Hydroxylation techniques

The use according to any preceding claim, wherein the cytochrome P-450 enzyme is used to catalyse the hydroxylation of a propyl group or a butyl group, preferably to catalyse the hydroxylation of an isopropyl or isobutyl group, more preferably to catalyse the hydroxylation of a tert-butyl group.

The use according to any preceding claim, wherein the compound to be hydroxylated is of formula (II), where R represents the rest of the compound and where R1 is CH3 or H:

The use according to any preceding claim, wherein the compound to be hydroxylated is bosentan, diclofenac, buparvaquone, BIRB796 or ritonavir.

The use according to any preceding claim, wherein the cytochrome P-450 enzyme is used in combination with reductase components, preferably with ferredoxin and ferredoxin reductase components.

The use according to any preceding claim, wherein the cytochrome P-450 enzyme comprises a sequence having at least 90% identity thereto, preferably 95% identity thereto, more preferably 96% identity thereto, even more preferably 97% identity thereto, most preferably 100% identity thereto.

The use according to any preceding claim, wherein the P-450 enzyme is in purified or part-purified form, crude enzyme extract, in a recombinant host cell or a natural host cell.

The use according to any preceding claim, wherein the amino acid at position 291 is Aspartic acid or Glutamic acid.

The use according to any preceding claim, wherein the cytochrome P-450 enzyme or variant thereof is expressed by at least one recombinant microorganism comprising heterologous nucleic acid encoding the enzyme, derived from Amycolatopsis lurida (NRRL 2430).

A method for the production of a hydroxylated organic compound, comprising reacting the organic compound with a cytochrome P-450 enzyme comprising SEQ ID NO:

• 110, or a variant enzyme having at least 70% identity thereto and having CYP-450 activity, wherein the amino acid residue at position 291 is not threonine.

A kit comprising i) a cytochrome P-450 enzyme comprising SEQ ID NO:

• 110, or a variant enzyme having at least 70% identity thereto and having CYP-450 activity, or ii) a microorganism that expresses a cytochrome P-450 enzyme comprising SEQ ID NO:
• 110, or a variant enzyme having at least 70% identity thereto and having CYP-450 activity, wherein the amino acid at position 291 is not threonine and wherein the kit further comprises instructions for use for the hydroxylation of an organic compound.