Abstract: The present invention relates to a coupled biotransformation process of converting chenodeoxycholic acid (CDCA) and related compounds to ursodeoxycholic acid (UDCA) and related compounds. It also relates to the cloning, expression, and biochemical characterization of a novel NADP+-dependent 7?-hydroxysteroid dehydrogenase (7?-HSDH) from Clostridium difficile, cofactor switch mutants thereof, and their application for the oxidation of bile acids. A further aspect of the invention relates to novel NADP-dependent cofactor switch mutants of the NADP+-dependent 7?-HSDH of E. coli and their application for the oxidation of bile acids.

Abstract: The invention provides novel 3?-hydroxysteroid dehydrogenase mutants, sequences that code for these enzyme mutants, methods for producing the enzyme mutants, and the use thereof in enzymatic reactions of cholic acid compounds, and in particular in the production of ursodeoxycholic acid (UDCA). The invention further provides processes for the synthesis of UDCA using the enzyme mutants and the production of UDCA using recombinant microorganisms that have been subjected to multiple modifications.

Abstract: One aspect of the invention provides a nucleic acid encoding a 7?-hydroxysteroid dehydrogenase (7?-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid. The enzyme includes a mutation at position 64 of SEQ ID NO:2 or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 90% sequence identity to SEQ ID NO:2. The mutation at position 64 is the mutation R64X1, wherein X1 represents E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I or Y. The enzyme shows the following property profile in comparison with the 7?-HSDH with SEQ ID NO:2: (a) an increased specific activity (Vmax [U/mg]) for NADPH in the enzymatic reduction of dehydrocholic acid (DHCA) with NADPH as cofactor.

Abstract: In various aspects and embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a 7?-hydroxysteroid dehydrogenase (7?-HSDH) mutant that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the wildtype 7?-HSDH of SEQ ID NO:2, a decreased substrate inhibition and/or an altered cofactor usage, and the mutant has, in comparison with the wildtype 7?-HSDH of SEQ ID NO:2, 1 to 15 amino acid additions, substitutions, deletions and/or inversions in the sequence motif VMVGRRE corresponding to positions 36 to 42 of SEQ ID NO:2.

Abstract: The invention relates to novel 7ß-hydroxysteroid dehydrogenase mutants, to the sequences which encode these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic reactions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCS). The invention also relates to novel processes for the synthesis of UDCS using the enzyme mutants; and to the preparation of UDCS using recombinant, multiply-modified microorganisms.

Abstract: The present invention relates to a coupled biotransformation process of converting chenodeoxycholic acid (CDCA) and related compounds to ursodeoxycholic acid (UDCA) and related compounds. It also relates to the cloning, expression, and biochemical characterization of a novel NADP+-dependent 7?-hydroxysteroid dehydrogenase (7?-HSDH) from Clostridium difficile, cofactor switch mutants thereof, and their application for the oxidation of bile acids. A further aspect of the invention relates to novel NADP-dependent cofactor switch mutants of the NADP+-dependent 7?-HSDH of E. coli and their application for the oxidation of bile acids.

Abstract: One aspect of the invention provides a nucleic acid encoding a 7?-hydroxysteroid dehydrogenase (7?-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid. The enzyme includes a mutation at position 64 of SEQ ID NO:2 or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 90% sequence identity to SEQ ID NO:2. The mutation at position 64 is the mutation R64X1, wherein X1 represents E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I or Y. The enzyme shows the following property profile in comparison with the 7?-HSDH with SEQ ID NO:2: (a) an increased specific activity (Vmax [U/mg]) for NADPH in the enzymatic reduction of dehydrocholic acid (DHCA) with NADPH as cofactor.

Abstract: The invention relates to novel 7?-hydroxysteroid dehydrogenase mutants, to the sequences which code for these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic conversions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCA); subject-matter of the invention is also novel processes for the synthesis of UDCA using enzyme mutants; and the preparation of UDCA using recombinant, multiply modified microorganisms.

Abstract: The invention provides novel 3?-hydroxysteroid dehydrogenase mutants, sequences that code for these enzyme mutants, methods for producing the enzyme mutants, and o the use thereof in enzymatic reactions of cholic acid compounds, and in particular in the production of ursodeoxycholic acid (UDCA). The invention further provides processes for the synthesis of UDCA using the enzyme mutants and the production of UDCA using recombinant microorganisms that have been subjected to multiple modifications.

Abstract: The invention relates to novel 3-hydroxysteroid dehydrogenase mutants, to the sequences which code for these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic conversions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCA); subject-matter of the invention is also novel processes for the synthesis of UDCA using enzyme mutants; and the preparation of UDCA using recombinant, multiply modified microorganisms.

Abstract: The invention relates to novel 7?-hydroxysteroid dehydrogenase mutants, to the sequences which code for these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic conversions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCA); subject-matter of the invention is also novel processes for the synthesis of UDCA using enzyme mutants; and the preparation of UDCA using recombinant, multiply modified microorganisms.

Abstract: The invention relates to novel 7?-hydroxysteroid dehydrogenase mutants, to the sequences which code for these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic conversions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCA); subject-matter of the invention is also novel processes for the synthesis of UDCA using enzyme mutants; and the preparation of UDCA using recombinant, multiply modified microorganisms.

Abstract: The invention relates to novel 3-hydroxysteroid dehydrogenase mutants, to the sequences which code for these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic conversions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCA); subject-matter of the invention is also novel processes for the synthesis of UDCA using enzyme mutants; and the preparation of UDCA using recombinant, multiply modified microorganisms.

Abstract: The invention relates to novel 7?-hydroxysteroid dehydrogenase mutants, to the sequences which code for these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic conversions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCA); subject-matter of the invention is also novel processes for the synthesis of UDCA using enzyme mutants; and the preparation of UDCA using recombinant, multiply modified microorganisms.

Abstract: The invention relates to novel 7?-hydroxysteroid dehydrogenase mutants, to the sequences which encode these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic reactions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCS). The invention also relates to novel processes for the synthesis of UDCS using the enzyme mutants; and to the preparation of UDCS using recombinant, multiply-modified microorganisms.

Abstract: The invention relates to novel 7?-hydroxysteroid dehydrogenase mutants, to the sequences which encode these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic reactions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCS). The invention also relates to novel processes for the synthesis of UDCS using the enzyme mutants; and to the preparation of UDCS using recombinant, multiply-modified microorganisms.