Method for producing rare fatty acid using novel enzyme, and novel rare fatty acid

Abstract

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 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 20 carbon atoms and a carbonyl group at the 12-position, comprising subjecting an unsaturated fatty acid having 18 or 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 . 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.    
     
     
         16 . 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 . 
     
     
         17 . 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.    
     
     
         18 . 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 . 
     
     
         19 . 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 . 
     
     
         20 . 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 . 
     
     
         21 . 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 . 
     
     
         22 . 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 . 
     
     
         23 . 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 . 
     
     
         24 . 14-Oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, which is produced by the method according to  claim 15 . 
     
     
         25 . 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, or 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid.