METHOD FOR PRODUCING RARE FATTY ACID USING NOVEL ENZYME, AND NOVEL RARE FATTY ACID
The present invention provides production of hydroxylated fatty acid by a hydration reaction using a novel enzyme derived from lactobacillus and using fatty acid as a substrate, and further, a production method of oxo fatty acid by an enzyme reaction or chemical oxidation reaction using the hydroxylated fatty acid as a substrate. In addition, a valuable novel rare fatty acid obtained by such production method is also provided.
1 . An enzyme protein of any of the following (a)-(c):
(a) the enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 2,
(b) a protein comprising an amino acid sequence wherein one or plural amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted and/or substituted and/or inserted and/or added, and having an enzyme activity that catalyzes a hydration reaction,
(c) a protein encoded by a base sequence that hybridizes to a nucleic acid consisting of a chain sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions, and having an enzyme activity that catalyzes a hydration reaction.
2 . Lactobacillus acidophilus fungus or fungal debris thereof, comprising the enzyme protein according to claim 1 .
3 . A nucleic acid encoding the enzyme protein according to claim 1 .
4 . A vector comprising the nucleic acid according to claim 3 .
5 . A host cell transformed with the vector according to claim 4 .
6 . A method of producing the enzyme protein according to claim 1 , comprising culturing the host cell according to claim 5 and recovering the enzyme from the culture.
7 . A method of producing a hydroxylated fatty acid having 18 carbon atoms and a hydroxyl group at the 13-position, comprising subjecting an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position to a hydration reaction using the enzyme protein according to claim 1 , wherein the unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position is linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid, 10-hydroxy-cis-12-octadecenoic acid, 10-hydroxy-cis-6,cis-12-octadecadienoic acid, 10-hydroxy-cis-12,cis-15-octadecadienoic acid, 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid, 10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid, or columbinic acid.
8 . A method of producing an oxo fatty acid having 18 carbon atoms and a carbonyl group at the 13-position, comprising subjecting an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 18 carbon atoms and a hydroxyl group at the 13-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position is linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid, 10-hydroxy-cis-12-octadecenoic acid, 10-hydroxy-cis-6,cis-12-octadecadienoic acid, 10-hydroxy-cis-12,cis-15-octadecadienoic acid, 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid, 10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid, or columbinic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.
9 . A method of producing a hydroxylated fatty acid having 16 carbon atoms and a hydroxyl group at the 10-position, comprising subjecting an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position to a hydration reaction using the enzyme protein according to claim 1 , wherein the unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position is pulmitoleic acid.
10 . A method of producing an oxo fatty acid having 16 carbon atoms and a carbonyl group at the 10-position, comprising subjecting an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 16 carbon atoms and a hydroxyl group at the 10-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position is pulmitoleic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.
11 . A method of producing a hydroxylated fatty acid having 20 carbon atoms and a hydroxyl group at the 15-position, comprising subjecting an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position to a hydration reaction using the enzyme protein according to claim 1 , wherein the unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position is cis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic acid, dihomo-γ-linolenic acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid, sciadonic acid, or juniperonic acid.
12 . A method of producing an oxo fatty acid having 20 carbon atoms and a carbonyl group at the 15-position, comprising subjecting an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 20 carbon atoms and a hydroxyl group at the 15-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position is cis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic acid, dihomo-γ-linolenic acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid, sciadonic acid, or juniperonic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.
13 . A method of producing a hydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxyl group at the 12-position, comprising subjecting an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position to a hydration reaction using the enzyme protein according to claim 1 , wherein the unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position is cis-vaccenic acid, cis-11-cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic, dihomo-γ-linolenic acid, mead acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, or arachidonic acid.
14 . A method of producing an oxo fatty acid having 18 or carbon atoms and a carbonyl group at the 12-position, comprising subjecting an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxyl group at the 12-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position is cis-vaccenic acid, cis-11-cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic, dihomo-γ-linolenic acid, mead acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, or arachidonic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.
15 .- 18 . (canceled)
19 . A method of producing 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, comprising subjecting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to a hydration reaction using the enzyme protein according to claim 1 .
20 . A method of producing 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, comprising subjecting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to a hydration reaction using the enzyme protein according to claim 1 to induce 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.
21 . A method of producing 10-hydroxy-tetradecanoic acid, comprising subjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydration reaction using the enzyme protein according to claim 1 .
22 . A method of producing 10-oxo-tetradecanoic acid, comprising subjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydration reaction using the enzyme protein according to claim 1 to induce 10-hydroxy-tetradecanoic acid, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.
23 .- 24 . (canceled)
25 . 13-Hydroxy-cis-9,cis-15-octadecadienoic acid, 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dihydroxy-cis-6-octadecenoic acid, 10,13-dihydroxy-cis-15-octadecenoic acid, 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid, 10-oxo-13-hydroxy-cis-6-octadecenoic acid, 10-oxo-13-hydroxy-cis-15-octadecenoic acid, 10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid, 13-hydroxy-cis-5,cis-9-octadecadienoic acid, or 13-hydroxy-trans-5,cis-9-octadecadienoic acid, which is produced by the method according to claim 7 .
26 . 15-Hydroxy-cis-11,cis-17-eicosadienoic acid, 15-hydroxy-cis-8, cis-11-eicosadienoic acid, 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-hydroxy-cis-5,cis-11-eicosadienoic acid, or 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by the method according to claim 11 .
27 . 12-Hydroxy-cis-14-eicosenoic acid, 12-hydroxy-cis-14,cis-17-eicosadienoic acid or 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced by the method according to claim 13 .
28 . 13-Oxo-cis-6,cis-9-octadecadienoic acid, 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoic acid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid, 13-oxo-cis-5,cis-9-octadecadienoic acid or 13-oxo-trans-5,cis-9-octadecadienoic acid, which is produced by the method according to claim 8 .
29 . 15-Oxo-cis-11,cis-17-eicosadienoic acid, 15-oxo-cis-8,cis-11-eicosadienoic acid, 15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-oxo-cis-5,cis-11-eicosadienoic acid or 15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by the method according to claim 12 .
30 . 12-Oxo-cis-14-eicosenoic acid, 12-oxo-cis-14,cis-17-eicosadienoic acid, 12-oxo-cis-8,cis-14-eicosadienoic acid, 12-oxo-cis-5,cis-8-eicosadienoic acid or 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced by the method according to claim 14 .
31 . 14-Hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid produced by the method according to claim 19 .
32 . 14-Oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, which is produced by the method according to claim 20 .
33 . A fatty acid-containing substance comprising 13-hydroxy-cis-9,cis-15-octadecadienoic acid, 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dihydroxy-cis-6-octadecenoic acid, 10,13-dihydroxy-cis-15-octadecenoic acid, 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid, 10-oxo-13-hydroxy-cis-6-octadecenoic acid, 10-oxo-13-hydroxy-cis-15-octadecenoic acid, 10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid, 13-hydroxy-cis-5,cis-9-octadecadienoic acid, 13-hydroxy-trans-5,cis-9-octadecadienoic acid, 15-hydroxy-cis-11,cis-17-eicosadienoic acid, 15-hydroxy-cis-8,cis-11-eicosadienoic acid, 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-hydroxy-cis-5,cis-11-eicosadienoic acid, 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid, 12-hydroxy-cis-14-eicosenoic acid, 12-hydroxy-cis-14,cis-17-eicosadienoic acid, 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid, 13-oxo-cis-6,cis-9-octadecadienoic acid, 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoic acid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid, 13-oxo-cis-5,cis-9-octadecadienoic acid, 13-oxo-trans-5,cis-9-octadecadienoic acid, 15-oxo-cis-11,cis-17-eicosadienoic acid, 15-oxo-cis-8,cis-11-eicosadienoic acid, 15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-oxo-cis-5,cis-11-eicosadienoic acid, 15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, 12-oxo-cis-14-eicosenoic acid, 12-oxo-cis-14,cis-17-eicosadienoic acid, 12-oxo-cis-8,cis-14-eicosadienoic acid, 12-oxo-cis-5,cis-8-eicosadienoic acid, 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid, 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, or 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid.
34 . (canceled)