3-alpha-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

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 a mutation selected from the group consisting of:

• a) single mutations
• R34X1 and
• L68X2;
• and
• b) dual mutations
• R34X1/L68X2,
• L68X2/T188X3 and
• L68X2/P185X1;
• wherein for the single mutations R34X1 and L68X2 and for the dual mutation R34X1/L68X2,
• X1 represents N, C, or S;
• X2 represents A, C, K, S or V;
• wherein for the dual mutants L68X2/T188X3 and L68X2/P185X1,
• 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;
• c) the mutations of a) or b), wherein the 3α-HSDH has a C-terminal extension of 1 to 10 histidine residues and/or an N-terminal extension of 1 to 10 histidine residues;
• d) single mutation R34L, wherein the 3α-HSDH has a C-terminal extension of 1 to 10 histidine residues;
• and
• e) dual mutation R34L/L6SX2, wherein X2 represents A, C, K, S or V, and wherein the 3α-HSDH has a C-terminal extension of 1 to 10 histidine residues;
• and
• wherein the amino acid numbering correspond to the amino acid sequence of SEQ ID NO:
• 4;
• and
• wherein the 3α-HSDH shows the following property profile in comparison with a 3α-HSDH consisting of the amino acid sequence of SEQ ID NO:4:
• i) an increased specific activity (vmax [U/mg]) for dehydrocholic acid (DHCA) in the enzymatic reduction of DHCA with NADH as cofactor, or
• ii) a reduced substrate inhibition by DHCA;
• or
• iii) an increased specific activity (vmax [U/mg]) for NADH in the enzymatic reduction of DHCA with NADH as cofactor;
• or iv) any combination of properties i), ii) and iii).