Characterization of a novel cellobiose phosphorylase with broad optimal pH range from a tailings pond macrogenomic library

Glucose-1-phosphate, a crucial pharmaceutical intermediate, can be generated through the synergistic transformation of lignocellulose using cellulase and cellobiose phosphorylase (CBP), thus holding promise for a profitable biorefinery process. However, the reported optimal pH range of CBPs (6.0–7.5) does not align with that of cellulase (4.8), resulting in suboptimal efficiency in the one-pot conversion of cellulose to glucose-1-phosphate. In this study, we identified a novel cellobiose phosphorylase, CBP1942, comprising 811 amino acid residues (92.5 kDa), in tailing pond macrogenomes, exhibiting identity of 60% to 75% with reported CBPs. CBP1942 was efficiently expressed in E. coli BL21 and purified with His tagging. It maintains over 90% enzyme activity across pH 4–9. Compared to the benchmark CtCBP (GenBank ID: AAL67138.1), CBP1942 displays a 1.2-fold increase in specific enzyme activity at the optimal cellulase temperature (50 °C), indicating its superior suitability for cellulase compounding. CBP1942 exhibits Km and kcat values of 6.69 mM and 8.83 s−1, respectively, with a kcat/Km ratio 6.6 times higher than CtCBP, suggesting superior catalytic efficiency. Notably, CBP1942 shows remarkable thermal stability, retaining 94.67% enzyme activity after 3 h at 50 °C, while CtCBP retains only 78.33%. Additionally, CBP1942 complexed with cellulase enhances the conversion of corn stover, resulting in a 6.2-fold increase in glucose-1-phosphate yield compared to CtCBP. These findings indicate CBP1942's potential as an industrial enzyme for corn stover conversion to glucose-1-phosphate.