Production of fatty acid estolides

Patent metadata

Field	Value
Publication number	US-10920252-B2
Application number	US-201615561644-A
Country	US
Kind code	B2
Filing date	Mar 24, 2016
Priority date	Mar 26, 2015
Publication date	Feb 16, 2021
Grant date	Feb 16, 2021

Abstract

It has been found that esterification of a hydroxy-fatty acid by a lipase can be coupled with oleate hydratase (OHase) generation of that hydroxy-FA from an unsaturated FA with a cis C9-C10 double bond, e.g. oleic acid, in a single aqueous buffered reaction medium at low temperature, e.g. 30° C. A simple one-pot enzymatic method to produce fatty acid estolides from one or more triglycerides, e.g. starting from a natural plant oil, is thereby enabled in which the same lipase catalyses both the initial hydrolysis of triglyceride and the final esterification step.

First claim

The invention claimed is:

1. A one-pot method for producing one or more esters of one or more hydroxy-fatty acids in a single aqueous buffered, cell-free reaction medium said method comprising:
(a) providing in said single aqueous buffered, cell-free reaction medium at least one unsaturated fatty acid with a cis C9-C 10 double bond;
(b) reacting said at least one unsaturated fatty acid with a cis C9-C10 double bond in said single aqueous buffered, cell-free reaction medium with an isolated cell-free oleate hydratase to produce at least one hydroxy-fatty acid; and
(c) producing one or more esters of said at least one hydroxy-fatty acid in said single aqueous buffered, cell-free reaction medium by means of an isolated lipase, wherein the isolated lipase and the isolated cell-free oleate hydratase are contemporaneously present in the single aqueous buffered, cell-free reaction medium, wherein to the extent the single aqueous buffered, cell-free reaction medium comprises an organic solvent, the organic solvent is not present in an amount such that the hydratase enzyme is rendered inactive in the reaction medium to produce the at least one hydroxy-fatty acid.

2. The one-pot method as claimed in claim 1 wherein said isolated cell-free oleate hydratase is recombinant oleate hydratase of Elizabethkingia meningoseptica.

3. The one-pot method as claimed in claim 1 wherein said isolated cell-free oleate hydratase is a recombinant oleate hydratase with an N-terminal His tag covalently linked to glutaraldehyde-activated magnetic chitosan composite particles in which magnetic iron oxide particles are dispersed in a chitosan matrix.

4. The one-pot method as claimed in claim 1 wherein the single aqueous buffered, cell-free reaction medium is further supplemented by one or more triglycerides, said one or more triglycerides being hydrolysed by said isolated lipase to provide said at least one unsaturated fatty acid with a cis C9-C10 double bond which is a substrate for said isolated cell-free oleate hydratase.

5. The one-pot method as claimed in claim 4 wherein the single aqueous buffered, cell-free reaction medium is continuously mixed to aid dispersion of said one or more triglycerides in said single aqueous buffered, cell-free reaction medium.

6. The one-pot method as claimed in claim 4 , wherein the isolated cell-free oleate hydratase and the isolated lipase are contacted with triolein or an oil comprising triolein dispersed in the single aqueous buffered, cell-free reaction medium whereby the following steps occur consecutively without any separation step:
(i) hydrolysis of triolein to generate oleic acid;
(ii) conversion of oleic acid to 10-hydroxystearic acid (10-HSA) by said isolated cell-free oleate hydratase and
(iii) conversion of 10-HSA to one or more estolides by esterification with itself and/or oleic acid.

7. The one-pot method as claimed in claim 6 wherein said isolated lipase is selected from the lipase of Candida rugosa or a Pseudomonas species such that said one or more fatty estolides consist of, or essentially consist of, the monoestolide of 10-HSA with oleic acid or said monoestolide and a 10-HSA ester dimer respectively.

8. The one-pot method as claimed in claim 1 wherein said isolated lipase is a lipase with non-1,3 positional specificity for hydrolysis of triglyceride.

9. The one-pot method as claimed in claim 8 wherein said isolated lipase is selected from microbial lipases of Candida species, Pseudomonas species, Chromobacterium species, Geotrichum species, Alcaligenes species and Thermomyces species.

10. The one-pot method as claimed in claim 1 which is carried out at about pH 6 to 8 and at about 20-40° C.

11. The one-pot method as claimed in claim 10 which is carried out at about pH 6.5 and about 30° C.

12. The one-pot method as claimed in claim 1 wherein the single aqueous buffered, cell-free reaction medium is an aqueous buffered solution containing NaCl at a concentration up to about 150-200 mM.

13. The one-pot method as claimed in claim 1 , wherein said one or more esters are one or more fatty acid estolides.

14. The one-pot method as claimed in claim 13 wherein said one or more hydroxy-fatty acids are selected mono-10-hydroxy fatty acids.

15. The one-pot method as claimed in claim 14 wherein 10-hydroxystearic acid is the sole hydroxy-fatty acid provided for esterification by said isolated lipase.

16. The one-pot method as claimed in claim 1 , which further comprises extraction of one or more ester products from the single aqueous buffered, cell-free reaction medium or extraction and further purification of one or more ester products and/or incorporation into a composition.

17. The one-pot method as claimed in claim 16 , wherein one or more ester products of lipase esterification are further reacted.

18. The one-pot method as claimed in claim 1 , wherein one or more ester products of lipase esterification are further reacted.

19. The one-pot method as claimed in claim 1 , wherein the single aqueous buffered, cell-free reaction medium comprises less than 5% v/v organic solvent.

20. A one-pot enzymic method for producing one or more esters of one or more hydroxy-fatty acids wherein at least one such hydroxy-fatty acid is a hydroxy-fatty acid obtained by action of an isolated cell-free oleate hydratase on an unsaturated fatty acid substrate with a cis C9-C10 double bond, said method comprising incubating an isolated cell-free oleate hydratase and an isolated lipase in a single aqueous buffered, cell-free reaction medium to carry out the following steps without any separation step:
(i) hydrolysis of one or more triglycerides by said isolated lipase to generate one or more unsaturated fatty acids with a cis C9-C 10 double bond as substrate for said isolated cell-free oleate hydratase;
(ii) conversion of said one or more unsaturated fatty acids to one or more hydroxy-fatty acids by said isolated cell-free oleate hydratase; and
(iii) conversion of said one or more hydroxy-fatty acids by said isolated lipase to one or more esters, wherein to the extent the single aqueous buffered, cell-free reaction medium comprises an organic solvent, the organic solvent is not present in an amount such that the hydratase enzyme is rendered inactive in the reaction medium to produce the one or more hydroxy-fatty acids.

21. The one-pot method as claimed in claim 20 , which further comprises extraction of one or more ester products from the single aqueous buffered, cell-free reaction medium or extraction and further purification of one or more ester products and/or incorporation into a composition.

22. The one-pot method as claimed in claim 21 , wherein one or more ester products of lipase esterification are further reacted.

23. The one-pot method as claimed in claim 20 , wherein one or more ester products of lipase esterification are further reacted.

24. The one-pot method as claimed in claim 20 , wherein the single aqueous buffered, cell-free reaction medium comprises less than 5% v/v organic solvent.

25. A one-pot method for producing fatty acid estolides, said method comprising incubating an isolated cell-free oleate hydratase and an isolated lipase in a single aqueous buffered, cell-free reaction medium to carry out the following steps without any separation step:
(i) providing one or more triglycerides to said single aqueous buffered, cell-free reaction medium;
(ii) hydrolysis of the one or m

Assignees

• Stichting Wageningen Res
• Univ Delft Tech
• Stichling Wageningen Research Technische Univ Delft

Inventors

• Boeriu Carmen Gabriela
• Todea Anamaria
• Arends Isabella Wilhelmina Christina Everdina
• Otten Ludwina Gerharda

Classifications

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12P7/42

  Hydroxy-carboxylic acids · CPC title

• C12N9/20

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C12P7/64

  Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats · CPC title

• C12P7/6418

  by hydrolysis of fatty acid esters · CPC title