Unveiling GH7 cellulases from Malbranchea cinnamomea for enhanced saccharification of biorefinery relevant pretreated lignocellulosic biomass

Malbranchea cinnamomea harbors a large yet under-explored cellulolytic repertoire. Two GH7 core enzymes, endo-β-1,4-glucanase EG7A_MALCI and cellobiohydrolase CBH7A_MALCI were heterologously expressed in Pichia pastoris and purified as ~ 48 kDa and ~ 51 kDa proteins, respectively. EG7A_MALCI displayed maximal activity at pH 7.0 and 60°C, hydrolysing barley β-glucan most efficiently (K _m = 2.27 mg ml⁻¹, V _max = 81 U mg⁻¹) while retaining appreciable activity on carboxymethyl-cellulose, lichenan and phosphoric-acid-swollen cellulose. CBH7A_MALCI operated optimally at pH 7.0 and 40°C, showed highest turnover on p-nitrophenyl-β-lactoside (K _m = 0.284 mM, V _max = 156 U mg⁻¹), and exhibited moderate activity towards crystalline substrates, consistent with its processive mode of action. Homology models confirmed conserved GH7 catalytic triads and revealed endo- versus exo-orientated substrate tunnels. Replacing 10–20% of a commercial cellulase cocktail (Celluclast 1.5 L + β-glucosidase) with either recombinant enzyme enhanced glucose release from acid- or alkali-pre-treated rice straw and bagasse by up to 1.5-fold. Cocktail prepared by replacing two parts of commercial CellicCTec2 with either EG or CBH enhanced the hydrolytic potential resulting in 1.30, 1.50, 1.35 and 1.31 folds increase in release of sugars after saccharification of acid and alkali treated rice straw and bagasse, respectively, using EG7A_MALCI. These results provide the first functional description of M. cinnamomea GH7 cellulases and introduce robust, thermostable biocatalysts for designing high-performance enzyme blends in lignocellulose biorefineries.