Cellobiose phosphorylase (CBP) phosphorolyzes cellobiose into glucose-1-phosphate and plays a crucial role in the conversion of lignocellulose to starch.The cbp gene derived from Clostridium thermocellum YM4 was utilized to express CBP by engineering the recombinant Escherichia coli, Rosetta(DE3)/pET-28a-CBP, with the aid of a pET-28a(+) expression vector.After the induced expression conditions were optimized, the enzyme activity increased from 0.18 IU/mL to 0.47 IU/mL when the induction temperature, isopropylthio-β-D-galactoside (IPTG) concentration, and induction time were set at 15 ℃, 0.2 mmol/L, and 12 h, respectively.Subsequently, the His-tagged CBP was isolated and purified using a Ni column.Results revealed that the recombinant enzyme had a molecular mass of 95 kDa, a specific enzyme activity of 1.03 IU/mL, an optimal reaction temperature of 60 ℃, an optimal reaction pH of 6, and a half-inactivation time of 60 min.Furthermore, Cu2+ exhibited the greatest inhibitory effect on the enzyme, followed by Zn2+, Fe3+, Ca2+, and Mn4+ in descending order (P<0.05).In contrast, no significant difference in enzyme activity was observed with K+, Mg2+, and Na+ (P>0.05).Finally, the kinetic parameters of the recombinant CBP were determined, with a Km value of 19.61 μmol/mL, a Vmax value of 1.19 μmol/(mL·min), and a Kcat value of 21.38 s-1.
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