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

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 using agro-industrial residues. β-Mannanase from Clostridium acetobutylicum 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 (K m = 30.7 mg/mL, V max = 7.88 µmol mL⁻¹ min⁻¹) indicated strong substrate affinity and catalytic efficiency. Application of the purified enzyme to pretreated palm kernel substrate, followed by fermentation of the supernatant containing fermentable sugars, yielded biobutanol, ethanol, and acetone, as confirmed by GC–MS and FTIR analyses. This study highlights a novel thermostable and pH-tolerant β-mannanase from C. acetobutylicum 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.