A novel cellobiose 2-epimerase gene ( CsCEase1 ) from Clostridium sp. TW13 was successfully overexpressed. Cs CEase1, with a molecular mass of 45.0 kDa, exhibited a specific activity of 254.75 U/mg and showed optimal activity at 40 °C and pH 7.5. Using Cs CEase1, 44.30 % of lactose was converted into epilactose, yielding a production level of 26.58 g/L. An efficient purification method was developed, incorporating crystallization and β-galactosidase treatment to remove the unconverted lactose, while Saccharomyces cerevisiae was used to consume the produced glucose. Final purification with activated carbon resulted in epilactose with 98 % purity and a recovery rate of 66.47 %. Furthermore, Cs CEase1 was incubated with whey power and expired milk, achieving epilactose conversion rates of 29.65 % and 32.69 %, respectively. These findings highlight the potential of Cs CEase1 for the cost-effective and environmentally sustainable production of epilactose from dairy waste.
Cellobiose 2-epimerase (CE) catalyzes the conversion of the lactose into its high-value derivatives, epilactose and lactulose, which has great prospects in food applications. In this study, CE sequences from the Qinghai-Tibet Plateau gene catalogue, we screened these for structural flexibility through molecular dynamics simulation to identify potential psychrophilic CE candidates. One such psychrophilic CE we termed psyCE demonstrated exceptional epimerization activity, achieving an optimum activity of 122.2 ± 1.6 U/mg. Its kinetic parameters (K cat and K m ) for epimerization activity were 219.9 ± 5.6 s −1 and 261.9 ± 18.1 mM, respectively, representing the highest K cat recorded among known cold-active CEs. Notably, this is the first report of a psychrophilic CE. The psyCE can effectively produce epilactose at 8 °C, converting 20.3 % of 200 mM lactose into epilactose within four hours. These findings suggest that psyCE is highly suitable for cryogenic food processing, and glaciers may serve as a valuable repository of psychrophilic enzymes.
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