[Directed evolution of cellobiose 2-epimerase and its application in the synthesis of lactulose]

We had engineered Caldicellulosiruptor morganii CE (CmCE) to construct the enzyme CmCE/D226G for catalyzing the isomerization of lactose to lactulose. However, the poor thermostability and catalytic efficiency of CmCE/D226G limits its application under high substrate concentrations. To address these issues, we employed directed evolution to improve the catalysis performance of CmCE/D226G. First, we used error-prone PCR to construct a variant library and identified the positive variants through cysteine-tryptophan chromogenic high-throughput screening. Then, aiming the crucial residue positions, we conducted saturation mutagenesis to obtain the variants with improved thermostability and catalysis. Accordingly, we successfully obtained a variant named CmCE/D226G/S180T. Its half-life and catalysis efficiency (k_cat/K_m) at 70 ℃ were 184.2 min and 36.5 L/(mmol·s), respectively, which were higher than those (117.7 min and 14.96 L/(mmol·s), respectively) of CmCE/D226G. The improvement of isomerization efficiency resulted from the shortening distance between His377 and the O1 of substrate enol intermediate. The improvement of thermostability was attributed to the rigidness enhancement of a flexible loop. With 350 g/L lactose as the substrate, CmCE/D226G/S180T achieved a lactulose yield of 54.8% and an epilactose yield of 9.2%, while CmCE/D226G only reached a lactulose yield of 50% and an epilactose yield of 10.3%. Taken together, this work provides an elite enzyme for catalyzing the transformation of lactose to lactulose, which especially facilitates the innovation of eco-friendly synthesis of lactulose.