Discovery and characterization of a thermophilic endo-inulinase with exceptional thermostability for efficient inulooligosaccharide production.

Endo-inulinases are key biocatalysts for converting inulin into prebiotic inulooligosaccharides (IOS). However, industrial implementation is hindered by a mismatch between inulin solubility and enzyme operating temperatures. Natural inulin dissolves well at high temperatures (≥70 °C) but poorly at the moderate temperatures (30-50 °C) typical for most endo-inulinases. Therefore, identifying thermophilic endo-inulinases is crucial. Herein, a novel thermophilic endo-inulinase InuG with exceptional thermostability was identified. Our strategy used a known endo-inulinase as a molecular probe to source homologs, prioritizing candidates from their source microorganisms with high optimal growth temperatures. Recombinant InuG was gene-synthesized, expressed in Escherichia coli and purified. InuG-His6 hydrolyzed inulin, yielding inulotriose (F3) as the dominant product. The enzyme exhibited superior properties to all reported endo-inulinases, with an optimal temperature of 70 °C and an exceptional half-life of 82 h at 70 °C. Molecular-dynamics simulations revealed that tight hydrophobic packing, reduced flexible segments, and rigid loop regions collectively underpin this extreme thermostability. Furthermore, we elucidated the functional role of the conserved bacterial domain LamG and the structural mechanism conferring InuG's product preference for F3. These properties position InuG as a robust biocatalyst for IOS production at high-temperature, offering immediate advantages for industrial food and feed applications.