Pharmazell Gmbh

Daniel Bakonyi

Werner Hummel

Dirk Weuster-Botz

Arno Aigner

Boqiao Sun

Michael Braun

Christina Kantzow

Sven Bresch

Ralf Gross

In various aspects and embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a 7β-hydroxysteroid dehydrogenase (7β-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 7β-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 7β-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.

7β-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

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&#x3b1;-hydroxysteroid dehydrogenase (7&#x3b1;-HSDH) from, 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&#x3b1;-HSDH ofand their application for the oxidation of bile acids.

Coupled, self-sufficient biotransformation of chenodeoxcholic acid to ursodeoxycholic acid and novel enzyme mutants applicable in said process

The invention provides novel 3&#x3b1;-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.

3&#x3b1;-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

One aspect of the invention provides a nucleic acid encoding a 7&#x3b2;-hydroxysteroid dehydrogenase (7&#x3b2;-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 R64X, wherein Xrepresents 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&#x3b2;-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.

7-beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

The invention relates to novel 7&#xdf;-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.

7Beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

The invention relates to novel 7&#x3b2;-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.

7-&#x3b2;-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

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.

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Cologne, Germany

Daniel Bakonyi