阅读说明：本技术 用于生产甘油二酯的方法 (Process for producing diglycerides ) 是由 J·A·克拉洛维奇 P·F·穆格福德 A·罗尔 于 2019-06-04 设计创作，主要内容包括：提供了一种用于生产甘油二酯的方法。所述方法包括将(i)包含至少一种呈乙酯形式的多不饱和脂肪酸、游离脂肪酸、和/或其组合的油,(ii)脂肪酶,和(iii)在水中的甘油组合以产生具有高纯度水平的甘油二酯。还提供了根据所述方法获得的高纯甘油二酯。(A process for producing diglycerides is provided. The method comprises combining (i) an oil comprising at least one polyunsaturated fatty acid in the form of an ethyl ester, free fatty acids, and/or combinations thereof, (ii) a lipase, and (iii) glycerol in water to produce diglycerides having a high level of purity. Also provided is a high purity diglyceride obtained according to the method.)

1. A process for producing one or more diglycerides comprising combining (i) an oil comprising at least one polyunsaturated fatty acid in the form of ethyl esters, free fatty acids, and/or combinations thereof, (ii) a lipase, and (iii) an alcohol in water.

2. The method of claim 1, wherein the lipase is derived from Candida antarctica (Candida antarctica).

3. The method of claim 1 or 2, wherein the lipase is lipase B.

4. The method of any one of claims 1-3, wherein the alcohol is glycerol.

5. The method of any one of claims 1-4, wherein the ethyl ester comprises an omega-3 ethyl ester.

6. The method of any one of claims 1-5, wherein the ethyl ester is eicosapentaenoic acid (EPA) ethyl ester, docosahexaenoic acid (DHA) ethyl ester, or a combination thereof.

7. The method of any one of claims 1-6, wherein the free fatty acids comprise omega-3 free fatty acids.

8. The method of any one of claims 1-7, wherein the free fatty acid is eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or a combination thereof.

9. The method of any one of claims 1-8, wherein the combination of ethyl esters, free fatty acids, and/or combinations thereof, lipase, and alcohol in water is reacted for between about 2 hours and about 24 hours.

10. The process of any one of claims 1-9, wherein the reaction is carried out at reduced pressure and at a sufficient temperature to evaporate the ethanol and/or water from the reaction.

11. The process of any one of claims 1-10, wherein the temperature during the reaction is from about 30 ℃ to about 90 ℃.

12. The process of any one of claims 1-11, wherein water is added back to the reaction mixture one or more times during the reaction.

13. The process of any one of claims 1-12, wherein water is continuously supplied during at least a portion of the reaction.

14. The method of any one of claims 1-13, wherein the reaction mixture is washed one or more times.

15. The method of any one of claims 1-14, wherein the reaction mixture is dried after the reaction is complete.

16. The process of any one of claims 1-15, wherein any residual ethyl esters, monoacylglycerols, and/or free fatty acids are separated from the reaction mixture.

17. A method for producing one or more diglycerides, comprising:

(a) combining (i) an oil comprising at least one polyunsaturated fatty acid in the form of an ethyl ester, free fatty acids, and/or combinations thereof, (ii) a lipase, and (iii) an alcohol in water;

(b) incubating the combination of (a) at about 30 ℃ to about 90 ℃ for about 2 to about 24 hours under reduced pressure to produce a reaction mixture;

(c) washing the reaction mixture obtained in (b);

(d) drying the reaction mixture;

(e) separating residual ethyl esters, one or more monoacylglycerols, and/or free fatty acids from the reaction mixture.

18. The method of claim 17, wherein the lipase is lipase B from candida antarctica.

19. The method of claim 17 or 18, wherein the alcohol is glycerol.

20. A process for producing diglycerides comprising combining (i) an oil comprising at least one polyunsaturated fatty acid in the form of ethyl esters, free fatty acids, and/or combinations thereof, (ii) a lipase, and (iii) glycerol in water to produce diglycerides having a high level of purity.

21. The method of any one of claims 1-20, wherein the amount of diacylglycerols as a percentage of the total product is from about 95% to about 100%.

22. The method of any one of claims 1-21, wherein the amount of diacylglycerols as a percentage of the total product is from about 96.5% to about 100%.

23. A diglyceride obtained according to the method of any one of claims 1-22.

24. Use of a diglyceride obtained according to the method of any one of claims 1-23, in a food product, a dietary supplement, a pharmaceutical product, or a cosmetic product.

Background

The present disclosure relates to a process for producing diglycerides. The method includes combining marine oil ethyl esters, free fatty acids, and combinations thereof, a lipase, and an alcohol in water to produce diglycerides having high purity levels.

The beneficial effects of long chain polyunsaturated fatty acids (PUFAs), particularly cis-5, 8,11,14, 17-eicosapentaenoic acid (EPA) and cis-4, 7,10,13,16, 19-docosahexaenoic acid (DHA), characteristic of marine lipids, on lowering serum triglycerides are now well established. Other cardioprotective benefits and other biological effects of these compounds are also known. The most commonly mentioned benefits are those associated with the prevention and treatment of inflammation, neurodegenerative diseases, and cognitive dysplasia. The public is becoming increasingly aware of the health benefits of fish oils and DHA and EPA concentrates as evidenced by the global marketing of polyunsaturated fatty acids (PUFAs).

Several methods are known for producing PUFA concentrates from marine oils, such as selective lipase hydrolysis, PUFA complexation using urea (or more complex molecular guest-host frameworks involving metric control), and physical removal of unwanted components by fractionation. U.S. publication No. 2004/0236128 describes the separation of EPA from DHA by precipitation of EPA magnesium salts.

Diacylglycerols (DAGs) are widely used in various applications, such as additives for improving the plasticity of oils and fats and edible oils in the food industry and as base materials for the production of cosmetics and pharmaceuticals. Recently, foods that focus on the beneficial physiological activities of diacylglycerols have attracted attention.

However, there are few examples of methods for producing PUFA-containing diglycerides.

U.S. patent No. 6,361,980 describes a process for preparing diacylglycerols comprising performing an esterification reaction between (1) an acyl donor selected from the group consisting of fatty acids, lower alcohol esters thereof, and mixtures thereof, and (2) an acyl acceptor selected from the group consisting of glycerol, monoacylglycerols, and mixtures thereof, using an enzyme packed column comprising an immobilized lipase preparation to obtain a reaction fluid from the enzyme packed column; reducing the water content or lower alcohol content in the reaction fluid; and recycling the reaction fluid to the enzyme-packed column after the reducing, wherein a residence time of the reaction fluid in the enzyme-packed column is 120 seconds or less; to obtain diacylglycerol, wherein the reducing comprises dehydrating or dealcoholizing the reaction fluid by feeding the reaction fluid through a nozzle during dehydration. However, this method is costly because it requires the use of expensive purified fatty acids as raw materials, immobilized lipase, and a dedicated packed enzyme column reactor. This process produces moderately pure diglycerides (88.6% -91.7%; DG purity ═ DG/(DG + TG), whose purity is reduced by the formation of triglycerides that are difficult to separate, also in this process.

JP 2004208539 describes a process for producing PUFA-containing diglycerides, wherein PUFA or lower alkyl esters thereof and glycerol are reacted in the presence of immobilized partial glyceride lipase while removing water produced during the reaction to the outside of the reaction system. However, the method requires monitoring of the acid value of the reaction, and the purity of the obtained diglycerides is low. Percent% DG purity is from 66% -85%; DG purity ═ DG/(DG + TG) (triglycerides are also formed)

JP 2004222594 describes a two-step process for producing PUFA-containing fats and oils, wherein glycerol is reacted in the presence of water and a lipase to carry out a glycerolysis reaction, and the resulting PUFA-containing partial glycerides and fatty acids or lower alkyl esters thereof are reacted in the presence of an immobilized partial glyceride lipase. However, the method requires two separate steps, and the purity of the obtained diglycerides is low. % DG purity 57% to 68% (triglyceride formation also

CN 101736044 describes a method for continuous enzymatic synthesis of n-3PUFA glycerides, which comprises mixing n-3PUFA (EPA, DHA) and glycerol into a reaction solution, and pumping the reaction solution into a device by a constant flow pump into an enzyme reaction column with immobilized lipase. The n-3PUFA glyceride products produced by the continuous synthesis process have an esterification rate of 30% to 50%, a monoester content of 20% to 30%, a diester content of 50% to 70%, and a triester content of 10% to 20%. Therefore, the amount and purity of diacylglycerols obtained by this method are low.

CN 101818176 describes a process for converting fatty acid ethyl esters into glycerides, comprising the steps of: mixing fatty acid ethyl ester and glycerol in a material tank; separating free glycerol by passing the material through a glycerol separator using a pump; then placing the material into a reactor filled with immobilized lipase; and passing the material through a packed column to remove ethanol; the material is finally returned to the material tank to carry out a cyclic reaction for 6 to 300 hours; the reaction product is then subjected to molecular distillation to remove unconverted reactants, thereby obtaining the glyceride product. Similar to the above process, a mixture of monoacylglycerols, diacylglycerols and triacylglycerols is obtained, wherein the concentration of diacylglycerols ranges from about 20% to 30%.

US 20070148745 describes the production of diacylglycerols, which comprises reacting triacylglycerols with water and an enzyme (such as an immobilized lipase) to obtain a mixture comprising diacylglycerols, monoacylglycerols and free fatty acids; removing the water content of the mixture by dewatering; and separating the monoacylglycerols, the free fatty acids and the residual triacylglycerols by at least one separation method to obtain the high-purity diacylglycerols. Thus, this process actually results in obtaining a mixture of glycerides as described above, and requires an additional step to separate the diacylglycerols from the monoacylglycerols, triacylglycerols, and free fatty acids. In particular, the reaction of triacylglycerols with water and immobilized lipase results in compositions comprising about 41% -44% diacylglycerol. After separation, a composition comprising about 88% -90% diacylglycerol is obtained.

Therefore, there is still a need to provide an improved process for producing high purity diglycerides.

The embodiments characterized below provide a solution to this technical problem.

Disclosure of Invention

The present application provides a process for producing diglycerides. In particular, the process of the present invention comprises combining (i) an oil comprising at least one polyunsaturated fatty acid in the form of an ethyl ester, free fatty acid, or a combination thereof, (ii) a lipase, and (iii) glycerol in water to produce diglycerides having a high level of purity.

In some embodiments, the lipase is lipase B derived from Candida antarctica (Candida antarctica).

In some embodiments, water is added back to the reaction at a rate sufficient to replace water evaporated under vacuum and such water levels are maintained for about the first 4-12 hours of the reaction.

In some embodiments, the reaction is carried out under reduced pressure. In some embodiments, the reaction is performed at about 20 mtorr.

After completion of the reaction, the reaction mixture may be washed one or more times with, for example, water, brine, or any combination thereof.

The reaction mixture may be dried, for example under vacuum, until all or substantially all of the residual water is removed from the reaction mixture.

Any remaining ethyl esters, monoacylglycerols, and/or free fatty acids may be separated from the diacylglycerols, for example, by distillation.

It should be understood that the steps of the method of the present invention may be performed in any order. In some embodiments, one or more steps of the methods of the present invention may be performed more than once. In a preferred embodiment, the steps of the method of the invention are performed in the order listed above.

Also provided is a diglyceride obtained according to the method of the present invention.

Further provided is the use of the diglycerides obtained according to the method of the present invention in food products, dietary supplements, pharmaceutical products, or cosmetic products.

Detailed Description

Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments of the disclosure described below, as modifications may be made to the particular embodiments and still fall within the scope of the appended claims. It is also to be understood that the terminology used is for the purpose of describing particular embodiments, and is not intended to be limiting. Rather, the scope of the disclosure is to be determined by the claims that follow.

In this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes mixtures of two or more such compounds, reference to "an unsaturated fatty acid" includes mixtures of two or more such unsaturated fatty acids, reference to "a substrate" includes mixtures of two or more such substrates, and the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

"optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that a number of values are disclosed herein, and that each value is also disclosed herein as "about" that particular value, in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It will also be understood that when a value is disclosed, as is well understood by those skilled in the art, "less than or equal to" the value, "greater than or equal to the value," and possible ranges between the values are also disclosed. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout this application, data is provided in a number of different formats, and that such data represents endpoints and starting points, and ranges for any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15 and between 10 and 15 are disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

Reference in the specification and the claims to parts by weight of a particular element or component in a composition means the weight relationship between the element or component and any other element or component in the composition or article expressed as a part by weight. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a 2:5 weight ratio, and are present in such a ratio regardless of whether additional components are contained in the compound.

Unless specifically stated to the contrary, the weight percentages of components are based on the total weight of the formulation or composition in which they are included.

Unless stated to the contrary, a formula having chemical bonds shown only in solid lines rather than wedges or dashed lines contemplates each possible isomer (e.g., each enantiomer and diastereomer) as well as mixtures of isomers, such as racemic or non-racemic (scalemic) mixtures.

Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying examples and figures.

Disclosed herein are molecules, materials, compounds, compositions, and components that can be used for, can be used in conjunction with, can be used in the preparation of, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a compound is disclosed and a number of modifications that can be made to a number of components or residues of the compound are discussed, each and every combination and permutation that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of components or residues A, B and C and a class of components or residues D, E and F are disclosed and examples of combinations of compounds a-D are disclosed, then each is individually and collectively contemplated even if each is not individually recited. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are specifically contemplated and should be considered to be from A, B and C; D. e and F; and example combinations a-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the subgroups of A-E, B-F and C-E are specifically contemplated and should be considered to be from A, B and C; D. e and F; and example combinations a-D. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

Certain materials, compounds, compositions, and components disclosed herein are commercially available or can be readily synthesized using techniques generally known to those skilled in the art. For example, starting materials and reagents for preparing the disclosed compounds and compositions can be obtained from commercial suppliers such as Aldrich Chemical Co. (milwaukee, wisconsin), Acros Organics (Morris Plains, new jersey), Fisher Scientific (pittsburgh, pa) or Sigma (st louis, missouri), or prepared by methods known to those skilled in the art following procedures such as those set forth in the following references: fieser and Fieser's Reagents for Organic Synthesis, Vol.1-17 (John Wiley and Sons, 1991); rodd's Chemistry of Carbon Compounds, Vol.1-5 and supple (Elsevier Science Publishers, 1989); organic Reactions, Vol.1-40 (John Wiley and Sons, 1991); march's Advanced Organic Chemistry, (John Wiley and Sons, 4 th edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

As used herein, the terms "diacylglycerol", "diglyceride", and "diester" are used interchangeably to refer to two fatty acids attached to a glycerol backbone. Similarly, the terms "monoacylglycerol" and "monoglyceride" are used interchangeably to refer to one fatty acid attached to the glycerol backbone, and the terms "triacylglycerol", "triglyceride" and "triester" are used interchangeably to refer to three fatty acids attached to the glycerol backbone.

Enzyme

Enzymes useful herein are any naturally occurring or synthetic enzyme that can be used to esterify a carboxylic acid or a transesterification ester. The term "esterification" is defined herein as the production of an ester (e.g., RCOOH + R) by reacting a carboxylic acid with an alcohol¹OH→RCOOR¹+H₂O) and the carboxylic acid is converted into the corresponding ester. The term "transesterification" is defined herein as the reaction of one ester with an alcohol to produce a different ester (e.g., RCOOR)¹+R²OH→RCOOR²+R¹OH) to convert the ester to another ester. The term "transesterification" is defined herein as the conversion of an ester moiety between two or more separate independent esters. The interesterification between two esters is depicted in scheme 1A, where the starting material (i.e., R) is¹COOR²And R³COOR⁴) In R²And R⁴And (4) converting groups. Scheme 1B depicts the reaction at carboxylic acid (R)¹COOH) and ester (R)³COOR⁴) Which produces new carboxylic acids and esters. The term "ester internalization" is defined herein as the conversion of a lactone moiety of the same molecule. Ester internalization is depicted in scheme 1C, wherein R²And R³The groups are converted in triesters. Scheme 1D depicts ester internalization between carboxylic acid groups and esters within the same molecule, where the hydrogen of the carboxylic acid is in contact with the R of the ester group³And (4) converting.

R¹CO₂R²+R³CO₂R⁴→R¹CO₂R⁴+R³CO₂R² 1A

R¹CO₂H+R³CO₂R⁴→R¹CO₂R⁴+R³CO₂H 1B

Scheme 1

Suitable enzymes may be derived from microorganisms. Examples of microorganisms that can produce enzymes useful herein include, but are not limited to, Burkholderia species (Burkholderia sp.), Candida antarctica B, Candida rugosa (Candida rugosa), Candida cylindracea (Candida cylindracea), Pseudomonas species (Pseudomonas sp.), Candida antarctica A, Porcine pancreas (Porcine pancreas), Humicola species (Humicola sp.), Humicola lanuginosa (Humicola lanuginosa), Mucor miehei (Mucor miehei), Rhizopus javanicus (Rhizopus javanicus.), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas cepacia (Pseudomonas cepacia), Candida cylindracea (Candida cylindracea), Aspergillus niger (Aspergillus niger), Rhizopus oryzae (Rhizopus oryzae), Rhizopus solani (Rhizopus), Rhizopus nizopus niveus sp), Rhizopus niveus (Rhizopus niveus sp), Rhizopus niveus sp) Or Penicillium camembertii (also Rhizopus deleman, Pseudomonas aeruginosa).

In one example, the enzyme is produced by candida antarctica. NOVOZYME^TMCALB L is a lipase (lipase B) produced from candida antarctica by submerged fermentation of a genetically modified Aspergillus oryzae (Aspergillus oryzae) microorganism. NOVOZYME^TMCALB L is a highly versatile catalyst with activity on a number of different substrates. The enzymes are particularly useful as strong enantioselective catalysts in the synthesis of optically active alcohols, amines and carboxylic acids. Candida antarctica lipase B is known to efficiently convert ethyl esters or free fatty acids to triglycerides. This enzyme is a protein having 317 amino acid residues and a molecular weight of 33,008 daltons. The amino acids were assembled into 14 alpha helices and 9 beta sheets. Amino acid sequence of candida antarctica lipase B andthe secondary structure is provided in SEQ ID NO 1. LPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTGPQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQVNTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQWGLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTNLYSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYVVGRSARSTTGQARSADYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP

It is also contemplated that derivatives of enzymes produced by microorganisms may be used in the methods described herein. It is understood that the structure of many enzymes as disclosed herein are known and can be found, for example, in Genbank and are incorporated herein by reference.

As all microbial lipases, CALB belongs to the α/β hydrolase, whose folding comprises an eight-chain β -sheet sandwiched between two layers of amphipathic α -helices. The ester hydrolysis mechanism of these enzymes generally involves binding to an ester substrate, forming a first tetrahedral intermediate by catalyzing nucleophilic attack by serine, in which oxygen anions are stabilized by two or three H bonds, so-called oxygen anion holes. In the final step, the ester bond is cleaved and the acylase is hydrolyzed. Nucleophilic attack by catalytic serine is mediated by catalytic histidine and aspartic or glutamic acid residues. In some instances, the longest fatty acid chain that is fully bound within the binding pocket of CALB is C13; thus, the cleavable fatty acid binding site of this enzyme is relatively shortThe binding site of CALB is relatively short and has a small hydrophobic region located at the binding funnel wall. The structure of CALB has been disclosed in The Protein database (The Protein Data Bank: a computer-based architecture file for cellular structures. Bernstein et al, J.Mal.biol.112:525- "542, 1977). It is also understood that conserved catalytic cores thereof understood in the art and disclosed herein may define the disclosed enzymes.

Sequence similarity

It is understood that the use of the terms "homology" and "identity" as discussed herein means the same as similarity. Thus, for example, if the use of the word homology is used between two non-native sequences, it will be understood that this does not necessarily indicate an evolutionary relationship between the two sequences, but rather looks at the similarity or relatedness between the sequences. For the purpose of measuring sequence similarity, many methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins, whether or not they are evolutionarily related. In general, it will be understood that one way to define any known variants and derivatives, or those that may arise from the genes and proteins disclosed herein (such as SEQ ID NO:1), is by defining variants and derivatives on homology to specific known sequences. Such identity of particular sequences disclosed herein is also discussed elsewhere herein. In general, variants of the genes and proteins disclosed herein typically have at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology relative to the recited or native sequence. One skilled in the art readily understands how to determine the homology of two proteins or nucleic acids, such as genes. For example, homology can be calculated after aligning the two sequences such that the homology is at its highest level.

Another method of calculating homology can be performed by the disclosed algorithm. Optimal sequence alignments for comparison can be performed by the local homology algorithm of Smith and Waterman, adv.Appl.Math.2:482,1981, by the homology alignment algorithm of Needleman and Wunsch, J.Mol.biol.48:443,1970, by search of the similarity method of Pearson and Lipman, Proc.Natl.Acad.Sci.U.S.A.85:2444,1988, by computerized implementation of these algorithms (GAP, BESTFIT, PASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group 575, Science Dr., Madison, Wisconsin), or by inspection.

The same type of homology for nucleic acids can be obtained by algorithms such as those disclosed in Zuker, Science 244:48-52,1989, Jaeger et al, Proc. Natl. Acad. Sci. U.S. A.86: 7706-. It is understood that any method can typically be used, and in some cases the results of these various methods can differ, but those skilled in the art understand that if identity is found with at least one of these methods, the sequences can be considered to have the stated identity and are disclosed herein.

For example, as used herein, a sequence recited as having a specified percentage homology to another sequence refers to a sequence having the recited homology as calculated by any one or more of the calculation methods described above. For example, if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method, even if the first sequence does not have 80 percent homology to the second sequence as calculated by any other calculation method, then the first sequence has 80 percent homology to the second sequence as defined herein. As another example, a first sequence has 80 percent homology to a second sequence as defined herein if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method and the Pearson and Lipman calculation method, even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation method, or any other calculation method. As yet another example, a first sequence has 80 percent homology to a second sequence as defined herein if the first sequence is calculated to have 80 percent homology to the second sequence using each calculation method (although in practice, different calculation methods will typically result in different calculated homology percentages).

hybridization/Selective hybridization

It is also understood that the enzymes disclosed herein, such as SEQ ID NO:1, can be classified by the ability of the nucleic acids encoding them to hybridize to other nucleic acids. The term "hybridization" typically means a sequence-driven interaction between at least two nucleic acid molecules, such as a primer or probe and a gene. The phrase "sequence-driven interaction" means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide-specific manner. For example, G-to-C interactions or a-to-T interactions are sequence driven interactions. Typically, sequence-driven interactions occur on the Watson-Crick face or Hoogsteen face of nucleotides. Hybridization of two nucleic acids is affected by a number of conditions and parameters known to those skilled in the art. For example, salt concentration, pH, and reaction temperature all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acid molecules are well known to those skilled in the art. For example, in some instances, selective hybridization conditions can be defined as stringent hybridization conditions. For example, the stringency of hybridization is controlled by both the temperature and salt concentration of one or both of the hybridization and wash steps. For example, hybridization conditions to achieve selective hybridization can involve hybridization in a high ionic strength solution (6X SSC or 6X SSPE) at a temperature from about 12 ℃ to about 25 ℃ below Tm (the melting temperature at which half of the molecule dissociates from its hybridization partner), followed by washing at a combination of temperature and salt concentration selected such that the washing temperature is from about 5 ℃ to about 20 ℃ below Tm. Temperature and salt conditions can be readily determined empirically in preliminary experiments in which a sample of reference DNA immobilized on a filter is hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringency.

For DNA-RNA and RNA-RNA hybridization, the hybridization temperature is typically higher. The conditions may be used as described above to achieve stringency or as known in the art. (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor, New York, 1989; Kunkel et al, Methods enzymol.1987:154:367,1987, which is incorporated herein by reference for at least material relevant to nucleic acid hybridization). DNA hybridization conditions can be preferably stringent hybridization conditions in 6X SSC or 6X SSPE at about 68 deg.C (in aqueous solution), followed by washing at 68 deg.C. The stringency of hybridization and washing (if desired) can be reduced accordingly with the desired degree of complementarity and further depends on the G-C or a-T abundance of any region of the sought variability. Also, hybridization and washing stringency (if desired) can be increased accordingly with the desired homology, and further depends on the abundance of G-C or A-T in any region where high homology is desired, as is known in the art.

Another way to define selective hybridization is to look at the amount (percentage) of one nucleic acid that binds to another nucleic acid. For example, in some instances, selective hybridization conditions will be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the restriction nucleic acid binds to the non-restriction nucleic acid. Typically, the non-limiting primer is, for example, in a 10 or 100-fold or 1000-fold excess. This type of assay can be performed under the following conditions: both the limiting and non-limiting primers are compared to k_dE.g.10-fold or 100-fold or 1000-fold lower, or only one nucleic acid molecule 10-fold or 100-fold or 1000-fold lower, or one or two nucleic acid molecules higher than k_d。

Another way to define selective hybridization is by looking at the percentage of primers that are enzymatically manipulated under conditions that require hybridization to facilitate the desired enzymatic manipulation. For example, in some instances, selective hybridization conditions will be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions that promote the enzymatic manipulation; for example, if the enzymatic manipulation is DNA extension, the selective hybridization conditions will be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended. Preferred conditions also include conditions suggested by the manufacturer or indicated in the art as being suitable for the enzyme to be manipulated.

As with homology, it is understood that there are a variety of methods disclosed herein for determining the level of hybridization between two nucleic acid molecules. It will be understood that these methods and conditions may provide different percentages of hybridization between two nucleic acid molecules, but unless otherwise indicated, meeting the parameters of either method will be sufficient. For example, if 80% hybridization is desired and as long as hybridization occurs within the desired parameters in any of these methods, it is considered to be disclosed herein.

It is understood that one skilled in the art understands that a composition or method is a composition or method disclosed herein if it meets any of these conditions for determining criteria for hybridization collectively or individually.

Peptides

As discussed herein, many variants and strain derivatives of the disclosed enzymes (such as SEQ ID NO:1) are known and are contemplated herein. Enzymes can be made from proteins or peptides. Protein variants and derivatives are well known to those skilled in the art and may involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of the following three categories: substitution variants, insertion variants, or deletion variants. Insertions include amino-and/or carboxy-terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions will typically be smaller than those of amino-or carboxy-terminal fusions, e.g., on the order of 1 to 4 residues. Immunogenic fusion protein derivatives, such as those described in the examples, are made by: the polypeptide large enough to confer immunogenicity is fused to the target sequence by in vitro cross-linking or by recombinant cell culture transformed with DNA encoding the fusion.

Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about 2 to 6 residues are deleted at any site within the protein molecule. Typically, these variants are prepared by: site-specific mutagenesis is performed on nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter the DNA is expressed in recombinant cell culture.

Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of a single residue, but may occur at a time in many different positions; insertions will typically be on the order of about 1 to 10 amino acid residues; and deletions will range from about 1 to 30 residues.

Deletions or insertions, i.e. deletion of 2 residues or insertion of 2 residues, are preferably made in adjacent pairs. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final construct. The mutation must not place the sequence out of reading frame and preferably will not produce a complementary region that can give rise to secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions are generally made according to tables a and B below, and are referred to as conservative substitutions.

Table a: amino acid abbreviations

Table B: amino acid substitutions

A significant change in functional or immunological identity is achieved by: substitutions were chosen that were less conservative than those in table B, i.e., residues were chosen that differ significantly in their effect in maintaining: (a) the structure of the polypeptide backbone in the substituted region, e.g., in a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chain. It is generally expected that substitutions that produce the greatest change in protein properties will be those in which (a) a hydrophilic residue (e.g., seryl or threonyl) is substituted for (or by) a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl); (b) cysteine or proline for (or by) any other residue; (c) a residue with an electropositive side chain (e.g., lysyl, arginyl, or histidine) is substituted for (or substituted by) an electronegative residue (e.g., glutamyl or aspartyl), or (d) a residue with a bulky side chain (e.g., phenylalanine) is substituted for (or substituted by) a residue without a side chain (e.g., glycine), in which case (e) by increasing the number of sulfation and/or glycosylation sites.

For example, the substitution of one amino acid residue with another amino acid residue that is biologically and/or chemically similar is a conservative substitution known to those skilled in the art. For example, conservative substitutions would be the substitution of one hydrophobic residue for another, or one polar residue for another. Such substitutions include combinations, such as, for example, Gly, Ala; val, lie, Leu; asp and Glu; asn, Gin; ser, Thr; lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each of the specifically disclosed sequences are included within the mosaic polypeptides provided herein.

Substitution or deletion mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr). Deletion of cysteine or other labile residues may also be desirable. Deletion or substitution of potential proteolytic sites (e.g., Arg) is accomplished, for example, by deleting a basic residue or substituting a basic residue with a glutaminyl or histidyl residue.

Certain post-translational derivatizations are the result of the action of the recombinant host cell on the expressed polypeptide. Glutaminyl and asparaginyl residues are usually post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the amino groups of lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francis. co., pp.79-86 (1983)), acetylation of the N-terminal amine, and in some cases amidation of the C-terminal carboxyl group.

It will be appreciated that one way to define variants and derivatives of the proteins disclosed herein is by defining variants and derivatives on homology/identity to particular known sequences. For example, SEQ ID NO 1 lists the specific sequences of lipases. Specifically disclosed are variants of these and other proteins disclosed herein that have at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 homology with respect to the recited sequences. One skilled in the art would readily understand how to determine the homology of two proteins. For example, homology can be calculated after aligning the two sequences such that the homology is at its highest level.

Another method of calculating homology can be performed by the disclosed algorithm. Optimal sequence alignments for comparison can be performed by the local homology algorithm of Smith and Waterman, adv.Appl.Math.2:482,1981, by the homology alignment algorithm of Needleman and Wunsch, J.Mal.biol.48:443,1970, by search of the similarity methods of Pearson and Lipman, Proc.Natl.Acad.Sci.U.S.A.85:2444,1988, by computerized implementation of these algorithms (GAP, BESTFIT, PASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group 575, Science Dr., Madison, Wisconsin), or by inspection.

The same type of homology for nucleic acids can be obtained by algorithms such as those disclosed in Zuker, Science 244:48-52,1989, Jaeger et al, Proc. Natl. Acad. Sci. U.S. A.86: 7706-.

It is understood that the descriptions of conservative mutations and homology may be combined together in any combination, such as embodiments having at least 70% homology with respect to a particular sequence in which the variant is a conservative mutation.

While the present specification discusses various proteins and protein sequences, it is understood that nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a particular protein sequence, i.e., all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids encoding the disclosed variants and derivatives of the protein sequence, including degenerate nucleic acids. Thus, although each particular nucleic acid sequence may not be written out herein, it is understood that each is actually disclosed and described herein by the disclosed protein sequences.

It is also understood that, while no amino acid sequence indicates which particular DNA sequence encodes the protein in vivo, where particular variants of the disclosed proteins are disclosed herein, the known nucleic acid sequences encoding the proteins in the particular strains producing the proteins are also known and disclosed and described herein.

It is understood that there are many amino acids and peptide analogs that can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids with different functional substituents, which are then shown in table a and table B. Opposite stereoisomers of naturally occurring peptides are disclosed, as well as stereoisomers of peptide analogs. These amino acids can be easily incorporated into polypeptide chains by: the tRNA molecule is filled with selected amino acids and a genetic construct is engineered that inserts analog amino acids into the peptide chain in a site-specific manner using, for example, amber codons (Thorson et al, meth.mol.biol.77:43-73,1991; Zoller, Curr. opinion Biotechnology.3: 348; 1992; lbba, Biotechnology.Gen. Eng.Rev.13:197-216, 1995; Cahill et al, TIBS 14(10):400-403, 1989; Benner, TIB Tech 12:158-163, 1994; lbba and Hecke, Bio/technology 12:678-682,1994, at least the materials related to amino acid analogs are incorporated herein by reference).

Molecules can be produced that resemble peptides but are not linked by natural peptide bonds. For example, linkages for Amino Acids or Amino acid analogs may include, but are not limited to, CH2NH-, -CH2S-, -CH2-CH2-, -CH ═ CH (cis and trans), -COCH2-, -CH (OH) CH2-, and-CHH 2SO- (these and others may be found in Spatola, Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.Weinstein editor, Marcel Dekker, New York, pp 267, 1983; Spatola, Vega Data (3.1983), Vol.1, No. 3, Peptide trakbone Modifications (general review), Morley, Trends rm.Sci.463-468,1980; Int.J. Prat. Res.14: 177.185: 1249-, CH2-CH2 469- (-CH2 W.1983), WO 35-CH 2-CH 589-) -CH 2-1983; Wolk.31.9, WO 35, W.9, W.31, W.3, W., cis and trans }; almquist et al, J Med. chem.23: 1392-; Jennings-White et al Tetrahedron Lett.23:2533,1982(-COCH 2-); szelke et al, European application No. EP 45665CA (1982):97:39405(1982) (-CH (OH) CH 2-); holladay et al tetrahedron.Lett.24:4401-4404,1983(-C (OH) CH 2-); and Hruby, Life Sci.31: 189-; each of which is incorporated herein by reference. A particularly preferred non-peptide bond is-CH 2 NH-. It is understood that peptide analogs may have more than one atom between the bond atoms, such as (1-alanine, -y-aminobutyric acid, etc.

Amino acid analogs and peptide analogs generally have enhanced or desirable properties, such as more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, and the like.

D-amino acids can be used to produce more stable peptides because D-amino acids are not recognized by peptidases and the like. Systematic substitution of one or more amino acids of the consensus sequence with the same type of D-amino acid (e.g., D-lysine instead of L-lysine) can be used to produce more stable peptides. Cysteine residues may be used to cyclize or attach two or more peptides together. This may be beneficial for confining the peptide to a particular conformation. (Rizo and Gierasch, Ann. Rev. biochem.61:387,1992, incorporated herein by general reference).

Use of enzymes

Described herein is a process for esterifying a carboxylic acid comprising reacting a carboxylic acid with an alcohol in the presence of any of the enzymes described herein. In another aspect, described herein is a method for transesterifying an ester comprising reacting the ester with an alcohol in the presence of any of the enzymes described herein. In yet another aspect, described herein is a method for transesterifying two or more different carboxylic acids or esters thereof, comprising reacting the carboxylic acids or esters with each other in the presence of any of the enzymes described herein. In yet another aspect, described herein is a method for ester internalization of a compound comprising at least two ester groups or a compound comprising at least one carboxylic acid group and one ester group, comprising contacting the compound with any of the enzymes described herein. A schematic of the transesterification of Ethyl Esters (EE) to triglycerides or the esterification of Free Fatty Acids (FFA) to triglycerides is shown below.

Although esterification of any carboxylic acid, transesterification of any ester, transesterification of two or more different carboxylic acids/esters, or ester internalization of a compound is contemplated using the methods described herein, in many instances, a fatty acid or ester thereof can be used in any method. In some examples, the ester of a fatty acid is C₁-C₆Branched or straight chain alkyl esters such as, for example, methyl, ethyl, propyl, butyl, pentyl, and the like.

In other specific examples, fatty acids or esters thereof may be used in the methods described herein. "fatty acid" means a carboxylic acid having at least 10 carbon atoms. In one aspect, the fatty acid or ester thereof can comprise at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20 carbon atoms. In some specific examples, a fatty acid or ester thereof can contain 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 carbon atoms, where any value stated where appropriate can form an upper or lower endpoint. In other examples, the fatty acid or ester thereof may comprise a mixture of fatty acids or esters thereof having a range of carbon atoms. For example, the fatty acid or ester thereof can comprise from about 10 to about 40, from about 12 to about 38, from about 14 to about 36, from about 16 to about 34, from about 18 to about 32, or from about 20 to 30 carbon atoms.

The fatty acid or ester thereof may be a saturated fatty acid, an unsaturated fatty acid, or a mixture of a saturated fatty acid and an unsaturated fatty acid. "saturated" means that the molecule or residue does not contain carbon-carbon double or triple bonds. By "unsaturated" is meant that the molecule or residue contains at least one carbon-carbon double or triple bond.

In a particular example, prior to esterification, the fatty acids or esters thereof may be derived from marine oils, such as fish oils. Such oils typically contain a mixture of saturated and unsaturated fatty acids, but can be processed into specific fatty acid mixtures (e.g., containing all saturated fatty acids, all unsaturated fatty acids, a mixture of both, or a mixture of fatty acids having a certain chain length or a range of chain lengths).

Any fish oil can be used in the disclosed compounds and methods. Examples of suitable fish oils include, but are not limited to, atlantic fish oil, pacific fish oil, mediterranean fish oil, light pressed fish oil, alkali treated fish oil, heat treated fish oil, light and heavy brown fish oil, bonito oil, pilchard (pilchard) oil, tuna oil, sea bass oil, halibut oil, spearfish oil, barracuda oil, cod oil, menhaden (menhaden) oil, sardine oil, anchovy oil, capelin oil, atlantic cod oil, atlantic herring (herring) oil, atlantic mackerel oil, atlantic menhaden (menhaden) oil, salmon oil, and shark oil, including mixtures and combinations thereof. Non-alkaline treated fish oils are also suitable. Other marine oils suitable for use herein include, but are not limited to, squid oil, cuttlefish oil, octopus oil, krill oil, seal oil, whale oil, and the like, including mixtures and combinations thereof. Any marine oil and combination of marine oils can be used in the disclosed compositions and disclosed methods of making the same. Additional oils include microbial oils, algal oils (e.g., oils from dinoflagellates (such as Crypthecodinium cohnii) or, for example, oils from chytrium (Thraustochytrium), Schizochytrium (Schizochytrium), or mixtures thereof), fungal oils (e.g., oils from Mortierella alpina (Mortierella alpina)), and/or vegetable oils, including mixtures and combinations thereof.

Examples of specific saturated fatty acids or esters thereof useful herein include, but are not limited to, capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), margaric acid (C17), stearic acid (C18), arachidic acid (C20), behenic acid (C22), lignoceric acid (C24), cerotic acid (C26), montanic acid (C28), and melissic acid (C30), including branched and substituted derivatives thereof.

Unsaturated fatty acids or esters thereof suitable for use in the methods disclosed herein can comprise at least one unsaturated bond (i.e., a carbon-carbon double or triple bond). In one example, the unsaturated fatty acid or ester thereof can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 carbon-carbon double bonds, triple bonds, or any combination thereof. In another example, an unsaturated fatty acid or ester thereof can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unsaturated bonds, where any stated value can form an upper or lower endpoint, as appropriate.

In one example, the unsaturated fatty acid or ester thereof may contain a carbon-carbon double bond (i.e., a monoenoic acid or residue). Examples of unsaturated fatty acids or esters thereof suitable for use in the methods disclosed herein include, but are not limited to, those in table 1 below.

Table 1: examples of monoenoic acids

In other examples, the unsaturated fatty acid or ester thereof may contain at least two unsaturated bonds (e.g.E.g., polyenoic acids or residues). In some examples, the unsaturated fatty acid or ester thereof may contain at least one methylene interrupted unsaturated bond. "methylene interrupted unsaturated bond" means that one carbon-carbon double or triple bond is interrupted by at least one methylene group (i.e., CH) from another carbon-carbon double or triple bond₂) And (4) separating. Specific examples of unsaturated fatty acids or esters thereof containing at least one pair of methylene interrupted unsaturated bonds include, but are not limited to, those from the n-1 family of 9, 12, 15-16: 3; n-2 family derived from 9, 12, 15-17:3, 15:3, 17:3, 17:4, 20: 4; from 9, 12, 15-18:3, 15:2, 15:3, 15:4, 16:3, 16:4, 18:3 (alpha-linolenic acid), 18:4, 18:5, 20:2, 20:3, 20: 4; 20:5(EPA), 21:5, 22:3, 22:5(DPA), 22:6(DHA), n-3 family of 24:3, 24:4, 24:5, 24:6, 26:5, 26:6, 28:7, 30: 5; n-4 family derived from 9, 12-16:2, 16:2, 16:3, 18:2, 18: 3; n-5 families derived from 9, 12-17:2, 15:2, 17:2, 17:3, 19:2, 19:4, 20:3, 20:4, 21:4, 21: 5; from 9, 12-18:2, 15:2, 16:2, 18:2 (linoleic acid), 18:3 (gamma-linolenic acid); n-6 family of 20:2, 20:3, 20:4 (arachidonic acid), 22:2, 22:3, 22:4 (adrenalic acid), 22:5, 24:2, 24:4, 25:2, 26:2, 30: 4; n-7 family derived from 9-16:1, 15:2, 16:2, 17:2, 18:2, 19: 2; n-8 families derived from 9-17:1, 15:2, 16:2, 17:2, 18:2, 19: 2; n-9 family derived from 9-18:1, 17:2, 18:2, 20:2, 20:3, 22:3, 22: 4; n-11 family 19:2, and n-12 family 20: 2.

The numbering scheme begins at the end of the fatty acid, where, for example, the group at the terminal CH3 is designated as position 1. In this sense, the n-3 family will be omega-3 fatty acids as described herein. The next number identifies the total number of carbon atoms in the fatty acid. The third number after the colon specifies the total number of double bonds in the fatty acid. Thus, for example, in the n-1 family, 16:3 refers to a 16 carbon long fatty acid with 3 double bonds, each separated by a methylene group, where the first double bond begins at position 1, the end of the fatty acid. In another example, in the n-6 family, 18:3 refers to an 18 carbon long fatty acid with 3 methylene separated double bonds starting at position 6, the sixth carbon from the end of the fatty acid, and so on.

Some other examples are fatty acids or esters thereof containing at least one pair of unsaturated bonds interrupted by more than one methylene group. Suitable examples of such acids and esters include, but are not limited to, those in table 2 below:

table 2: examples of polyenoic acids

Still other examples of unsaturated fatty acids or esters thereof suitable for use in the methods disclosed herein are those containing at least one conjugated unsaturated bond. "conjugated unsaturated bond" means that at least one pair of carbon-carbon double and/or triple bonds are bonded together without a methylene group (CH) between them₂) A group (e.g., -CH ═ CH-). Specific examples of the unsaturated fatty acid or ester thereof containing a conjugated unsaturated bond include, but are not limited to, those in table 3 below.

Table 3: examples of conjugated polyenoic acids

Omega-3 fatty acids and esters thereof may also be used in the methods described herein. Omega-3 fatty acids are unsaturated fatty acids that are particularly useful in the compounds and methods disclosed herein. Not only do omega-3 fatty acids exhibit the demonstrated effect of lowering serum triglyceride levels, but they have a strong association with diabetes. For example, docosahexaenoic acid (DHA) also has a strong insulin permeability enhancing effect and is considered as a potential absorption enhancer for insulin delivered enterally (Onuki et al, int.J.Pharm.198:147-56, 2000). DHA intake prevents certain biochemical processes that originate from insulin deficiency (Ovide-Bordeaux and Grynberg, am.J.Physiol.Regul.lntegr.Comp.Physiol.286: R519-27,2003), and both DHA and BPA (eicosapentaenoic acid) significantly increase fasting insulin levels (Mori et al, am.J.Clin.Nutr.71:1085-94, 2000).

Omega-3 fatty acids are unsaturated fatty acids containing CH₃-CH₂-CH-as its terminal. Specific examples of omega-3 fatty acids and esters thereof suitable for use herein include, but are not limited to, linolenic acid (18:3 omega 3), stearidonic acid (18:4 omega 3), eicosapentaenoic acid (20:5 omega 3) (EPA), docosahexaenoic acid (22:6 omega 3) (DHA), docosapentaenoic acid (22:6 omega 3) (DPA), derivatives thereof, and mixtures thereof.

In still other examples, the unsaturated fatty acids and esters thereof may be derived from compounds having the formula:

wherein R is¹Is C containing at least one double bond₃-C₄₀An alkyl or alkenyl group. The term "alkane" or "alkyl" as used herein is a saturated hydrocarbon group. The term "alkene" or "alkenyl" as used herein is a hydrocarbon group of at least 2 carbon atoms, the structural formula of which contains at least one carbon-carbon double bond. Asymmetric structures such As (AB) C ═ C (cd) are intended to include both E and Z isomers (cis and trans). This may be presumed in the formulae herein where an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C ═ C. In another example, R¹May be C₅-C₃₈、C₆-C₃₆、C₈-C₃₄、C₁₀-C₃₂、C₁₂-C₃₀、C₁₄-C₂₈、C₁₆-C₂₆Or C₁₈-C₂₄An alkenyl group. In yet another example, R¹The alkenyl group of (a) may have from 2 to 6, from 3 to 6, from 4 to 6, or from 5 to 6 double bonds. Still further, R¹The alkenyl group of (a) may have 1, 2, 3, 4, 5, or 6 double bonds, where any stated value may form an upper or lower endpoint, as appropriate.

Some specific examples of unsaturated fatty acids and esters thereof that may be used in the methods disclosed herein include, but are not limited to, linoleic acid, linolenic acid, gamma-linolenic acid, arachidonic acid, mead acid (mead acid), stearidonic acid (stearidonic acid), alpha-eleostearic acid, pinolenic acid (pinolenic acid), docosadienic acid (docosadienic acid), docosatetraenoic acid, docosapentaenoic acid, docosahexaenoic acid, octadecadienoic acid, octadecatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, or any combination thereof. In one aspect, the unsaturated fatty acid ester can be derived from linolenic acid (18:3 ω 3), stearidonic acid (18:4 ω 3), eicosapentaenoic acid (20:5 ω 3) (EPA), eicosatetraenoic acid (20:4 ω 3), heneicosapentaenoic acid (21:5 ω 3), docosahexaenoic acid (22:6 ω 3) (DHA), docosapentaenoic acid (22:5 ω 3) (DPA), including derivatives and mixtures thereof.

Further examples of suitable unsaturated fatty acids and esters thereof suitable for use in the process include, but are not limited to, dienoic acids (allenic) and acetylenic acids such as C14:2, 4, 5; c18:5, 6 (octadecadienoic acid (labellenic)); 5. 6, 16 (octadecatrienoic acid (lamenaleninic)); c18:6a (tarinic); 9 a; 9a, 11t (ximenynic acid); 9a, 11 a; 9a, 11a, 13c (bolekic); 9a, 11a, 13a, 15e, 8a, 10t (pyrulic)9c, 12a (crepenolic acid); 9c, 12a, 14c (dehydroandroandrographolide acid); 6a, 9c, 12 c; 6a, 9c, 12c, 15c, 8a, 11c, 14c and the corresponding Δ 17e derivatives, 8-OH derivatives, and Δ 17e,8-OH derivatives.

Branched acids and esters thereof, particularly iso and trans iso (anteiso) acids, polymethylbranched acids, phytal-based acids (e.g., phytanic acid, pristanic acid), furan-based acids, are also fatty acids suitable for use in the methods disclosed herein.

Still further, suitable fatty acids and esters thereof include, but are not limited to, cyclic acids such as cyclopropane fatty acids, cyclopropenoic acids (e.g., lactobacillic acid), sterculic acid (sterulic), malvalic acid (malvalic), sterculic acid (sterculic), 2-hydroxy sterculic acid, aleprolic acid (alprolic), alepramic, aleprolic acid (alepressic), aleprolic acid (aleprolic), hydnocarpic acid (hydnocarpic), chaulmoogric acid (chalomogenous) homoelic, manaoic, allic acid (garlic), oncobic, cyclopentenoic acid, and cyclohexanoic acid.

Hydroxy acids and esters thereof, particularly butyric acid, ricinoleic acid, isoricinoleic acid, densipolic, lesquerolic, and auriolic, are also suitable fatty acids that, after esterification, can be used in the processes disclosed herein.

Epoxy acids and esters (particularly epoxidized C18:1 and C18:2) and furan-based acids and esters are additional examples that may be used in the disclosed process.

In some embodiments, the oil comprising at least one polyunsaturated fatty acid in ethyl ester form, free fatty acid, and/or combinations thereof is docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or combinations thereof.

In some embodiments, the amount of DHA (mg/g oil) in the oil comprising at least one polyunsaturated fatty acid in the form of ethyl esters, free fatty acids, and/or combinations thereof is from about 100mg to about 950mg, about 100mg to about 800mg, about 100mg to about 700mg, about 100mg to about 600mg, about 100mg to about 500mg, about 100mg to about 400mg, about 100mg to about 300mg, about 100mg to about 200mg, or about 0 to about 100 mg.

In some embodiments, the amount of EPA (mg/g oil) in an oil comprising at least one polyunsaturated fatty acid in the form of ethyl esters, free fatty acids, and/or combinations thereof is from about 100mg to about 950mg, about 100mg to about 800mg, about 100mg to about 700mg, about 100mg to about 600mg, about 100mg to about 500mg, about 100mg to about 400mg, about 100mg to about 300mg, about 100mg to about 200mg, or about 0 to about 100 mg.

In some embodiments, the amount of DHA (mg/g oil) and EPA (mg/g oil) in the oil comprising at least one polyunsaturated fatty acid in ethyl ester form, free fatty acid, and/or combinations thereof is from about 100mg to about 950mg, about 100mg to about 800mg, about 100mg to about 700mg, about 100mg to about 600mg, about 100mg to about 500mg, about 100mg to about 400mg, about 100mg to about 300mg, about 100mg to about 200mg, or about 0 to about 100 mg.

In some embodiments, the amount of DHA in the oil (mg/g oil) in the diglycerides produced is from about 100mg to about 950mg, about 100mg to about 800mg, about 100mg to about 700mg, about 100mg to about 600mg, about 100mg to about 500mg, about 100mg to about 400mg, about 100mg to about 300mg, about 100mg to about 200mg, or about 0 to about 100 mg.

In some embodiments, the amount of EPA in the oil (mg/g oil) in the diglyceride produced is from about 100mg to about 950mg, about 100mg to about 800mg, about 100mg to about 700mg, about 100mg to about 600mg, about 100mg to about 500mg, about 100mg to about 400mg, about 100mg to about 300mg, about 100mg to about 200mg, or about 0 to about 100 mg.

In some embodiments, the amount of DHA (mg/g oil) and EPA (mg/g oil) in the oil in the diglyceride produced is from about 100mg to about 950mg, about 100mg to about 800mg, about 100mg to about 700mg, about 100mg to about 600mg, about 100mg to about 500mg, about 100mg to about 400mg, about 100mg to about 300mg, about 100mg to about 200mg, or about 0 to about 100 mg.

The alcohol used in any of the methods disclosed herein can be any alcohol. In one example, the alcohol is a polyol, which is defined as a compound having two or more hydroxyl groups. Examples of polyols useful herein include, but are not limited to, pentaerythritol, dipentaerythritol, tripentaerythritol, tetrapentaerythritol, tris (hydroxymethyl) ethane, or tris (hydroxymethyl) propane. In other examples, the alcohol is a sugar, such as, for example, glucosamine, methylglucoside; or other sugars, such as sucrose, for example. In another example, the polyol is glycerol.

The amount of carboxylic acid/ester and alcohol used will vary depending on the acid, ester and alcohol selected. In one example, a stoichiometric amount of carboxylic acid or ester relative to the number of hydroxyl groups present on the alcohol may be used. For example, if the alcohol is a diol, two molar equivalents of the carboxylic acid or ester may be esterified or transesterified, respectively, with one molar equivalent of the diol. An excess of alcohol can be used to achieve maximum esterification or transesterification and to reduce the total reaction time. In one aspect, when the alcohol is glycerol, the molar ratio of carboxylic acid or ester to alcohol is from 0.1:1 to 6:1, from 1:1 to 3:1, from 1.5:1 to 2.5:1, or from 2:1 to 3: 1.

The amount of enzyme may also vary. In one example, the enzyme is from 0.1% to 20% by weight based on the total weight of carboxylic acid/ester and alcohol. In other examples, the enzyme is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16; 17%, 18%, 19%, 20%, any value therein may form an end point of a range.

The amount of water may also vary. In one example, the ratio of carboxylic acid/ester to water is from 1:1 to 15:1, from 1:1 to 12:1, from 1:1 to 10:1, from 1:1 to 9:1, from 1:1 to 8:1, from 1:1 to 7:1, from 1:1 to 6:1, from 1:1 to 5:1, from 1:1 to 4:1, from 1:1 to 3:1, or from 1:1 to 2: 1. In another example, the ratio of carboxylic acid/ester to water is from 1:1 to 15:1, from 2:1 to 15:1, from 3:1 to 15:1, from 4:1 to 15:1, from 5:1 to 15:1, from 6:1 to 15:1, from 7:1 to 15:1, from 8:1 to 15:1, from 9:1 to 15:1, from 10:1 to 15:1, or from 12:1 to 15: 1.

The carboxylic acid/ester, alcohol, enzyme and water may be mixed with each other in any order. Depending on the choice of carboxylic acid/ester and alcohol, it may be desirable to perform the esterification or transesterification while stirring the reaction mixture. For example, a solution of the ester and the alcohol may be added to each other with stirring, followed by addition of the enzyme.

In one aspect, water is added during the reaction to replace water lost due to evaporation or the like. Water may be added continuously or aperiodically throughout the duration of the reaction. For example, water may be added when the reaction volume falls below a predetermined threshold. Water may be added during the entire reaction or during a portion of the reaction. For example, water may be added during the start of the reaction and discontinued late in the reaction; for example, water may be added during the first quarter, the first third, or the first half of the reaction.

In certain aspects, the esterification, transesterification, and interesterification/ester internalization reactions can be carried out at elevated temperatures. The exact elevated temperature may depend on the particular carboxylic acid or ester used, the particular alcohol used, the amount or concentration of reagents, preferences, and the like. Suitable temperatures at which esterification and transesterification reactions can occur include, but are not limited to, from about 30 ℃ to about 90 ℃, from about 60 ℃ to about 90 ℃, from about 80 ℃ to about 90 ℃, or about 85 ℃.

In another example, the esterification temperature can be from about 50 ℃ to about 70 ℃, or about 60 ℃. By varying the temperature, the reaction time can be reduced depending on the concentration of the starting material. Thus, the reaction time may vary from 2 hours to 72 hours, 2 hours to 48 hours, 2 hours to 24 hours, 6 hours to 48 hours, 6 hours to 36 hours, 8 hours to 24 hours, 8 hours to 16 hours, or 8 hours to 12 hours.

The esterification, transesterification, and interesterification/ester internalization reactions may be carried out under reduced pressure. For example, the esterification, transesterification, and interesterification/ester internalization reactions can be performed at a pressure of from about 1 mtorr to about 200 mtorr, from about 5 mtorr to about 100 mtorr, from about 10 mtorr to about 50 mtorr, from about 15 mtorr to about 30 mtorr, or about 20 mtorr.

In other examples, the process involves esterifying eicosapentaenoic acid 20:5 ω 3(EPA), docosahexaenoic acid 22:6 ω 3(DHA), docosapentaenoic acid 22:5 ω 3(DPA), or any mixture thereof, with glycerol, wherein the acid and alcohol are present in a molar ratio of from about 1:1 to about 3:1, wherein the reaction is stirred in the presence of an enzyme and water at a temperature of from about 30 ℃ to about 90 ℃ for about 2 hours to about 24 hours under reduced pressure, wherein the enzyme comprises an enzyme derived from candida antarctica.

In another aspect, the process involves transesterifying an ethyl ester of eicosapentaenoic acid 20:5 ω 3(EPA), docosahexaenoic acid 22:6 ω 3(DHA), docosapentaenoic acid 22:5 ω 3(DPA), or any mixture thereof, with glycerol, wherein the ester and alcohol are present in a molar ratio of from about 1:1 to about 3:1, wherein the reaction is stirred in the presence of an enzyme and water at a temperature of from about 30 ℃ to about 90 ℃ for about 2 hours to about 24 hours under reduced pressure, wherein the enzyme comprises an enzyme derived from candida antarctica.

The methods described herein are effective for producing mainly diacylglycerols while forming very little triacylglycerols. This process can be used to obtain high purity diacylglycerols since any remaining ethyl esters, monoacylglycerols, and free fatty acids can be easily removed (e.g., by distillation). Additionally, the amount of diacylglycerols as a percentage of the total product is from about 80% to about 100%; optionally from about 90% to about 100%; optionally from about 95% to about 100%; optionally from about 96% to about 100%; optionally from about 96.5% to about 100%; optionally from about 97% to about 100%; optionally from about 97.5% to about 100%; optionally from about 98% to about 100%; optionally from about 98.5% to about 100%; optionally from about 99% to about 100%; optionally from about 99.5% to about 100%.

Separation methods for separating monoacylglycerols, free fatty acids, and residual triacylglycerols from diacylglycerols include, but are not limited to, deodorization, short path distillation, steam distillation, molecular distillation, adsorption chromatography, or any combination thereof. The separation process can be carried out batchwise, continuously and semi-continuously.

The resulting product may be further processed. The refined product may also be stabilized, for example by the addition of a-tocopherol.

The following examples are provided to illustrate, but not to limit, the claimed invention.

Examples

Example 1

Preparation of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) -diacylglycerol ester (DAG)

Diacylglycerol production from high concentrations of ethyl esters is facilitated by one-pot hydrolysis followed by re-esterification under reduced pressure in the presence of lipase B from candida antarctica (CAL-B).

A mixture of omega-3 concentrated ethyl ester (EPA: 439 mg/g; DHA: 405 mg/g; 300g), glycerol (60g) and CAL-B (5000LU/g, 1.9g) in water (75.0g) was added to a 1L flask equipped with a concentrator condenser and stirred under vacuum (20 mTorr) at 60 ℃ for 8-12 hours. During the first 4-6 hours, the water was replaced at a rate of 15 ml/h.

After completion, the reaction mixture was washed with water (45 ℃, 300ml), followed by brine washing (45 ℃, 300ml) and final water washing (45 ℃, 300 ml). The mixture was dried under high vacuum (100 mtorr) at a temperature of about 65 ℃ overnight (12-15 hours). Unreacted ethyl ester, the resulting monoacylglyceride, and other low boiling components were removed by short path distillation (200 ℃, 100 mtorr). The residue obtained is further processed by bleaching treatment at 90 ℃ for 2 hours. The refined product was stabilized by the addition of 500ppm alpha-tocopherol and the final formulation was analyzed for fatty acids (gas chromatography with flame ionization detector, GC-FID) and lipid based compositions (h high performance liquid chromatography-size exclusion chromatography-refractive index detector, HPLC-SEC-RI), respectively.

The final product composition was 96.5% DAG obtained in 49.6% yield, where PV ═ 0.1 and pAV ═ 4.2. The amount of EPA in the DAG fraction was 453mg/g and the amount of DHA in the DAG fraction was 370 mg/g.

Example 2

Preparation of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) -diacylglycerol ester (DAG)

Diacylglycerol production from high concentrations of ethyl esters is facilitated by one-pot hydrolysis followed by re-esterification under reduced pressure in the presence of lipase B from candida antarctica (CAL-B).

A mixture of 50g of omega-3 concentrated ethyl ester (EPA: 380mg/g and DHA: 260mg/g), glycerol (4.5g) and CAL-B (0.5%, 0.25 ml-reaction A and 0.25%, 0.125 ml-reaction B) in water (10ml) was added and stirred under vacuum (20 mTorr) at 65 ℃. After 2 and 4 hours, 5ml of water were added to each reaction. After 6 hours, 10ml of water were added to each reaction. The reaction was stirred overnight. After 24 hours, 10ml of water was added to each reaction and continued under vacuum. The reaction was stopped at 30 hours.

After completion, the oil was analyzed for lipid-like composition (LC-SEC-RI). Tables 4 and 5 show the results for the lipid class compositions.

Table 4: lipid composition, reaction A

Time (hours)	TG(％)	DG(％)	MG(％)	FFA+EE(％)
					6	0.3	7.2	3.9	88.7
24	0	21.7	12.4	66
					27	0	28.9	15	56
30	0	53.1	13.9	30

Table 5: lipid composition, reaction B

Time (hours)	TG(％)	DG(％)	MG(％)	FFA+EE(％)
					6	0	18.3	8.2	73.6
24	0	31.9	16.3	51.8
					27	0	36.3	18	45
30	0	44.7	17.3	37.9

In reaction A, the amount of TG in the sample was 0% after 24 hours. In reaction B, no TG was present in the starting oil. The short path distillation removes unreacted ethyl esters, the resulting monoacylglycerides, and other low boiling components, as shown in example 1.

Example 3

Preparation of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) -diacylglycerol ester (DAG)

Diacylglycerol production from high concentrations of ethyl esters is facilitated by one-pot hydrolysis followed by re-esterification under reduced pressure in the presence of lipase B from candida antarctica (CAL-B).

A mixture of 200g of omega-3 concentrated ethyl ester (1:1EPA: DHA), glycerol (14g) and CAL-B (2ml) in water (55ml) was added and stirred under vacuum (20 mTorr) at 69 ℃. 5-35ml of water are added over 1-7 hours. After completion, the oil was analyzed for lipid-like composition (LC-SEC-RI). Table 6 shows the results for the lipid class composition.

Table 6: lipid composition

The amount of TG in the sample was 0%. The short path distillation removes unreacted ethyl esters, the resulting monoacylglycerides, and other low boiling components, as shown in example 1.

Example 4

Preparation of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) -diacylglycerol ester (DAG)

Diacylglycerol production from high concentrations of ethyl esters is facilitated by one-pot hydrolysis followed by re-esterification under reduced pressure in the presence of lipase B from candida antarctica (CAL-B).

A mixture of 200g of omega-3 concentrated ethyl ester (1:1EPA: DHA), glycerol (16g) and CAL-B (1ml) in water (55ml) was added and stirred under vacuum (20 mTorr) at 69 ℃. 5-35ml of water are added over 1-7 hours. After completion, the oil was analyzed for lipid-like composition (LC-SEC-RI). Table 7 shows the results for the lipid class composition.

Table 7: lipid composition

The amount of TG in the sample was 0%. The short path distillation removes unreacted ethyl esters, the resulting monoacylglycerides, and other low boiling components, as shown in example 1.

All references cited in this specification are herein incorporated by reference as if each reference were specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

It will be understood that each of the elements described above, or two or more together, may also find a useful application in other types of methods differing from the types described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure as set forth in the appended claims. The foregoing embodiments are provided by way of example only; the scope of the present disclosure is limited only by the following claims.