Characterization of novel cellobiose 2-epimerases from Teredinibacter Haidensis and Cellvibrio Japonicus

Cellobiose 2-epimerases (CEs) are promising enzymes that catalyze the conversion of lactose to the bioactive disaccharides epilactose and lactulose. Low-temperature (≤8 °C) lactose bioconversion is essential in dairy applications to prevent microbial contamination. Unfortunately, most CEs are not particularly active at these low temperatures. Therefore, this work aims to discover and characterize novel cold-active CEs.

Two novel mesophilic CEs from Teredinibacter haidensis (ThCE) and Cellvibrio japonicus (CjCE) were characterized, which showed both epi- and isomerization activity additionally at low temperatures. ThCE showed a maximum epimerization activity (595.4 ± 4.4 nkat/mg protein) at pH 7.5 and 35 °C, while CjCE achieved maximum epimerization activity (887.4 ± 3.9 nkat/mg protein) at pH 9.5 and 40 °C. In addition, CjCE maintained an >80% activity across a wide pH (6-9.5) and temperature range (30-45 °C). Both CEs exhibited significant activity and stability at 8 °C. Specifically, ThCE and CjCE retained around 20 and 40% of their epimerization activity at 8 °C and showed half-lives of 96.3 and 72.2 days, respectively. Moreover, CjCE was also found to be capable of catalyzing the isomerization reaction at 8 °C. The isomerization activity of ThCE and CjCE increased ~370-fold (70.8 ± 2.7 nkat/mg protein) and ~230-fold (66.1 ± 0.3 nkat/mg protein), respectively, at lactose concentrations up to 600 mM (35 °C for ThCE and 40 °C for CjCE). This demonstrates how important it is to conduct investigations of CEs for lactose isomerization to lactulose up to the solubility limit of lactose.

This study identified two new CEs that exhibit interesting catalytic properties for possible application in the generation of disaccharides with prebiotic properties from lactose at low temperatures.