3-Alpha-Hydroxysteroid Dehydrogenase Mutants and Process for the Preparation of Ursodeoxycholic Acid
AssigneePharmaZell GmbH
InventorsWerner Hummel, Daniel Bakonyi
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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.
Claims20
A 3α-hydroxysteroid dehydrogenase (3α-HSDH), which catalyzes at least the stereospecific enzymatic reduction of a 3-ketosteroid to the corresponding 3-hydroxysteroid, in particular under stoichiometric consumption of NADH, wherein the enzyme possesses at least 80% sequence identity to SEQ ID NO:4;
- and is selected among:
- a) the single mutants
- R34X1 and
- L68X2 and
- b) the dual mutants
- R34X1/L68X2,
- wherein
- X1 represents N, C, S or L;
- X2 represents A, C, K, S or V
- c) addition mutants which are C-terminally extended by a residue comprising 3 to 10 histidine residues;
- and
- d) mutants as per a) and b), in each case additionally C-terminally extended by a residue comprising 1 to 10 histidine residues and/or N-terminally extended by a residue comprising 1 to 10 histidine residues;
- wherein this 3α-HSDH shows the following property profile in comparison with the 3α-HSDH with 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;
- ii) a reduced substrate inhibition by DHCA,
- iii) an increased specific activity (vmax [U/mg]) for NADH in the enzymatic reduction of DHCA with NADH as cofactor
- iv) it being possible for these properties i) to iii) to be present individually or in any combination.
A 3α-hydroxysteroid dehydrogenase (3α-HSDH), which catalyzes at least the stereospecific enzymatic reduction of a 3-ketosteroid to the corresponding 3-hydroxysteroid, in particular under stoichiometric consumption of NADH, wherein the enzyme possesses at least 80% sequence identity to SEQ ID NO:4;
- and is selected among the dual mutants
- P185X1/T188X3;
- L68X2/T188X3;
- L68X2/T185X1,
- 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;
- optionally additionally with C-terminal extension comprising 1 to 10 histidine residues;
- and/or optionally additionally with N-terminal extension comprising 1 to 10 histidine residues;
- wherein the 3α-HSDH shows the following property profile in comparison with the 3α-HSDH with SEQ ID NO:4;
- a) an increased specific activity (vmax [U/mg]) for dehydrocholic acid (DHCA) in the enzymatic reduction of DHCA with NADH as cofactor;
- b) a reduced substrate inhibition by DHCA,
- c) an increased specific activity (vmax [U/mg]) for NADH in the enzymatic reduction of DHCA with NADH as cofactor,
- it being possible for these properties a) to c) to be present individually or in any combination.
A process for the preparation of ursodeoxycholic acid (UDCA) of the formula (1)
- in which
- R represents alkyl, H, an alkali metal ion or N(R3)4+, wherein the radicals R3 are identical or different and represent H or alkyl, or the group —CO2R is replaced by the acid amide group —CONR1R2, in which R1 and R2 independently of one another represent an alkyl radical;
- in which
- a) optionally a cholic acid (CA) of the formula (2)
- in which R has the abovementioned meanings or the group —CO2R is replaced by the acid amide group —CONR1R2 as defined hereinabove is oxidized chemically to dehydrocholic acid (DHCA) of the formula (3)
- in which R has the abovementioned meanings and the group —CO2R is replaced by the acid amide group —CONR1R2 as defined hereinabove;
- b) DHCA is reduced in the presence of at least one 3α-HSDH mutant selected among
- (i) the single mutants
- R34X1 and
- L68X2 and
- (ii) the dual mutants
- R34X1/L68X2
- wherein
- X1 represents N, C, S or L;
- X2 represents A, C, K, S or V
- (iii) addition mutants which are C-terminally extended by a residue comprising 1 to 10 histidine residues;
- (iv) addition mutants which are N-terminally extended by a residue comprising 1 to 10 histidine residues;
- and
- (v) mutants as per a) and b), in each case additionally comprising the addition mutation as per (c) and/or (d);
- or selected among
- (vi) the single mutants
- P185X1
- T188X3
- (vii) the dual mutants
- P185X1/T188X3;
- L68X2/T188X3;
- L68X2/T185X1;
- 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;
- (viii) optionally additionally with C-terminal extension comprising 1 to 10 histidine residues;
- and/or optionally additionally with N-terminal extension comprising 1 to 10 histidine residues;
- and in the presence of at least one 7β-HSDH to the corresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of the formula (5)
- in which R has the abovementioned meanings or the group —CO2R is replaced by the acid amide group —CONR1R2 as defined hereinabove, in particular in the presence and with the consumption of NADH and/or NADPH, and subsequently
- c) 12-keto-UDCA of the formula (5) is reduced chemically to UDCA;
- and
- d) optionally, the reaction product is purified further.
Dependencies are inferred from the claim text.