Method for biocatalytic whole cell reduction of dehydrocholic acid compounds, and 7-β-hydroxysteroid dehydrogenase mutants
AssigneePharmaZell GmbH
InventorsDirk Weuster-Botz, Boqiao Sun, Christian Quirin
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Abstract
The invention relates to novel biocatalytic processes comprising the whole cell reduction of dehydrocholic acid (DHCA) compounds, novel 7β-hydroxy steroid dehydrogenase mutants, the sequences coding for these enzyme mutants, methods for producing the enzyme mutants and use thereof in enzymatic conversions of cholic acid compounds, and in particular in the production of ursodesoxycholic acid (UDCA); also a subject of the invention are novel methods for the synthesis of UDCA using the enzyme mutants; and in particular a further improved method for producing UDCA using recombinant whole cell biocatalysts.
Claims37
A 7β-hydroxysteroid dehydrogenase (7β-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the enzyme has an amino acid sequence having at least 90% sequence identity to SEQ ID NO:
- 2 and comprises:
- a) the double mutation G39D/R40F, wherein the amino acid numbering of said mutation corresponds to positions 39 and 40 of SEQ ID NO:
- 2:
- b) the triple mutation G39D/R40F/R41X1, wherein X1 represents any other amino acid residue:
- or
- c) the quadruple mutation G39D/R40F/R41X1/K44X2, wherein X1 represents any other amino acid residue,
- wherein the amino acid numbering of said triple and quadruple mutations corresponds to positions 39, 40, 41 and 44 respectively of SEQ D NO:
- 2.
The method of 23, wherein, if GDH is not added to the liquid reaction medium, the one or more whole cell biocatalysts express the enzyme activity of GDH.
Dependencies are inferred from the claim text.