A thermostable and alkaline β-mannanase from Clostridium chauvoei isolated from the ruminant gut exhibits potential for bioethanol production

Background The ruminant gut microbiome represents a valuable source of lignocellulolytic enzymes, particularly β-mannanases that hydrolyze mannan into fermentable sugars. However, few studies have characterized β-mannanases from Clostridium species with respect to their catalytic stability and potential for biofuel production from agro-industrial residues. Results β-Mannanase from Clostridium chauvoei was purified from the crude extract through ammonium sulfate precipitation, dialysis, and ion-exchange and size-exclusion chromatography, achieving a 36% yield and 9-fold purification. The enzyme exhibited optimal activity at pH 4.0 and 60°C and maintained stability over a broad pH range (2–12 for 6 h) and temperature range (30–80°C). Enzyme activity was enhanced by Mg²⁺, Zn²⁺, and Mn²⁺, while inhibited by EDTA, SDS, and cysteine; among organic solvents, only formaldehyde stabilized the enzyme. It showed a half-life of 216 min at 70°C, with thermodynamic parameters ΔG = 63 kJ/mol, ΔH = 23 kJ/mol, and ΔS = − 134.4 J/mol·K. Kinetic constants (Km = 30.7 mg/mL, Vmax = 7.88 µmol/mL/min) indicated strong substrate affinity and catalytic efficiency. Application of the purified enzyme to pretreated palm kernel substrate yielded substantial biobutanol (55 g/L), ethanol (60 g/L), and acetone (70 g/L) confirmed by GC–MS and FTIR analyses. Conclusion This study highlights a novel thermostable and pH-tolerant β-mannanase from C. chauvoei capable of efficiently hydrolyzing lignocellulosic biomass into fermentable sugars for acetone-butanol-ethanol (ABE) production. The enzyme’s robust catalytic properties and high saccharification efficiency position it as a promising biocatalyst for sustainable biofuel production and other industrial bioprocess applications.