No abstract

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7β-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7β-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7β-HSDH. The best variant (V) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V increased to 942 g L day, in contrast to 141 g L day with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction of UDCA.

Dehydrogenases are widely employed as biocatalysts for the production of optically pure chemicals under mild conditions. Most dehydrogenases are nicotinamide cofactor (NADPH or NADH)-dependent oxidoreductases. 7β-Hydroxysteroid dehydrogenase (7β-HSDH) is a key enzyme for the biochemical synthesis of ursodeoxycholic acid (UDCA). To date, all reported 7β-HSDHs are strictly NADPH-dependent enzymes. However, compared with NADPH, NADH is much more economical, making it the preferential cofactor for synthetic applications of dehydrogenases. In this work, a recombinant 7β-HSDH originating from Ruminococcus torques was rationally engineered to alter its cofactor dependence using a strategy referred to as Cofactor Specificity Reversal: Small-and-Smart Library Design (CSR-SaSLiD), which is based on structural information and conservative sequence alignment. We rationally designed a small-and-smart library containing only five mutants that enabled the quick identification of target variants. Compared with the wild type, the resultant mutant, G39D, showed a 953 000-fold switch in cofactor specificity from NADPH to NADH, and another mutant, G39D/T17A, resulted in 223-fold enhanced activity with NADH. The structural mechanism regarding the effect of mutation on the reversal of cofactor preference and improvement of catalytic activity was elucidated with the aid of molecular dynamics simulation. Furthermore, it was confirmed that the CSR-SaSLiD strategy can be extended to other 7β-HSDHs. This work provides an efficient approach to altering cofactor preference and subsequently recovering the enzymatic activity of dehydrogenases for cost-effective biotechnical applications.

Ursodeoxycholic acid (UDCA) is an effective drug for the treatment of hepatitis. In this study, 7α-hydroxysteroid dehydrogenase (7α-HSDH) and lactate dehydrogenase (LDH), as well as 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH), were co-immobilized onto an epoxy-functionalized resin (ES-103) to catalyze the synthesis of UDCA from chenodeoxycholic acid (CDCA). Through optimizing the immobilization pH, time, and loading ratio of enzymes to resin, the specific activities of immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 were 43.2 and 25.8 U g , respectively, which were 12- and 516-fold higher than that under the initial immobilization conditions. Continuous production of UDCA from CDCA was subsequently achieved by using immobilized LDH-7αHSDH@ES-103 and 7βHSDH-GDH@ES-103 in two serial packed-bed reactors. The yield of UDCA reached nearly 100 % and lasted for at least 12 h in the packed-bed reactors, which was superior to that of the batchwise reaction. This efficient continuous approach developed herein might provide a feasible route for large-scale biotransformation of CDCA into UDCA.

An eco-friendly and convenient preparation method for notoginsenoside ST-4 has been established by completely transforming vina-ginsenoside R7 using a recombinant glycosidase hydrolyzing enzyme (HaGH03) from Herpetosiphon aurantiacus. This enzyme specifically hydrolyzed the glucose at the C-20 position but not the external xylose or two inner glucoses at position C-3. Protein sequence BLAST revealed that HaGH03, composed of 749 amino acids and presumptively listed as a member of the family 3 glycoside hydrolases, has highest identity (48 %) identity with a thermostable β-glucosidase B, which was not known of any functions for ginsenoside transformation. The steady state kinetic parameters for purified HaGH03 measured against p-nitrophenyl β-D-glucopyranoside and vina-ginsenoside R7 were K M = 5.67 ± 0.24 μM and 0.59 ± 0.23 mM, and k cat = 69.2 ± 0.31/s and 2.15 ± 0.46/min, respectively. HaGH03 converted 2.5 mg/mL of vina-ginsenoside R7 to ST-4 with a molar yield of 100 % and a space-time yield of 104 mg/L/h in optimized conditions. These results underscore that HaGH03 has much potential for the effective preparation of target ginsenosides possessing valuable pharmacological activities. This is the first report identifying an enzyme that has the ability to transform vina-ginsenoside R7 and provides an approach to preparing rare notoginsenoside ST-4.