Characterization of a Cellobiose Phosphorylase Derived from a Compost Metagenomic Library and Enhancement of Its Thermostability by Introducing Artificial Disulfide Bonds

A novel cellobiose phosphorylase, CBP1775, comprising 811 amino acid residues (92.5 kDa) and belonging to glycoside hydrolase family 94 was identified from a compost metagenomic library. Its homology with commonly employed cellobiose phosphorylases typically ranges from 62 to 73%. CBP1775 was efficiently expressed in Escherichia coli BL21 and purified using a His-tag. The optimal pH and temperature of this recombinant enzyme were determined to be 5.5 and 55°C, respectively, with 10 mM cellobiose as substrate. Notably, its optimal pH is lower than that of other reported cellobiose phosphorylases, resembling the pH optimum of cellulases. Additionally, a mutant of cellobiose phosphorylase, K323C/A681C, engineered to enhance thermal stability via an extra non-consecutive disulfide bond, was successfully developed. This modification led to 45.9% increase in the enzyme’s half-inactivation time, while molecular dynamics simulations showed that the introduction of disulfide bonds stabilized the overall protein conformation. The research progress lays the technical foundation for the conversion of straw into high-value-added chemicals such as straight-chain starch and inositol.