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

Abstract Background: 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. Results Two novel mesophilic CEs from Teredinibacter haidensis ( Th CE) and Cellvibrio japonicus ( Cj CE) were characterized, which showed both epi- and isomerization activity additionally at low temperatures. Th CE showed a maximum epimerization activity (595.4 ± 4.4 nkat/mg protein) at pH 7.5 and 35 °C, while Cj CE achieved maximum epimerization activity (887.4 ± 3.9 nkat/mg protein) at pH 9.5 and 40 °C. In addition, Cj CE 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, Th CE and Cj CE 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, Cj CE was also found to be capable of catalyzing the isomerization reaction at 8 °C. The isomerization activity of Th CE and Cj CE 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 Th CE and 40 °C for Cj CE). This demonstrates how important it is to conduct investigations of CEs for lactose isomerization to lactulose up to the solubility limit of lactose. Conclusions 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.