Enzymatic hydroxylation of aliphatic hydrocarbon

Abstract

The invention relates to enzymatic methods for hydroxylation in position 2 or 3 of substituted or unsubstituted, linear or branched aliphatic hydrocarbons.

Claims

1. A method for hydroxylation in position 2 or 3 of either end of a substituted or unsubstituted, linear or branched, aliphatic hydrocarbon having at least 3 carbons and having a hydrogen attached to the carbon in position 2 or 3, comprising contacting the aliphatic hydrocarbon with hydrogen peroxide and a polypeptide having peroxygenase activity; wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:2.

2. The method of claim 1, wherein the carbon in position 2 or 3, which is hydroxylated, is unsubstituted until it is contacted with the peroxygenase.

3. The method of claim 1, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:9.

4. The method of claim 1, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:10.

5. The method of claim 1, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:11.

6. The method of claim 1, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:12.

7. The method of claim 1, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:13.

8. The method of claim 1, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:14.

9. The method of claim 1, wherein the polypeptide has at least 95% sequence identity to SEQ ID NO:2.

10. The method of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:2.

11. The method of claim 1, wherein the polypeptide is a fragment of SEQ ID NO:2 having peroxygenase activity.

12. The method of claim 1, wherein the substituents of the aliphatic hydrocarbon are selected from the group consisting of halogen, hydroxyl, carboxyl, amino, nitro, cyano, thiol, sulphonyl, formyl, acetyl, methoxy, ethoxy, phenyl, benzyl, xylyl, carbamoyl and sulfamoyl.

13. The method of claim 1, wherein the substituents are selected from the group consisting of chloro, hydroxyl, carboxyl and sulphonyl; in particular chloro and carboxyl.

14. The method of claim 1, wherein the aliphatic hydrocarbon is unsubstituted.

15. The method of claim 1, wherein the aliphatic hydrocarbon is linear.

16. The method of claim 1, wherein the aliphatic hydrocarbon is an alkane.

17. The method of claim 16, wherein the alkane is propane, butane, pentane, hexane, heptane, octane, nonane or decane, or isomers thereof.

18. The method of claim 1, wherein the aliphatic hydrocarbon is part of a fatty acid.

19. A method for hydroxylation in position 2 or 3 of the terminal end of an acyl group of a lipid, comprising contacting the lipid with hydrogen peroxide and a polypeptide having peroxygenase activity; wherein the polypeptide has at least 90% sequence identity to SEQ ID NO:2.

20. The method of claim 19, wherein the polypeptide has at least 95% identity to the polypeptide of SEQ ID NO:2.

Description

EXAMPLES

(1) The polypeptide having peroxygenase activity from Agrocybe aegerita, which is shown as SEQ ID NO:2, is referred to as AaeAPO in the following examples.

Example 1

(2) Hydroxylation of N-Hexane

(3) Enzymatic hydroxylation of hexane was performed in the pure substrate (n-hexane, >97%, Sigma Aldrich) containing 2 U ml.sup.1 (0.31 nmol) AaeAPO added as aqueous enzyme solution (10 l). H.sub.2O.sub.2 (4 mM) was added by syringe pumps over 1 hour. The experiment was done in 200 l scale (total volume) in 1 ml glass vials stirred with a magnetic stirrer. Products were analyzed by GC-MS (Varian) by direct injection of the reaction mixture. Controls were processed identically except that water (10 pl) was added instead of enzyme solution.

(4) The gas chromatogram and mass spectra of the sample with active enzyme (AaeAPO) and n-hexane showed formation of high amounts of 2-hexanol, and 3-hexanol; the control without enzyme did not contain any of these peaks.

Example 2

(5) Hydroxylation of N-Decane

(6) Enzymatic conversion (in 200 l total volume) was done in pure n-decane (>97%, Sigma Aldrich) supplemented with 2 U ml.sup.1 (0.31 nmol) AaeAPO. H.sub.2O.sub.2 (8 mM) was added by syringe pumps over 2 hours and the sample was stirred with a magnetic stirrer. Products were measured by GC-MS. Controls were processed identically except that water (10 l) was added instead of enzyme.

(7) The gas chromatogram and mass spectra of the sample with active enzyme (AaeAPO) and n-decane showed formation of high amounts of two n-alkanols, 3-decanol and 2-decanol; the control without enzyme did not contain these peaks.

Example 3

(8) Enzymatic Hydroxylation of Lauric Acid

(9) Enzymatic hydroxylation of lauric acid was performed using a total reaction mixture of 4 ml containing 50 mM potassium phosphate buffer, 40 v/v % acetronitrile, 1 mM lauric acid (>98% pure, Aldrich W261408 was dissolved in acetonitrile), 0.01 mg peroxygenase protein/ml (the peroxygenase shown as SEQ ID NO:4) and 2 mM ascorbic acid was added according to the table below.

(10) The reaction was started by addition of hydrogen peroxide corresponding to a concentration of 0.5 mM in the reaction mixture. The reaction mixtures were incubated for 60 minutes at 35 C. using a heat block. A second addition of peroxide was added after 30 minutes incubation to a total concentration of 1 mM. The reactions were stopped by a heat treatment of 85 C. in a water bath for 5 minutes. Products were measured by GC-FID (Varian 3900) by injection at 100 C. in split mode with ratio of 10:1 (helium was used as carrier gas at a constant flow of 25 ml/min). A temperature gradient were applied heating to 200 C. at a rate of 10 C./min, then proceeding to 360 C. at a rate of 50 C./min. The results were recorded as peak area (see Table 1).

(11) TABLE-US-00006 TABLE 1 Lauric acid Product Treatment (Area@3.2 min) (Area @4.3 min) Peroxygenase + H.sub.2O.sub.2 No peak No peak Lauric acid + H.sub.2O.sub.2 + ascorbic acid 16011 91 Lauric acid + peroxygenase + 14406 395.5 H.sub.2O.sub.2 + ascorbic acid

(12) A product peak appeared in the presence of the peroxygenase. The elution time of the product was slightly shifted compared to 12-Hydroxydodecanoic acid, which is an iso-form of 2-hydroxy lauric acid and 3-hydroxy lauric acid. Hence, the elution time was in accordance to the expected product hydroxylated in the 2 or 3 position.

Example 4

(13) Enzymatic Hydroxylation of Palmitic Acid

(14) Enzymatic hydroxylation of palmitic acid was performed using a total reaction mixture of 4 ml containing 50 mM potassium phosphate buffer, 40 v/v % acetronitrile, 1 mM palmitic acid (>99% pure, Sigma P0500) and 0.01 mg peroxygenase protein/ml (the peroxygenase shown as SEQ ID NO:4).

(15) The reaction was started by addition of hydrogen peroxide corresponding to a concentration of 1 mM in the reaction mixture. The reaction mixtures were incubated for 1, 2, 3 and 10 minutes at 35 C. using a heat block. The reactions were stopped by a heat treatment of 85 C. in a water bath for 5 minutes. Products were measured by GC-FID (Varian 3900) by injection at 100 C. in split mode with ratio of 10:1 (helium was used as carrier gas at a constant flow of 25 ml/min). A temperature gradient were applied heating to 200 C. at a rate of 10 C./min, then proceeding to 360 C. at a rate of 50 C./min. The results were recorded as peak area (see Table 2).

(16) TABLE-US-00007 TABLE 2 Incubation time Palmitic acid Product (min) (Area) (Area) 0 1799.5 No peak 1 1481.2 82.8 2 979.1 191.3

(17) A product peak appeared already after 1 minutes of incubation, and increased after two minutes incubation. The elution profile was slightly shifted compared to 16-Hydroxyhexadecanoic acid, which is an iso-form of 2-hydroxy palmitic and 3-hydroxy palmitic. Hence, the elution time was in accordance to the expected product hydroxylated in the 2 or 3 position.