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Title: Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents

Abstract

Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme’s stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A fromTalaromyces emersonii(TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 fromStegonsporium opalus(SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T50) (15 and 19°C), and the melting temperature (Tm) (16.5 and 22.9°C) and extended half-lives (t1/2) (~240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 fromAspergillus niger(AnCel5), whichmore » demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both theSoCel5 andTeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2and α3is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parentSoCel5.Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure,SoCel5 andTeEgl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.« less

Authors:
 [1];  [2];  [3];  [3];  [3];  [1];  [4];  [3];  [2];  [3]
  1. Chinese Academy of Agricultural Sciences, Beijing (China); Beijing Forest Univ. (China)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  3. Chinese Academy of Agricultural Sciences, Beijing (China)
  4. Beijing Forest Univ. (China)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE National Renewable Energy Laboratory (NREL), Laboratory Directed Research and Development (LDRD) Program; USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office
OSTI Identifier:
1498081
Report Number(s):
NREL/JA-2700-72982
Journal ID: ISSN 0099-2240
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 85; Journal Issue: 5; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; chimera; molecular dynamics; thermostability

Citation Formats

Zheng, Fei, Vermaas, Josh V., Zheng, Jie, Wang, Yuan, Tu, Tao, Wang, Xiaoyu, Xie, Xiangming, Yao, Bin, Beckham, Gregg T., and Luo, Huiying. Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents. United States: N. p., 2019. Web. doi:10.1128/AEM.02079-18.
Zheng, Fei, Vermaas, Josh V., Zheng, Jie, Wang, Yuan, Tu, Tao, Wang, Xiaoyu, Xie, Xiangming, Yao, Bin, Beckham, Gregg T., & Luo, Huiying. Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents. United States. doi:10.1128/AEM.02079-18.
Zheng, Fei, Vermaas, Josh V., Zheng, Jie, Wang, Yuan, Tu, Tao, Wang, Xiaoyu, Xie, Xiangming, Yao, Bin, Beckham, Gregg T., and Luo, Huiying. Wed . "Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents". United States. doi:10.1128/AEM.02079-18. https://www.osti.gov/servlets/purl/1498081.
@article{osti_1498081,
title = {Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents},
author = {Zheng, Fei and Vermaas, Josh V. and Zheng, Jie and Wang, Yuan and Tu, Tao and Wang, Xiaoyu and Xie, Xiangming and Yao, Bin and Beckham, Gregg T. and Luo, Huiying},
abstractNote = {Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme’s stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A fromTalaromyces emersonii(TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 fromStegonsporium opalus(SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T50) (15 and 19°C), and the melting temperature (Tm) (16.5 and 22.9°C) and extended half-lives (t1/2) (~240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 fromAspergillus niger(AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both theSoCel5 andTeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2and α3is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parentSoCel5.Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure,SoCel5 andTeEgl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.},
doi = {10.1128/AEM.02079-18},
journal = {Applied and Environmental Microbiology},
number = 5,
volume = 85,
place = {United States},
year = {2019},
month = {2}
}

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