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 (??)<
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8<
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-barrel structure, where each (??) 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 from<
named-content content-type='genus-species'>
Talaromyces emersonii<
/named-content>
(<
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Te<
/italic>
Egl5A), which has an optimal temperature of 90�C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from<
named-content content-type='genus-species'>
Stegonsporium opalus<
/named-content>
(<
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So<
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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 optimum (10 and 20�C), the temperature at which the protein lost 50% of its activity (<
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T<
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<
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50<
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) (15 and 19�C), and the melting temperature (<
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T<
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m<
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<
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) (16.5 and 22.9�C) and extended half-lives (<
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t<
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<
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1/2<
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) (~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 from<
named-content content-type='genus-species'>
Aspergillus niger<
/named-content>
(<
italic>
An<
/italic>
Cel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the<
italic>
So<
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Cel5 and<
italic>
Te<
/italic>
Egl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between ?<
sub>
2<
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and ?<
sub>
3<
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is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent<
italic>
So<
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Cel5.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,<
italic>
So<
/italic>
Cel5 and<
italic>
Te<
/italic>
Egl5A, with different thermal properties, were hybridized to study the roles of different (??) 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.