Study Of Biocatalytic Production Of Ursodeoxycholic Acid And Molecular Modification Of Its Key Enzyme

Ursodeoxycholic acid is a bile acid that is widely used in clinical practice to dissolve cholesterol stones and improve liver function in cholestatic diseases.7β-Hydroxysteroid dehydrogenase（7β-HSDH）,a key enzyme for the enzymatic synthesis of ursodeoxycholic acid,can reduce the ketocarbonyl group at the C-7 site to aβ-oriented hydroxyl group.However,the number of reports on 7β-HSDH is limited,and there are only one or two sources of industrial application,and there is a lot of room for exploration,and the low catalytic efficiency and stability of 7β-HSDH have become the speed limit for the production of ursodeoxycholic acid.In this study,a 7β-HSDH＿Osp with high conversion rate was efficiently heterologously expressed,and its enzymatic properties were determined.Catalytic production methods of ursodeoxycholic acid were optimised,and its catalytic efficiency was improved by molecular modification.The main research contents are as follows:（1）A 7β-HSDH＿Osp with high conversion rate from Olsenella sp.An188 was efficiently heterologously expressed in E.coli BL21.Its optimum p H was 7.0（a substrate concentration was 10 m M）.The stable p H range was 6.5-9.0.The optimum temperature was 55°C.It had the high thermal stability.The catalytic constant k_cat and the specific constant k_cat/K_m were 558.72min^-1 and 49.80 L·mol^-1·min^-1,respectively.（2）In combination with 7α-hydroxysteroid dehydrogenase,the conversion rate of ursodeoxycholic acid from chenodeoxycholic acid reached 68.3%via“one-pot”catalysis under the optimum conditions.When 7β-HSDH＿Osp and 7α-HSDH＿Cd were co-expressed,the conversion rate of whole-cell catalysis was higher than that of free enzyme catalysis via the“one-pot”catalysis.When plasmid p RSFDuet-1 with high copy number was used to co-express7β-HSDH＿Osp and 7α-HSDH＿Cd,the conversion rate of ursodeoxycholic acid was higher than that of co-expression using the plasmids p RSFDuet-1 and p ETDuet-1,and its conversion rate reached 68.0%.（3）The conversion rate of“two-step”catalytic production of ursodeoxycholic acid by co-expression of enzymes based on cyclic regeneration of cofactors was higher than that of the“one-pot”catalysis.The conversion rate of cofactor cyclic regeneration based on 7α-HSDH＿Es and lactic dehydrogenase LDH and 7β-HSDH＿Osp and glucose dehydrogenase GDH via free enzyme catalysis reached 81.0%,which was higher than that of whole-cell catalysis.For cofactor cyclic regeneration system,the conversion rate of ursodeoxycholic acid via separate expression of enzymes was significantly higher than that of co-expression of enzymes,which reached 90.3%.Based on the above optimised method,the ursodeoxycholic acid conversion rate reached 84.0%when the concentration of chenodeoxycholic acid was 100 g·L^-1.（4）A compound mutant T189V/V207M with high catalytic efficiency was obtained by site-directed mutagenesis,and its k_cat/K_m value was increased by 7.1-fold over that of 7β-HSDH＿Osp.Based on tertiary structural homology modelling,25 key amino acid residues were selected for site-directed saturation mutagenesis and 5 mutants with high activity were obtained:T189V/V207M/V91P,T189V/V207M/M144H,T189V/V207M/G146A,T189V/V207M/A158S,T189V/V207M/V183I.Their specific activities were increased by 3.5-,3.6-,3.6-,3.5-and 3.8-fold that of 7β-HSDH＿Osp,respectively.The k_cat/K_m value of mutant T189V/V207M/G146A was significantly increased from 352.63 L·mol^-1·min^-1 of T189V/V207M to 654.42 L·mol^-1·min^-1.The substrate binding energy of the T189V/V207M/G146A mutant was lower than that of T189V/V207M,which verified its improved catalytic efficiency.The reasons for the improved catalytic domain of the enzymes and the substrate.Subsequently,the conversion rates of the different mutants were compared when they catalyzed high concentrations of substrate.Based on the above optimised method,the conversion rate of the mutant T189V/V207M/G146A increased to 91.2%when catalyzing100 g·L^-1 substrate.