Substrate-specific synergism between CTec 2 and galactoglucomannan-hydrolysing enzymes from Penicillium rotoruae improves softwood saccharification

The substrate-specific synergistic action of hemicellulolytic enzymes supplemented to the commercial cellulase cocktail CTec 2 was demonstrated using softwood galactoglucomannan (GGM) containing substrates. Submerged fermentation of the novel fungus Penicillium rotoruae produced three key GGM-hydrolysing enzymes, namely β-mannanase, β-mannosidase, and α-galactosidase, which were partially purified using sequential ultrafiltration and fast protein liquid chromatography (FPLC). A systematic enzyme combination screen was employed to evaluate dual and triple enzyme combinations supplemented to CTec 2 during the hydrolysis of fully characterized GGM-containing softwood substrates: (i) insoluble softwood pulp and (ii) purified soluble GGM. Supplementation of P. rotoruae enzymes to CTec 2 significantly enhanced total monomeric sugar yields by up to 50.7% and 104.4% for pulp and soluble GGM, respectively, compared with CTec 2 alone. The highest increases were observed for galactosyl (247% and 765%), mannosyl (147% and 130%), and glucosyl (44% and 94%) release compared to CTec 2 used alone, highlighting the importance of coordinated cellulose-hemicellulose deconstruction during softwood hydrolysis. Distinct substrate-dependent synergistic enzyme mechanisms were identified. For insoluble pulp, the combination of β-mannanase and β-mannosidase provided the greatest enhancement in hydrolysis efficiency, whereas β-mannosidase and α-galactosidase was most effective for soluble GGM hydrolysis. Molecular weight distribution analysis further confirmed synergistic enzyme mechanisms in depolymerisation of GGM-containing softwood substrates. These findings demonstrate that targeted supplementation of commercial cellulase systems with tailored combination of GGM-hydrolysing enzymes from P. rotoruae significantly improves softwood saccharification and provides a promising strategy for the development of efficient lignocellulosic biorefinery processes.