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.