Characterization of a novel halophilic and thermostable multifunctional cellulase from Ebinur Salt Lake

Cellulase is essential for cellulose hydrolysis and is used across industries such as food, feed, textiles, biofuel, and biomass pretreatment. However, its utility is restricted by high temperatures and salt concentrations. This study identified a novel cellulase gene (named c5-cel4) from Ebinur Salt Lake in Xinjiang, China using metagenomic technology. Its amino acid sequence has a 90.97% similarity to the GH5 family cellulase of Microbulbifer litoralis (WP_250463697.1). The gene was expressed in Escherichia coli, and the recombinant protein, C5-CEL4, was purified via Ni-NTA affinity chromatography. C5-CEL4, secreted extracellularly (0.886 U/mL), revealed a protein size 14 KDa smaller than predicted, with mass spectrometry indicating a truncated C-terminal of 135 amino acid residues. Optimal activity was observed at 50 °C and pH 7.0, maintaining over 80% activity at 40-65 °C and 45-50 °C for 2 h. The enzyme's half-life was 60 min at 55-60 °C, retaining over 90% activity after 24 h in pH 5.0-12.0 buffers. C5-CEL4 showed activity against CMC-Na, bagasse xylan, and beech xylan, with Kcat values of 98.20 s^{- 1} and 12.32 s^{- 1} for CMC-Na and bagasse xylan, respectively. It also hydrolyzed wheat bran and maize stalks into reducing sugars. Remarkably, C5-CEL4 exhibited high salt tolerance, maintaining 100% activity in 0.5 M-5.0 M NaCl and after 9 months in 5.0 M NaCl. It retained over 90% activity in ionic liquids (BMIM-Ac, EMIM-Cl, BMIM-BF4) at 50% concentration and showed resistance to heavy metal ions (Co²⁺, Cu²⁺, Ag⁺, Mn²⁺, Pb²⁺, and Ni²⁺) and inhibitors (PMSF, DTT, Tween80, and SDS). In conclusion, C5-CEL4 is a robust cellulase with heat, alkali, salt, ionic liquid, and inhibitor resistance, alongside cellulase and xylanase activity, presenting significant potential for feed, food, and bioenergy applications.