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Characterizing Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents

Published on Dec 14, 2018in Applied and Environmental Microbiology4.077
· DOI :10.1128/AEM.02079-18
Fei Zheng5
Estimated H-index: 5
(UMN: University of Minnesota),
Josh V. Vermaas10
Estimated H-index: 10
(NREL: National Renewable Energy Laboratory)
+ 7 AuthorsHuiying Luo33
Estimated H-index: 33
Abstract
Cellulases from glycoside hydrolase (GH) family 5 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 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 a previously characterized thermophilic GH5 endoglucanase from Talaromyces emersonii ( Te Egl5A, with an optimal temperature of 90°C), we created ten hybrid enzymes with the mesophilic endoglucanase from Stegonsporium opalus ( So Cel5) 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 optima (10 and 20°C), T 50 (15 and 19°C), T m (16.5 and 22.9°C), and extended half life, t 1/2 (∼240- and 650-fold at 55°C) relative to the mesophilic parent enzyme, and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase from Aspergillus nidulans ( An Cel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics simulations (MD) of both So Cel5 and Te Egl5A parent enzymes as well as their hybrids, we hypothesize that improved hydrophobic packing of the interface between α 2 and α 3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to the mesophilic parent So Cel5. IMPORTANCE 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, So Cel5 and Te Egl5A 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.
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