High-efficiency secretion expression of cellobiose 2-epimerase in Escherichia coli and its applications

Cellobiose 2-epimerase (CE) plays a crucial role in catalyzing the conversion of lactose. In this study, the N-terminal 20 amino acids of Lactobacillus amylovorus feruloyl esterase (N20) were employed as a signal peptide and fused with the CE gene from Caldicellulosiruptor bescii for recombinant expression. Following ligation with the pET-22b(+) vector, Escherichia coli BL21 (DE3) was transformed. SDS-PAGE analysis confirmed the extracellular secretion of the CE following fusion with the signal peptide. Following fermentation optimization to maximize extracellular protein secretion, the optimal conditions were identified as a 2 × YT medium, supplemented with 0.8 mM IPTG, 0.1 mM ferrous ion (Fe²⁺), and 25 mM glycine after a 2.5 h induction, with incubation at 37 °C and 200 rpm for 36 h. The CE was purified using ammonium sulfate precipitation at 60 % saturation, yielding 1529.61 mg of enzyme protein per liter of fermentation broth, with a specific activity of 19.25 U/mg. A lactose substrate at 40 % concentration was employed, with varying enzyme concentrations (0.3825 g/L, 0.765 g/L, 1.1475 g/L, and 1.53 g/L) and reaction times (3 h, 6 h, 9 h, 12 h, and 24 h). After reaction, high-performance liquid chromatography (HPLC) was used for analysis, determining that an enzyme concentration of 1.53 g/L reacting with the lactose substrate for 24 h achieved the highest lactulose conversion rate at 56 %. This constitutes the first study on the direct extracellular secretion of CE, laying the groundwork for its production and application.