Hydroxylation techniques
The use of 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, for the hydroxylation of an organic compound, wherein the amino acid residue at position 291 is not threonine.
1. A kit comprising:
i) an enzyme having cytochrome P-450 activity and comprising the amino acid sequence set forth in SEQ ID NO: 110, or a sequence having at least 90% identity thereto, wherein the amino acid residue at the position corresponding to position 291 of SEQ ID NO: 110 is aspartic acid, glutamic acid, asparagine, glutamine, valine, methionine, phenylalanine, tryptophan, isoleucine, proline or histidine; or
ii) a microorganism that expresses an enzyme as defined in (i); or
iii) an extract of the microorganism of (ii).
2. The kit of claim 1 , further comprising a reducing agent.
3. The kit of claim 1 , further comprising one or more other cytochrome P-450 enzymes.
4. The kit of claim 1 , wherein the cytochrome P-450 enzyme, microorganism or extract is lyophilised.
5. The kit of claim 1 , wherein the amino acid at the position of the enzyme corresponding to position 291 of SEQ ID NO: 110 is glutamic acid.
6. The kit of claim 5 , wherein the enzyme comprises the amino acid sequence set forth in SEQ ID NO: 12 or a sequence having at least 95% identity thereto.
7. The kit of claim 5 , wherein the amino acid at the position of the enzyme corresponding to position 47 of SEQ ID NO: 110 is isoleucine, or the amino acid at the position of the enzyme corresponding to position 315 of SEQ ID NO: 110 is leucine.
8. The kit of claim 7 , wherein the enzyme comprises the amino acid sequence set forth in SEQ ID NO: 84 or 108, or a sequence having at least 95% identity thereto.
9. The kit of claim 1 , wherein the enzyme comprises the amino acid sequence set forth in SEQ ID NO: 8, 16, 20, 28, 44, 48, 52, 56, 60 or 64, or a sequence having at least 95% identity thereto.
10. An enzyme having cytochrome P-450 activity and comprising the amino acid sequence set forth in SEQ ID NO: 110, or a sequence with at least 90% sequence identity thereto, wherein the amino acid residue at the position corresponding to position 291 of SEQ ID NO: 110 is aspartic acid, glutamic acid, asparagine, glutamine, valine, methionine, phenylalanine, tryptophan, isoleucine, proline or histidine.
11. The enzyme of claim 10 , wherein the amino acid at the position corresponding to position 291 of SEQ ID NO: 110 is glutamic acid.
12. The enzyme of claim 11 , comprising the amino acid sequence set forth in SEQ ID NO: 12 or a sequence having at least 95% identity thereto.
13. The enzyme of claim 11 , wherein the amino acid at the position corresponding to position 47 of SEQ ID NO: 110 is isoleucine, or the amino acid at the position corresponding to position 315 of SEQ ID NO: 110 is leucine.
14. The enzyme of claim 13 , comprising the amino acid sequence set forth in SEQ ID NO: 84 or 108, or a sequence having at least 95% sequence identity thereto.
15. The enzyme of claim 10 , comprising the amino acid sequence set forth in SEQ ID NO: 8, 16, 20, 28, 44, 48, 52, 56, 60 or 64, or a sequence having at least 95% identity thereto.
16. A method for the production of a hydroxylated organic compound, comprising reacting the organic compound with an enzyme according to claim 10 .
17. The method according to claim 16 , wherein the enzyme is used to catalyse the hydroxylation of a propyl group or a butyl group of the compound.
18. The method according to claim 16 , wherein the P-450 enzyme is in purified form, part-purified form, a crude enzyme extract or a recombinant host cell.
19. The kit of claim 2 , wherein the reducing agent is a ferredoxin reductase and a ferredoxin, wherein these are provided either as discrete components or linked within a single fusion protein construct.
20. The kit of claim 1 , further comprising a buffer.