Conversion of chenodeoxycholic acid to ursodeoxycholic acid by high-performance segmented continuous-flow with whole-cell catalysis

Ursodeoxycholic acid (UDCA) is widely used in treating liver and gallbladder diseases. Currently, the biosynthesis of UDCA faces several challenges, including the need for intermediate inactivation to prevent reverse reactions, reduced conversion efficiency due to byproduct interference, and the economic and environmental drawbacks of single-use catalytically active whole-cell catalysts. In this study, agar was identified as the optimal carrier material. Using 5.5% ( w / v ) agar, E. coli BL21(DE3) cells individually expressing 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase were co-immobilized, and E. coli BL21(DE3) cells co-expressing 7β-hydroxysteroid dehydrogenase and glucose dehydrogenase were immobilized separately. These immobilized cells were then used to catalyze the sequential conversion of chenodeoxycholic acid (CDCA) to 7-ketolithocholic acid (7-KLCA), and then to UDCA. Compared to the whole-cell catalyst, the immobilized catalyst showed comparable or superior performance, including excellent pH tolerance, thermostability, and storage stability. For each of the two reaction steps, continuous-flow catalysis was performed by using a 50 mL reaction system containing 50 mM substrate, and the efficiency of the second-step continuous-flow catalysis (7-KLCA → UDCA) was 15.6 times that of batch catalysis. Ultimately, two packed-bed reactor systems (tandem and segmented) were designed. The segmented system achieved 76.48% conversion after 40 h under optimized conditions and retained 59.2% after 72 h, showing excellent catalytic performance.