EP1913145A2

EP1913145A2 - Method for the chemoenzymatic production of fatty acid esters

Info

Publication number: EP1913145A2
Application number: EP06776548A
Authority: EP
Inventors: Sabine Both; Ulrich SCHÖRKEN; Carolin Meyer
Original assignee: Cognis IP Management GmbH
Current assignee: Cognis IP Management GmbH
Priority date: 2005-08-11
Filing date: 2006-08-02
Publication date: 2008-04-23
Also published as: JP2009504813A; CN101238219A; DE102005037989A1; BRPI0615156A2; WO2007017167A3; WO2007017167A2; US20100159538A1

Abstract

Disclosed is a method for producing fatty acid esters, in which (a) fatty acids are treated with aqueous aliphatic alcohols having a boiling point ranging between 60 and 120 °C in the presence of esterases so as to obtain a pre-esterification product, (b) water and non-reacted alcohol are separated from the pre-esterification product, (c) the pre-esterification product is subjected to a second, chemically catalyzed esterification process, the same aliphatic alcohol being added as in step (a), and (d) the separated aqueous alcohol from step (c) is reused for the enzymatic pre-esterification process in stage (a).

Description

Process for the chemo-enzymatic production of fatty acid esters

Field of the invention

The present invention is in the field of biotechnology and describes a process for the chemo-enzymatically catalyzed production of fatty acid esters whose alcohol components are aliphatic alcohols having a boiling point between 60 and 120 ^° C.

State of the art

In chemical and biochemical synthesis, enzymes are increasingly being used as catalysts. Thus, in many cases due to the often milder reaction conditions, esterases, especially lipases (EC 3.1.1.3) are already used in industrial processes for lipid cleavage, esterification and transesterification. These enzymes are produced by different microorganisms. To isolate the enzymes followed by the fermentation of microorganisms, a complex purification process.

The effectiveness of these catalysts often contrasts with the high costs of production and isolation, so that research groups are constantly striving to increase the yields of enzymes or to increase the productivity of the enzymes.

Suitable biocatalytic methods for synthesis are described, for example, in K. Drauz and H. Waldmann, Enzymes Catalysis in Organic Synthesis, WILEY-VCH, Volumes I to III, 2002; U. T. Bornscheuer, RJ. Kazlauskas in Hydrolases in Organic Synthesis. The technical implementation of biocatalytic processes is described by A. Liese, K. Seelbach and C. Wandrey in Industrial Biotransformations, WILEY-VCH, 2002.

Accordingly, enzymatically catalyzed esterifications represent state of the art. The use of immobilized enzymes with the background to increase the cost efficiency in a process are state of the art.

The disadvantage of biocatalytic reactions is often the availability and stability of the catalysts involved in the process. From the prior art are already enzymes or Microorganisms which are stabilized by immobilization, for example by microencapsulation, and can be used repeatedly.

Chemically catalyzed esterifications such as e.g. Acid catalysis is also state of the art. In these esterifications, a stoichiometric amount of water is released, which must be removed to drive the reaction towards the ester product.

Disadvantages of the purely chemical or purely enzymatically catalyzed esterification of fatty acids with aliphatic alcohols whose boiling point is between 60 and 120 ⁰ C, is the high amount of distillate, depending on the degree of conversion has different proportions of water. Also in the enzymatically catalyzed process, the water must be removed from the reaction mixture at high conversions, usually under vacuum and higher temperatures, which can lead to enzyme deactivation.

In particular, alcohols having a boiling point below that of water or forming a low boiling azeotrope with water (such as ethanol) are difficult to work up. Here, complicated processes such as membrane separation, molecular sieve drying or azeotropic distillation using entrainers must be used, which leads to high process costs.

If only the chemically catalyzed process is used, longer reaction times result. Due to the high thermal load, the color number of the product deteriorates considerably compared to enzymatically catalyzed esterifications.

The object of the present invention was thus to provide a method available in which fatty acids with aliphatic alcohols whose boiling point is between 60 and 120 ⁰ C, are esterified, without a complicated drying of the alcohol component must be performed. In addition, the process should be cost-efficient and recyclable. The enzymes are said to be only slightly impaired in their stability.

Description of the invention

The invention relates to a process for the preparation of fatty acid esters, in which

(a) treating fatty acids in the presence of esterases with hydrous aliphatic alcohols having a boiling point between 60 and 120 ° C, thereby producing a pre-esterification product,

(b) separating from the pre-esterification product water and the unreacted alcohol,

(c) subjecting the pre-esterification product to a second esterification, which is chemically catalysed, by re-adding the same aliphatic alcohol as used in step (a), and (d) re-using the separated hydrous alcohol of step (c) for enzymatic pre-esterification in step (a).

In a further embodiment, optionally during the pre-esterification stage (a), water is separated by phase separation.

Surprisingly, it has been found that with the aid of the process according to the invention, no work-up of the resulting aqueous fraction of aliphatic alcohol having a boiling point between 60 and 120 ° C., which forms a low-boiling azeotrope with water, is necessary. The aqueous alcohol resulting in the chemical esterification can be easily used in the enzymatic pre-esterification, since the enzymes catalyze a low-temperature esterification to the reaction equilibrium, which is far on the side of the ester. Since the reaction is carried out at low temperatures, no distillate is produced. After completion of the pre-reaction, the water and remaining alcohol is separated and disposed of. Since the reaction proceeds in the direction of ester synthesis, the loss of low-boiling alcohol is low. The reaction can be carried out by adding inert solvents, e.g. Hexane, isooctane or n-octane are moved towards ester, so that the loss of low-boiling alcohol can be kept even lower. A complex and energy-intensive workup of the aqueous alcohol coupled to the reaction is no longer necessary with this reaction. This saving represents an economic and ecological advantage. Accordingly, in a preferred embodiment of the method according to the invention of the enzymatic reaction, an inert organic solvent is added.

Advantage of the method according to the invention is that it is a chemo-enzymatic process. In a first stage, the fatty acid is esterified with an aliphatic alcohol / water mixture to a partial conversion. After completion of the reaction most of the water of reaction can be separated by phase separation from the product mixture. This is possible with mild temperatures and defined water / alcohol / ester compositions. Water separation is improved by adding a solvent such as n-octane in the enzymatic stage. After completion of the enzymatic stage, the solvent, unreacted alcohol and non-deposited water are distilled off. The remaining teilumgesetze material is then fed to a second esterification stage. This stage is carried out, for example, catalysed acid or tin salt. Here, up to 99 - 99.7% of sales esterified. The resulting alcohol / water of reaction distillate is collected and used completely in the first, enzymatically catalyzed pre-esterification stage. The combination of both processes and water separation by separation in the enzymatically catalyzed stage is an extremely synergistic process. Moreover, it must be emphasized that according to the reaction of the invention, with only slight variation of the conditions, a very wide range of different products can be produced in better yields under gentler conditions than is possible according to the processes known from the prior art.

Part of the conditions for the enzymatic pre-esterification according to step (a) can be listed as follows:

• Use of an alcohol / water mixture of 0.1 to 50% water content, wherein the pure alcohol has a boiling point between 60 and 12O ⁰ C. If appropriate, the use of an inert solvent such as n-octane for better separation of water in step (b) and for lowering the temperature of the enzymatic catalysis

• 1 to 5-fold molar excess of aliphatic alcohol with a boiling point between 60 and 120 ° C with a 1.1-fold excess is preferred. • Temperature between 20 and 70 ° C

• Prefers normal pressure

All other conditions and in particular the preferred conditions are described elsewhere in the specification.

In pre-esterification, final conversions of between 50 and 85% are achieved, depending on the reaction time. Depending on the carrier material, the initial amount of water and the fatty acid used, this is 8 to 16 hours for 80% conversion.

Part of the conditions for post-esterification under chemically catalyzed conditions can be listed as follows: • Use of aliphatic alcohol with at least 95% alcohol content

Use of the fatty acid / fatty acid ester / alcohol mixture from the pre-esterification wherein the optionally added solvent and the azeotrope are distilled off beforehand from water and aliphatic alcohol

• 1 to 4-fold molar excess of aliphatic alcohol with a boiling point between see 60 and 120 ⁰ C with a 1-fold excess is preferred.

• Temperature between 150 and 250 ° C

• The pressure should be reduced to 1 bar via a pressure ramp of 5 bar at the beginning of the reaction. Vacuum is applied towards the end of the reaction to separate the product mixture from unreacted aliphatic alcohol. Suitable catalysts are all esterification catalysts, tin-2 compounds, zinc compounds, sulfuric acid, para-toluenesulfonic acid or acidic ion exchangers are preferably used. • The reaction time for the chemically catalyzed reaction is only 10 to 12 hours, which is at least 50% lower than for a purely chemically catalyzed reaction without enzymatic pre-esterification.

For the purposes of the process according to the invention are used as fatty acids carboxylic acids of the general formula R-COOH, where R is a straight or branched chain optionally hydroxy-substituted alkyl or alkenyl radical having 6 to 32 carbon atoms, which contains up to 6 conjugated or non-conjugated double bonds.

In a particular embodiment of the process according to the invention, di- and / or polycarboxylic acids are used as fatty acids with straight-chain or branched, optionally hydroxy-substituted alkyl or alkenyl chains having 2 to 32 C atoms.

For the purposes of the process according to the invention, the fatty acids are selected from the group consisting of caproic, enanthic, caprylic, pelargonic, capric, lauric, lauric, myristic, palmitic, palmitoleic, stearic, petroselinic, petroselaidic, oleic, elaidic, ricinoleic , Linoleic, linoleic, linolenic, levostearic, arachidic, gadoleic, arachidonic, behenic, erucic, brassidic, clupanodonic, lignoceric, cerotic, melissic, eicosapentaenoic, docosahexaenoic, conjugated linoleic, isostearic, 2-ethylhexanoic. Especially preferred for the process according to the invention are myristic acid, oleic acid, lauric acid and palmitic acid.

Preferably, the molar ratio used in the process according to the invention between the fatty acid and the aliphatic alcohol deviates only minimally from 1 and is in particular in the range from 0.8 to 1.2, since then the highest yields of the desired product occur.

As the aliphatic alcohols, the advertising admixed during the pre- and the post-esterification the principle are those in question, which have a boiling point between 60 and 120 ⁰ C, so, for example, alcohols having 1 to 4 carbon atoms such as methanol (bp 64 ° C) , ethanol (boiling point of 78 ⁰ C), propanol (boiling point 97 ° C), isopropyl alcohol (boiling point 82 ° C) and 1-butanol (boiling point of 118 ⁰ C), isobutyl alcohol (boiling point 108 ⁰ C), sec-butyl alcohol (boiling point 99 ⁰ C) and tert. Butyl alcohol (boiling point 83 ⁰ C). In particular preferably for the present process are alcohols whose boiling point lying between 60 and 100 ⁰ C and is most preferably isopropyl alcohol. Enzymes used according to the invention are preferably enzymes from the group of esterases and especially lipases which can be used either alone or in combination with a plurality of enzymes.

Preferred within the meaning of the invention are esterases from organisms selected from the group formed by Thermomyces lanugenosus, Candida antarctica A, Candida antarctica B, Rhizomucor miehei, Candida cylindracea, Rhizopus javanicus, Porcine pancreas, Aspergillus niger, Candida rugosa, Mucor javanicus, Pseudomonas fluorescens, Rhizopus oryzae, Pseudomonas sp., Chromobacterium viscosum, Fusarium oxysporum and Penicillium camenberti. Particularly preferred as enzymes in the sense of biocatalysts are lipases from the organisms mentioned. In particular, the lipase from Candida antarctica B.

The enzymes to be used according to the invention can be used in various forms. In principle, all dosage forms of enzymes customary to the person skilled in the art can be used. The enzymes are preferably used in pure form or as a technical enzyme preparation either immobilized on a carrier material and / or in solution, in particular in aqueous solution, and reused in so-called repeated batches. Particularly preferred immobilized enzymes are adsorbed on hydrophobic supports such as polystyrenes, polyacrylamide or polypropylene support.

The esterase and especially the lipase is particularly preferably used in a stabilized form which is obtained by chemical modification with crosslinking reagents, in particular glutaraldehyde, or by chemical surface modification, for example with octanal. The reaction conditions of the biocatalytic reaction according to the invention depend on the optimum reaction range of the selected enzymes. In particular, there are conditions in which, inter alia, the reaction temperature between 20 and 70 ⁰ C, preferably a temperature between 35 and 55 ° C, in particular a temperature between 43 and 45 ⁰ C is selected.

Examples

Example 1: Stage 1, enzymatic pre-esterification:

Experimental apparatus: double jacket 4-neck round bottom flask with stirrer, internal thermometer,

Heating cryostat, bottom drain valve.

To 10 g immobilized enzyme on polypropylene pellets MP-100 (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 200 mg technical liquid preparation per g carrier) 125 g (0.548 mol) myristic acid, 62.5 g (1, 04 mol) isopropyl alcohol and 6.25 g of deionized water and stirred at 43 ⁰ C. After 24 h, a conversion of 55% is obtained. From about 40% conversion, a heavier water phase begins to precipitate with a maximum of 30% isopropyl alcohol. This is separated from the product mixture, so that the reaction can be started again. After a further 24 h, a final conversion of 70% is obtained. Here, after 57% of sales, another water phase has formed. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 10% when no solubilizer is used.

Example 2: Stage 1, enzymatic pre-esterification,

Experimental apparatus: Double-jacket 4-neck round bottom flask with stirrer, internal thermometer, heating cryostat, bottom drain valve

To 10 g immobilized enzyme polypropylene pellets on MP-100 (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 200 mg technical liquid preparation per g carrier) 125 g (0.548 mol) myristic acid, 62.5 g ( optionally 1.04 moles) of isopropyl alcohol and 11 g deionized water and stirred at 43 ⁰ C. After 24 h, a conversion of 55% is obtained. From about 40% conversion, a heavier water phase begins to precipitate with a maximum of 30% isopropyl alcohol. This is separated from the product mixture, so that the reaction can be started again. After a further 24 h, a final conversion of 70% is obtained. Here, after 57% of sales, another water phase has formed. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 10% when no solubilizer is used.

Example 3: Stage 1, enzymatic pre-esterification, experimental apparatus: double-jacket 4-neck round bottom flask with stirrer, internal thermometer, heating cryostat, bottom outlet valve.

To 10 g of immobilized enzyme on polypropylene pellets MP-100 (Candida antarctica B lipase, Novozymes adsorbed on polypropylene support, enzyme charge 200 mg technical liquid preparation per g of support) were added 125 g (0.548 mol) of myristic acid, 62.5 g (1.04 mol) of isopropyl alcohol and 11 g deionized water and stirred at 53 ⁰ C. After 24 h, a conversion of 55% is obtained. From about 40% conversion, a heavier water phase begins to precipitate with a maximum of 30% isopropyl alcohol. This is separated from the product mixture, so that the reaction can be started again. After a further 24 h, a final conversion of 70% is obtained. Here, after 57% of sales, another water phase has formed. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 10% when no solubilizer is used.

Example 4: Stage 1, enzymatic pre-esterification,

Experimental apparatus: double jacket 4-neck round bottom flask with stirrer, internal thermometer, heating cryostat, bottom drain valve

To 10 g of immobilized enzyme on polypropylene powder MP-1000 (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 500 mg technical liquid preparation per g carrier) 125 g (0.548 mol) myristic acid, 62.5 g (1, 04 mol) I- sopropylalkohol and 3.25 g of deionized water and stirred at 60 ⁰ C. After 8 h, a conversion of 70% is obtained. From about 60% conversion, a heavier water phase begins to precipitate with a maximum of 30% isopropyl alcohol. This is separated from the product mixture, so that the reaction can be restarted. After a further 4 h, a final conversion of 83% is obtained. It has formed another water phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 10% when no solubilizer is used.

Example 5: Stage 1, enzymatic pre-esterification,

To 10 g of immobilized enzyme on polypropylene powder on MP-1000 (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 500 mg technical liquid preparation per g carrier) 125 g (0.548 mol) of myristic acid, 62.5 g (1 , 04 mol) of isopropyl alcohol and 11 g of deionized water and stirred at 43 ⁰ C. After 8 h, a conversion of 70% is obtained. From about 60% conversion, a heavier water phase begins to precipitate with a maximum of 30% isopropyl alcohol. This is separated from the product mixture, so that the reaction can be restarted. After a further 4 h, a final conversion of 83% is obtained. It has formed another water phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 10% when no solubilizer is used. Example 6: Stage 1, enzymatic pre-esterification,

To 2 g of immobilized enzyme on polypropylene powder (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 500 mg technical liquid preparation per g carrier, crosslinked by standard methods with glutaraldehyde) are 7.5 g (32.9 mmol) myristic , 2.5 g (41.7 mmol) of isopropyl alcohol and 0.2 g of deionized water and 10 g of octane. The mixture is incubated at 45 ^° C. in a sealed Erlenmeyer flask on a shaker. After 4 h and 24 h, a sample is taken and the conversion is determined by acid number measurement. After completion of the reaction, the enzyme immobilizate is filtered off and reused in a new batch under identical conditions. In this form, the experiment is carried out over a period of 35 days.

Table 1: Activity determination of the enzyme immobilizate

Reaction Sales [%] Sales [%] Reaction Sales [%] Sales [%] (days) (after 4 h) after 24 h) (days) (after 4 h) after 24 h)

1 - 79.2 17 81.2 82.1

2 - 77.1 18 81.3 85.7

3 - 76.6 24 77.6 79.6

8 80.4 81.0 28 80.0 81.2

9 82.7 84.7 29 81.3 82.1

10 82.4 81.2 30 78.6 81.7

11 81.6 82.9 31 79.8 82.7

14 82.7 80.2 32 80.0 84.6

15 81.9 82.4 35 83.6

16 80.6 82.8

Over a period of 35 days no loss of activity of the enzyme immobilizate is observed. Without removal of a reaction product, a conversion of about 80% is achieved. In each batch, a water phase separates, which is sharply separated from the organic phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 3-5% when a solubilizer is used. Example 7: Stage 1, enzymatic pre-esterification,

To 2 g of immobilized enzyme (Immobilisat used in Example 6) are added 11.25 g (49.3 mmol) myristic acid, 3.75 g (62.5 mmol) of isopropyl alcohol and 0.2 g of deionized water and 5 g of octane. The mixture is incubated at 45 ^° C. in a sealed Erlenmeyer flask on a shaker. After 4 h and 24 h, a sample is taken and the conversion is determined by acid number measurement. After completion of the reaction, the enzyme immobilizate is filtered off and reused in a new batch under identical conditions. In this form, the experiment is carried out over a period of 115 days.

Table 2: Activity determination of the enzyme immobilizate - 2nd test

Reaction Turnover [%] Turnover [%] Reaction Turnover [%] Turnover [%]

(Days) (after 4 h) (after 24 h) (days) (after 4 h) (after 24 h)

1 - 80.8 49 50.6 80.0

2 69.6 79.2 50 52.6 79.2

3 67.9 80.3 51 50.2 79.9

4 67.2 78.1 52 52.4 80.0

6 68.6 78.8 53 50.5 79.8

7 66.4 74.5 56 53.3 79.5

9 68.5 78.9 57 49.0 79.6

10 69.1 78.7 65 48.5 79.1

11 68.5 78.7 66 50.8 80.2

14 66.6 79.9 67 49.6 79.3

15 66.6 79.9 70 47.2 79.4

16 67.3 80.1 71 48.4 79.4

17 66.1 79.1 72 44.7 78.9

21 65.0 79.1 74 45.3 79.0

22 65.2 79.5 78 48.7 79.2

23 63.9 79.4 85 42.0 79.0

24 64.6 79.2 86 41.3 79.3

25 62.9 79.4 87 38.6 79.1

28 63.5 79.9 91 44.1 79.6

29 62.0 79.9 92 41.5 79.4

30 60.2 79.5 93 40.9 78.9 31 61.9 78.6 94 39.2 78.9

32 61.3 76.5 95 41.1 79.7

36 57.4 79.5 98 38.3 79.9

37 60.5 81.2 99 38.2 78.7

43 49.8 79.2 100 38.1 78.8

44 55.5 79.7 101 34.7 79.2

45 54.4 79.2 113 33.1 79.0

46 55.5 79.6 115 36.2 78.2

Over a period of 115 days, a loss of activity of the enzyme immobilizate of about 50% is observed, the half-life of the immobilized enzyme under the above reaction conditions is 100-120 days. Without removal of a reaction product, a conversion of about 80% is achieved. In each batch, a water phase separates, which is sharply separated from the organic phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 3-5% when a solubilizer is used.

Example 8 Step 1, enzymatic pre-esterification:

To 2 g of immobilized enzyme (from Example 7, newly loaded with Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 1100 mg technical liquid preparation per g carrier, crosslinked by standard methods with glutaraldehyde) are 11.25 g (49.3 mmol) Myristic acid, 3.75 g (62.5 mmol) of isopropyl alcohol and 0.2 g of deionized water and 5 g of octane. The mixture is incubated at 45 ^° C. in a sealed Erlenmeyer flask on a shaker. After 4 h and 24 h, a sample is taken and the conversion is determined by acid number measurement. After completion of the reaction, the enzyme immobilizate is filtered off and reused in a new batch under identical conditions. In this form, the experiment is carried out over a period of 68 days.

Table 3: Activity determination of the enzyme immobilizate - 3rd test

Reaction Turnover [%] Turnover [%] Reaction Turnover [%] Turnover [%]

(Days) (after 4 h) after 24 h) (days) (after 4 h) after 24 h)

1 79.5 80.7 44 80.0 79.8

2 78.0 79.4 45 80.5 79.9

3 79.1 82.9 46 79.6 79.6

4 78.5 79.9 64 79.7 80.1

5 79.2 79.9 65 80.4 80.1

8 78.7 79.6 67 79.5 80.4

9 78.5 79.8 68 79.9 -

43 80.0 80.0

Over a period of 68 days no loss of activity of the enzyme immobilizate is observed. Without removal of a reaction product, a conversion of about 80% is achieved. In each batch, a water phase separates, which is sharply separated from the organic phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 3-5% when a solubilizer is used.

Example 9: Stage 1, enzymatic pre-esterification,

To 5 g immobilized enzyme on polypropylene powder (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 500 mg technical liquid preparation per g carrier) are 37.5 g (164.5 mmol) myristic, 10.5 g ( 175.0 mmol) isopropyl alcohol and 2.0 g of deionized water and 50 g of hexane are added. The mixture is incubated at 35 ^° C. in a sealed Erlenmeyer flask on a shaker. After 5 h and 22 h, a sample is taken and the conversion is determined by acid number measurement. After completion of the reaction, the enzyme immobilizate is filtered off and reused in a new batch under identical conditions. In this form, the experiment is carried out over a period of 77 days. Table 4: Activity determination of the enzyme immobilizate - 4th test

Reaction Turnover [%] Turnover [%] Reaction Turnover [%] Turnover [%]

(Days) (after 5 h) after 22 h) (days) (after 5 h) after 22 h)

1 61.2 72.8 46 33.7 77.4

4 51.2 80.6 47 28.3 76.7

5 42.8 82.1 48 29.5 78.8

6 40.7 78.4 49 31.4 76.4

12 47.3 83.3 50 26.7 85.1

13 39.0 82.3 53 21.4 74.1

14.44 82.4 54 23.8 73.5

15 82.2 55 23.2 68.8

18 39.8 80.7 56 22.3 68.1

19 37.6 81.9 57 20.7

26 30.9 81.3 62 25.0 68.8

27 30.9 80.6 63 21.3 66.2

28 36.3 80.9 64 18.7 -

29 23.7 83.1 67 15.9 60.7

32 19.8 78.0 68 10.4 54.5

33 30.6 79.9 69 16.9 57.3

34 30.0 79.6 70 14.8 56.2

35 28.0 79.4 74 15.6 55.4

39 16.1 77.3 75 13.3 52.7

40 18.2 81.9 76 10.7 51.8

41 30.0 79.5 77 11.7 -

A deactivation of the enzyme immobilizate is observed under the above conditions, which corresponds to a half-life of the enzyme of about 30 days. Without removal of a reaction product, a conversion of about 80% is achieved. In each batch, a water phase separates, which is sharply separated from the organic phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 3-5% when a solubilizer is used. Example 10: Esterification with lauric acid: stage 1, enzymatic pre-esterification, experimental apparatus: double jacket 4-neck round bottom flask with stirrer, internal thermometer, heating cryostat, bottom outlet valve

To 10 g of immobilized enzyme on polypropylene pellets on MP-100 (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 200 mg technical liquid preparation per g carrier) are 125 g (0.625 mol) of lauric acid, 62.5 g (1, 04 mol) isopropyl alcohol and 11 g of deionized water and stirred at 43 ⁰ C. After 24 h, a conversion of 55% is obtained. From about 40% conversion, a heavier water phase begins to precipitate with a maximum of 30% isopropyl alcohol. This is separated from the product mixture, so that the reaction can be started again. After a further 24 h, a final conversion of 71% is obtained. Here, after 57% of sales, another water phase has formed.

Example 11: Stage 1, enzymatic pre-esterification, to 3 g of immobilized enzyme on polypropylene powder (Candida antarctica B lipase, novozymes adsorbed on polypropylene support, enzyme charge 500 mg technical liquid preparation per g carrier, crosslinked by standard methods with glutaraldehyde) 30 g (150 , 0 mmol) of lauric acid, 9.9 g (165.0 mmol) of isopropyl alcohol, 0.5 g of demineralized water and 8.5 g of octane. The mixture is incubated at 45 ° C in a sealed Erlenmeyer flask on a shaker. After 5 h and 24 h, a sample is taken and the conversion is determined by acid number measurement. After completion of the reaction, the enzyme immobilizate is filtered off and reused in a new batch under identical conditions. In this form, the experiment is carried out over a period of 9 days.

Table 5: Activity determination of the enzyme immobilizate - 5th test

Over a period of 9 days, an enzyme immobilization activity loss of about 10% is observed. Without removal of a reaction product, a conversion of about 75% is achieved. In each batch, a water phase separates, which is sharply separated from the organic phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 3-5% when a solubilizer is used.

Example 12: Esterification with Palmitic Acid: Stage 1, Enzymatic Pre-esterification,

To 3 g of immobilized enzyme on polypropylene powder (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 500 mg technical liquid preparation per g carrier, crosslinked by standard methods with glutaraldehyde) are 30 g (117.2 mmol) of palmitic acid, 7 , 7 g (128.3 mmol) of isopropyl alcohol, 0.5 g of deionized water and 8.5 g of octane. The mixture is incubated at 45 ^° C. in a sealed Erlenmeyer flask on a shaker. After 5 h and 24 h, a sample is taken and the conversion is determined by acid number measurement. After completion of the reaction, the enzyme immobilizate is filtered off and reused in a new batch under identical conditions. In this form, the experiment is carried out over a period of 9 days.

Table 6: Activity determination of the enzyme immobilizate - 6th test

Over a period of 9 days, no loss of activity of the enzyme immobilizate is observed. Without removal of a reaction product, a conversion of about 78% is achieved. In each batch, a water phase separates, which is sharply separated from the organic phase. Studies on the composition of the water phase typically show a maximum isopropyl alcohol content of 3 - 5%, if a solubilizer is used.

Example 13 Esterification with Oleic Acid: Stage 1, Enzymatic Preesterification,

To 7 g of immobilized enzyme on polypropylene powder (Candida antarctica B lipase, Novozymes adsorbed on polypropylene carrier, enzyme charge 500 mg technical liquid preparation per g carrier, crosslinked by standard methods with glutaraldehyde) are 110.0 g (390.1 mmol) of oleic acid , 26.0 g (433.3 mmol) of isopropyl alcohol and 1.0 g of deionized water. The mixture is incubated at 60 ^° C. in a sealed Erlenmeyer flask on a shaker. The conversion is determined by acid number measurement.

Table 7: Determination of the conversion in the reaction of oleic acid with isopropyl alcohol

Example 14: Step 2, Chemically Catalyzed Reaction,

In the reaction kettle 100 kg of pre-esterified mixture IPM, IPA, octane, myristic acid are dosed. The mixture is heated with stirring and under a slight stream of nitrogen (4 l / h) to 225 ⁰ C. Here, the distillate obtained via a dephlegmator (Tset = 130 ⁰ C) is collected and reported. The distillate is used after completion of the reaction in the enzymatic pre-esterification. When the temperature is reached, a pressure of 5 bar is initially set. Subsequently, the catalyst is drawn in 200 g tin-2 compound. After adding the catalyst, a pressure ramp of 5 bar to 1 bar is run, reducing the pressure per hour by one bar. At 5 bar pressure, the isopropyl alcohol is started to be metered. 1.5 kg of isopropyl alcohol 99.9% are added per hour. During the reaction, an isopropyl alcohol / water distillate is continuously obtained. This is driven over the dephlegmator and completely condensed after the dephlegmator and collected. With the pressure reduction, a dephlegmatist temperature reduction is made.

Table 8: Pressure and temperature during the reaction

The turnover trend is determined by SZ. Therefore, every hour samples are taken for SC determination. The reaction is run for about 8 hours, the final acid number should be less than 2 after this time. Then the reaction is finished. The excess isopropyl alcohol is distilled off (at 200 ⁰ C reactor temperature, 100 ⁰ C dephlegmator temperature, vacuum ramp from 1000 mbar to 500 mbar within 30 min). The entire collected distillate is collected and fed to the enzymatic pre-esterification.

Table 9: Sales Control

After completion of the reaction, the excess isopropyl alcohol is distilled off (at 200

⁰ C reactor temperature, 100 ⁰ C Dephlegmator temperature, vacuum ramp of 1000 mbar

500 mbar within 30 min). The entire collected distillate is collected and fed to the enzymatic pre-esterification.

Analysis of the distillate showed the following composition:

5% water

94% IPA

1% fatty acid, IP ester mixture

Claims

claims

1. A process for the preparation of fatty acid esters which comprises: (a) treating fatty acids in the presence of esterases with water-containing aliphatic alcohols having a boiling point between 60 and 120 ° C, thereby producing a pre-esterification product,

(b) separating from the pre-esterification product water and unreacted alcohol,

(c) subjecting the pre-esterification product to a second esterification, which is chemically catalysed, by re-adding the same aliphatic alcohol as used in step (a);

(d) re-using the separated hydrous alcohol of step (c) for enzymatic pre-esterification in step (a).

2. The method according to claim 1, characterized in that are used as fatty acids carboxylic acids of the general formula R-COOH, where R is a straight or branched chain optionally hydroxy-substituted alkyl or alkenyl radical having 6 to 32 carbon atoms, which up to 6 conjugated or contains non-conjugated double bonds.

3. Process according to claims 1 and / or 2, characterized in that the fatty acids are selected from the group formed by caproic acid, oenanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, lauroleinic acid, myristic acid, Myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, petroselinic acid, petroselaidic acid, oleic acid, elaidic acid, ricinoleic acid, linoleic acid, linolaidic acid, linolenic acid, levostearic acid, arachidic acid, gadoleic acid, arachidonic acid, behenic acid, erucic acid, brassidic acid, clupanodonic acid, lignoceric acid, cerotic acid, melisinic acid, Eicosapentaenoic acid, docosahexaenoic acid, conjugated linoleic acid, isostearic acids, 2-ethylhexanoic acid.

4. The method according to any one of claims 1 to 3, characterized in that are used as fatty acids di- and / or polycarboxylic acids with straight or branched, optionally hydroxy-substituted alkyl or alkenyl chains having 2 to 32 carbon atoms.

5. The method according to any one of claims 1 to 4, characterized in that a 1 to 5-fold molar excess of aliphatic alcohol having a boiling point between 60 and 120 ⁰ C in the pre-esterification according to step (a) is present and a 1 to 4 fold molar excess of aliphatic alcohol having a boiling point between 60 and 120 ⁰ C in the post-esterification according to step (d).

6. The method according to any one of claims 1 to 5, characterized in that the aliphatic alcohols are selected from the group formed by methanol, ethanol, propanol, isopropyl alcohol, 1-butanol; Isobutyl alcohol, sec-butyl alcohol and tert. Butyl alcohol.

7. The method according to any one of claims 1 to 6, characterized in that the aliphatic alcohol is isopropyl alcohol.

8. Process according to claims 1 to 7, characterized in that one uses in step (a) esterases in free or immobilized form.

9. Process according to claims 1 to 8, characterized in that the esterases are derived from organisms selected from the group formed by

Thermomyces lanugenosus, Candida antarctica A, Candida antarctica B, Rhizomucor miehei, Candida cylindracea, Rhizopus javanicus, P ordne pancreas, Aspergillus niger, Candida rugosa, Mucor javanicus, Pseudomonas fluorescens, Rhizopus oryzae, Pseudomonas sp., Chromobacterium viscosum, Fusarium oxysporum and Penicilium cameraberti ..

10. The method according to any one of claims 1 to 9, characterized in that it is the lipases in the esterases.

11. The method according to at least one of claims 1 to 10, characterized in that one uses as the lipase Candida antarctica in immobilized form.

12. The method according to at least one of claims 1 to 11, characterized in that the enzymatic reaction of inert organic solvents are mixed.

13. The method according to at least one of claims 1 to 12, characterized in that in the chemically catalyzed post-esterification according to step (c) catalysts are used, which are selected from the group consisting of tin-2 compounds, zinc compounds, sulfuric acid , Para-toluenesulfonic acid and acidic ion exchangers.