type. Conclusions This study provides initial insight into the biochemical function of N-glycosylation in thermostable endoglucanases. Moreover, the design approach to the optimization of N-glycosylation sites presents an effective and feasible strategy to improve enzymatic activity and thermostability.
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spelling doaj.art-9047e22642994aaea4f7a9745ec7a1132022-12-22T00:26:17ZengBMCBiotechnology for Biofuels1754-68342020-02-0113111110.1186/s13068-020-1668-4Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sitesChao Han0Qunqing Wang1Yanxu Sun2Ruirui Yang3Mengyu Liu4Siqi Wang5Yifan Liu6Lifan Zhou7Duochuan Li8Shandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityShandong Key Laboratory for Agricultural Microbiology, College of Plant Protection, Shandong Agricultural UniversityAbstract Background Endoglucanase has been extensively employed in industrial processes as a key biocatalyst for lignocellulosic biomass degradation. Thermostable endoglucanases with high catalytic activity at elevated temperatures are preferred in industrial use. To improve the activity and thermostability, site-directed mutagenesis was conducted to modify the N-glycosylation sites of the thermostable β-1,4-endoglucanase CTendo45 from Chaetomium thermophilum. Results In this study, structure-based rational design was performed based on the modification of N-glycosylation sites in CTendo45. Eight single mutants and one double mutant were constructed and successfully expressed in Pichia pastoris. When the unique N-glycosylation site of N88 was eliminated, a T90A variant was active, and its specific activity towards CMC-Na and β-d-glucan was increased 1.85- and 1.64-fold, respectively. The mutant R67S with an additional N-glycosylation site of N65 showed a distinct enhancement in catalytic efficiency. Moreover, T90A and R67S were endowed with extraordinary heat endurance after 200 min of incubation at different temperatures ranging from 30 to 90 °C. Likewise, the half-lives (t 1/2) indicated that T90A and R67S exhibited improved enzyme thermostability at 80 °C and 90 °C. Notably, the double-mutant T90A/R67S possessed better hydrolysis activity and thermal stability than its single-mutant counterparts and the wild type. Conclusions This study provides initial insight into the biochemical function of N-glycosylation in thermostable endoglucanases. Moreover, the design approach to the optimization of N-glycosylation sites presents an effective and feasible strategy to improve enzymatic activity and thermostability.http://link.springer.com/article/10.1186/s13068-020-1668-4EndoglucanaseN-glycosylation siteThermostabilitySpecific activity
spellingShingle Chao Han
Qunqing Wang
Yanxu Sun
Ruirui Yang
Mengyu Liu
Siqi Wang
Yifan Liu
Lifan Zhou
Duochuan Li
Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites
Biotechnology for Biofuels
Endoglucanase
N-glycosylation site
Thermostability
Specific activity
title Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites
title_full Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites
title_fullStr Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites
title_full_unstemmed Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites
title_short Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing N-glycosylation sites
title_sort improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing n glycosylation sites
topic Endoglucanase
N-glycosylation site
Thermostability
Specific activity
url http://link.springer.com/article/10.1186/s13068-020-1668-4
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