3α-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid
AssigneePharmaZell GmbH
InventorsWerner Hummel, Daniel Bakonyi
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Abstract
The invention provides novel 3α-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.
Claims28
A 3α-hydroxysteroid dehydrogenase (3α-HSDH), which catalyzes at least the stereospecific enzymatic reduction of a 3-ketosteroid to the corresponding 3-hydroxysteroid, wherein the amino acid sequence of the 3α-HSDH has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 and has triple mutation P185X1/T188X3/L68X2;
- wherein
- X1 represents A, T, S, G or V,
- X2 represents A, G, C, K, S or V and
- X3 represents A, G, W, S or V;
- and
- wherein the amino acid numbering corresponds to the amino acid sequence of SEQ ID NO:
- 4.
A 3α-hydroxysteroid dehydrogenase (3α-HSDH), which catalyzes at least the stereospecific enzymatic reduction of a 3-ketosteroid to the corresponding 3-hydroxysteroid, wherein the amino acid sequence of the 3α-HSDH has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:4 and has dual mutation P185X1/T188X3;
- wherein X1 represents T, S, or V, and
- X3 represents G, W, or V;
- and wherein the amino acid numbering corresponds to the amino acid sequence of SEQ ID NO:
- 4.
Dependencies are inferred from the claim text.