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1. WO2020109776 - BIOCATALYTIC TECHNIQUES

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[ EN ]

CLAIMS

1. Use of a cytochrome P450 enzyme comprising any of SEQ ID NO: 1 -1 18, or mutants thereof or a variant enzyme having at least 70% identity thereto and having CYP450 activity, for the hydroxylation and or dealkylation of an organic compound.

2. The use according to claim 1 , wherein the cytochrome P450 enzyme used to catalyse the hydroxylation and/or dealkylation of an organic compound is any of SEQ ID NO: 2, 29, 34, 47, 51 or 109 or mutant or a variant enzyme having at least 70% identity thereto and having CYP450 activity.

3. The use according to claims 1 or 2, wherein the cytochrome P450 enzyme is used to catalyse the hydroxylation and/or removal of an alkyl or aryl group.

4. The use according to claims 1 , 2 or 3, wherein the compound to be hydroxylated is of formula (I), where R represents the rest of the compound and where R1 , R2 and R3 is alkyl or H:


5. The use according to claims 1 , 2 or 3, wherein the compound to be dealkylated is of formula (II), where where R represents the rest of the compound, y is the number of bonded moieties depending on the valency of X, X is oxygen or nitrogen, and where R1 is the leaving moiety and where R1 is alkyl:

R(y)-X-R1 (I I)

6. The use according to any preceding claim, wherein the compound to be hydroxylated is bosentan, cyclosporine A, diclofenac, ritonavir, valsartan or vanoxerine.

7. The use according to any preceding claim, wherein the compound to be dealkylated is bosentan, disopyramide or ritonavir.

8. The use according to any preceding claim, wherein the cytochrome P450 enzyme is used in combination with reductase components, preferably with ferredoxin and ferredoxin reductase components.

9. The use according to any preceding claim, wherein the cytochrome P450 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.

10. The use according to any preceding claim, wherein the P450 enzyme is in purified or part-purified form, crude enzyme extract, in a recombinant host cell or a natural host cell.

1 1. The use according to any preceding claim, wherein the cytochrome P450 enzyme or variant thereof is present in Streptomyces rimosus NRRL-2234 cells. 12. The use according to any preceding claim, wherein the cytochrome P450 enzyme or variant thereof is expressed by at least one recombinant

microorganism comprising heterologous nucleic acid encoding the enzyme, derived from Streptomyces rimosus NRRL-2234.

13. A method for the production of a hydroxylated and/or dealkylated organic compound, comprising reacting the organic compound with a cytochrome P450 enzyme comprising any of SEQ ID NO: 1 -1 18, or mutant or a variant enzyme having at least 70% identity thereto and having CYP450 activity.

14. The method according to claim 13, wherein the cytochrome P450 enzyme used to catalyse the hydroxylation and/or dealkylation of an organic compound is any of SEQ ID NO: 2, 29, 34, 47, 51 or 109 or mutant or a variant enzyme having at least 70% identity thereto and having CYP450 activity.

15. The method according to claim 13 or claim 14, wherein the 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.

16. The method according to any of claims 13 to 15, wherein the enzyme is used to catalyse the hydroxylation of a compound of formula (II), where R represents the rest of the compound and where R1 is CH3 or H:


17. The method according to any of claims 13 to 16, wherein the compound to be hydroxylated and/or dealkylated is bosentan, diclofenac, buparvaquone, BIRB796 or ritonavir.

18. The method according to claims 13 to 17, wherein the cytochrome P450 enzyme is used in combination with reductase components, preferably with ferredoxin and ferredoxin reductase components.

19. The method according to claims 13 to 18, wherein the cytochrome P450 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.

20. The method according to claims 13 to 19, wherein the P450 enzyme is in purified form, part-purified form, crude enzyme extract, in a recombinant host cell or a natural host cell.

21. A method according to claims 13 to 20, wherein the cytochrome P450 enzyme or variant thereof is present in Streptomyces rimosus NRRL-2234 cells and wherein said cells are dosed with an organic compound to be hydroxylated and/or dealkylated, optionally wherein the cells are subsequently harvested and purified to obtain the hydroxylated and/or dealkylated compound.

22. A method according to claims 13 to 21 , wherein the cytochrome P450 enzyme or variant thereof is expressed by at least one recombinant

microorganism comprising heterologous nucleic acid encoding the enzyme, derived from Streptomyces rimosus NRRL-2234, wherein the at least one recombinant microorganism is dosed with an organic compound to be

hydroxylated and/or dealkylated, optionally followed by a purification step to obtain the hydroxylated and/or dealkylated compound.

23. A kit comprising i) a cytochrome P450 enzyme comprising any of SEQ I D NOs: 1 -1 18, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, or ii) a microorganism that expresses a cytochrome P450 enzyme comprising any of SEQ ID Nos 1 -1 18, or a variant enzyme having at least 70% identity thereto and having CYP450 activity, and wherein the kit further comprises instructions for use for the hydroxylation of an organic compound.

24. The use according to claim 23, wherein the cytochrome P450 enzyme used to catalyse the hydroxylation and/or dealkylation of an organic compound is any of SEQ ID NO: 2, 29, 34, 47, 51 or 109 or mutant or a variant enzyme having at least 70% identity thereto and having CYP450 activity.

25. A kit for use according to claim 23 or 24, wherein the kit further comprises a reducing agent, preferably ferredoxin reductase and a ferredoxin, optionally wherein the kit further comprises a buffer.

26. A kit according to claims 23 or 25, further comprising one or more other CYP450 enzymes.

27. A kit according to claims 23 to 26 wherein the cytochrome P450 enzyme or microorganism is lyophilised and sealed with or without being under vacuum.