2025 AIChE Annual Meeting

Characterization of Novel Thermophilic Glycoside Hydrolases from Family GH43 for Biofuel Production from Lignocellulosic Biomass

The sustainable production of advanced biofuels relies on the efficient deconstruction of lignocellulosic biomass, a process often bottlenecked by the recalcitrance of complex hemicelluloses like glucuronoarabinoxylan (GAX). Industrial bioethanol production often requires robust and efficient enzyme mixtures capable of operating under high temperatures. The potent hemicellulolytic organism Acetivibrio thermocellus (basionym: Clostridium thermocellum) DSM1313 is known for its diverse cellulosome comprised of Carbohydrate-Active enZymes (CAZymes), particularly those of the glycoside hydrolase 43 (GH43) family, which are critical for hemicellulose degradation. To address this need, we heterologously expressed and purified three novel CAZymes (Clo1313_2857, Clo1313_2216, and Clo1313_2861) from this bacterium.

Initial characterization efforts included dinitrosalicylic acid (DNS) and p-nitrophenyl (pNP) glycoside assays to determine the substrate specificities of the three purified enzymes. Clo1313_2857 exhibited high specific activity on wheat arabinoxylan (WAX), suggesting it is a potent α-L-arabinofuranosidase. Optimization studies demonstrated a maximal operational window for Clo1313_2857 at 70−75 °C and pH 5. Additionally, Clo1313_2216 showed high activity specifically on sugar beet arabinan, suggesting it is an endo-arabinase with optimal conditions at 60 °C and pH 7. Clo1313_2861 was characterized as a β-galactosidase based on its strong activity on pNP-β-D-galactopyranoside.

We have extended this characterization by investigating the long-term thermostability of these enzymes, as well as for a recently-purified multi-domain enzyme Clo1313_2795. Residual activity on WAX showed the α-L-arabinofuranosidase (Clo1313_2857) maintained remarkable stability, retaining over 80% of its initial activity after a 4-hour incubation period at 65 °C. This stability declined sharply at 75 °C and above. In contrast, the β-galactosidase Clo1313_2795 exhibited poor stability even at its optimal working temperature of 70 °C, losing nearly all activity after a 4-hour incubation period.

These findings provide essential activity and stability information for novel thermophilic GH43 family members. This comprehensive characterization informs the rational design of robust enzyme cocktails for industrial lignocellulosic biomass processing, while simultaneously clarifying the molecular basis of A. thermocellus’ native hemicellulose degradation mechanism. This work advances both engineered biofuel production and fundamental enzyme biochemistry.