The technical area to which this invention belongs comprises immobilized lipase preparations and use thereof for ester synthesis. One of the immobilization methods for lipases comprises adsorption from an aqueous solution to a particulate, porous carrier with affinity for the iipase. The selection of a carrier is a delicate problem: in the first place the carrier has to exhibit a high selective affinity to the special lipase in question in the aqueous solution; in the second place the thus produced immobilized lipase preparation has to exhibit a high activity and stability during use conditions, whereby use conditions may be ester synthesis or ester hydrolysis, in the third place the carrier has to be relatively cheap, if the immobilized lipase preparation should be used on an industrial scale, and in the fourth place the carrier has to be non-toxic, and be able to be food approved. Candida antarctica lipase A and Candida antarctica lipase B are known lipases, vide e.g. EP 287.634, and also, it is described in EP 287.634 that these lipases can be immobilized by adsorption on anion exchange resins of the Duolite ^® type. However, for ester synthesis purposes these prior art preparations were unsatisfactory, as they exhibited rather low ester synthesis activities. Thus the purpose of the invention is the provision of an immobilized lipase preparation prepared by adsorption of the lipase on a carrier from an aqueous solution of the lipase in which the lipase is one or both of the lipases selected from Candida antarctica lipase A and Candida antarctica lipase B, whereby the immobilized lipase preparation should exhibit a higher ester synthesis activity under use conditions than the ester synthesis activity under use conditions of the prior art immobilized lipase preparations of this kind.

Now, surprisingly it has been found that it is possible to fulfil the purpose of the invention by selection of a specific carrier category chosen among a vast number of carrier categories, and by selection of a special pretreatment of the chosen carrier.

Thus, the immobilized lipase preparation for ester synthesis according to the invention is characterized by the fact that the lipase is one or

both of the lipases selected from the group consisting of Candida antarctica lipase A and Candida antarctica lipase B, and that the immobilization is performed by adsorption from aqueous solution on a particulate porous aliphatic olefinic polymer, said polymer being pretreated with a polar, organic, water miscible solvent, in which the polymer is insoluble and which does not inactivate the lipase. Surprisingly it has been found that the ester synthesis activity of the immobilized lipase preparation according to the invention is much higher than the corresponding prior art ester synthesis activity, which will be documented in detail in a later part of the specification. Reference is made to EP 232.933, which describes the special category of carriers used in the invention together with some other categories of carriers. It is indicated in the EP patent No. 232.933 that lipase originating from Candida cyiindracea can be used. No indication is made of the possibility of using Candida antarctica lipase A and Candida antarctica lipase B. In regard to structure and properties the lipase from Candida cyiindracea is completely different from the lipases from Candida antarctica A and/or B. Furthermore, the EP patent is concerned exclusively with hydrolysis of fats, i.e. with reaction systems with high water concentrations, whereas the invention is concerned with ester synthesis, i.e. with systems with low water concentrations to extreme low water concentrations.

In WO published patent application no. 89/02916 it is described that Candida lipase immobilized on adsorbing resins of the acrylate type ("Lewatit") exhibit a higher interesterification activity than Candida lipase immobilized on adsorbing resins of the phenol type and weak anion exchange resins. It has been found, however, that the immobilized lipase preparation according to the invention, especially the immobilized lipase preparation according to the invention with Candida antarctica lipase B, at low water concentrations (0-1% w/w) exhibit a much higher interesterification activity than the interesterification activity of the prior art preparation immobilized on Lewatit, vide Example 6 in the following. In relation to interesterification processes it is highly advantageous to work with low water concentrations due to the corresponding low formulation of byproducts.

In this specification with claims use is made of the following terminology.

The term "porous" is intended to mean that the carrier particles exhibit pores. The smaller the pores the better, as smaller pores is equivalent to bigger surface areas. The pores, however, should preferably not be smaller than

100 A in diameter, as the lipase molecules tend to be too large to enter such small pores. Thus, in accordance with the definition in "Colloid and Interface

Chemistry" Void and Void, 1983, Addison-Wesley Publishing Company, Inc., page

101 the preferred pore structure in the immobilized lipase preparation is to be classified as macropores and/or mesopores.

The term "ester synthesis" encompasses all kinds of ester synthesis, e.g. a reaction between an alcohol and an acid, a reaction between an ester and an alcohol (alcoholysis), a reaction between two esters (transesterification), or a reaction between an ester and an acid (acidolysis). The term "interesterification" covers transesterification, acidolysis, and alcoholysis. The term "ester synthesis" also covers the glycolipid synthesis, i.e. a reaction between the alcohol glucose and a fatty acid.

The LU which is the lipase unit used for evaluation of the activity in relation to fat hydrolysis, is described in the publication AF 95/5-GB, which is obtainable on request from Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark. The BIU which is the lipase unit used for evaluation of the interesterification activity, is described in the publication AF 206-2, which is obtainable on request from Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark.

The PLU, which is the lipase unit used for evaluation of the ester synthesis activity, is performed as follows:

Principle: The activity is measured approximately as initial rate of n-propyl laurate formation.

Lipase preparation: an immobilized preparation of a Candida antarctica lipase A and/or Candida antarctica lipase B is hydrated to 10% w/w (overnight).

Substrate:

250 mg enzyme preparation (dry substance)

40 mmole lauric acid (8.01 g)

40 mmole n-propanol (2.40 g)

Reaction:

60°C, 20 minutes, shaking in 20 ml glass vials (as in relation to BlU-assay).

Calculation of activity:

The acid value (AV) of the substrate is determined before and after reaction.

Degree of ester formation is calculated and initial activity is approximated:

40 mmole x (Degree of ester formation)

Activity = [mmole/h/g]

0.33 h x 0.250 g

AV ₀ -AV

Degree of ester formation =

AV,

In a preferred embodiment of the immobilized lipase preparation according to the invention the carrier is polypropylene. Practical experiments have shown that this olefinic polymer is very satisfactory as a carrier.

In a preferred embodiment of the immobilized lipase preparation according to the invention more than 90% of the particles exhibit particle sizes between 100 and 1000 μ . If the particle size is below 100 μm, the pressure loss in a column will tend to be too large, and if the particle size is above 1000 μm, the diffusion inhibition tends to be too high.

Also, the invention comprises a use for ester synthesis of the immobilized lipase preparation according to the invention.

In a preferred embodiment of the use according to the invention the lipase is Candida antarctica lipase A, and the ester synthesis is an

interesterification. Immobilization yield and interesterfication activity has been found to be most satisfactory.

In a preferred embodiment of the use according to the invention the lipase is Candida antarctica lipase B, and the ester synthesis is a reaction between an alcohol and an acid. Immobilization yield and ester synthesis activity has been found to be most satisfactory.

In a preferred embodiment of the use according to the invention the lipase is Candida antarctica lipase B, and the ester synthesis is an interesterification. Interesterification activity at low water concentration has been found to be most satisfactory.

The invention will be illustrated by means of the following examples. In these examples three preparations of Candida antarctica are investigated, i.e. Candida antarctica lipase A (abbreviated LA), Candida antartica lipase B (abbreviated LB) and the mixed preparation (abbreviated L(A+ B)). LA and LB are described and characterized in EP 287.634.

The carriers used according to the invention and the control carriers belong to the types ACCUREL ^® and DUOLITE ^® , respectively.

ACCUREL, which is a particulate polypropylene, is described in AKZO, Fibres and Polymers division, Accurel systems, datasheet, obtainable from Enka AG, Postfach D-8753 Obemburg, West Germany.

Duolite is a trademark from Rohm & Haas for anion and cation exchange resins and resinous adsorbent agents.

EXAMPLE 1

Comparison A suspension of 4.0 g (dry matter) of Duolite ES-568N was ad¬ justed to pH 7, and 0.80 g of LA (222,800 LU/g) dissolved in deionized water (total weight of LA solution 12 g) was added. pH was readjusted to 7, and the suspension was stirred for 2 hours at room temperature. The product was filtered and rinsed with 60 ml of deionized water and 60 ml of cold acetone. The product was vacuum dried at 40°C.

Immobilization yield = 34% (calculated from LU determinations) Load = 15,700 LU/g of dry matter BlU/g = 7; PLU/g = 0

EXAMPLE 2

According to the invention

4.0 g (dry matter) of Accurel EP 100 is slurried in 96% ethanol. The excess of ethanol is sucked away (residual ethanol 9 g). 0.80 g of LA (222,800 LU/g) dissolved in deionized water (total weight of LA solution is 20 g) is added. pH is adjusted to 7, and the suspension is stirred for 2 hours at room temperature. The product is filtered and rinsed with 60 ml of deionized water. The product is vacuum dried at 40°C.

Immobilization yield = 96% (calculated from LU determinations) Load = 38,000 LU/g of dry matter BlU/g = 56; PLU/g = 0

It appears from Examples 1 and 2 that the immobilized lipase preparation according to the invention is highly superior in comparison to the corresponding prior art preparation, both in regard to load ad to interesterification activity.

EXAMPLE 3

According to the invention

10 g (dry matter) of Accurel EP 100 is slurried in 96% ethanol. The excess of ethanol is sucked away (residual ethanol 23 g). 34.7 g of LB (14,400 LU/g) dissolved in deionized water (total weight of LB solution is 50 g) is added. pH is adjusted to 7, and the suspension is stirred overnight, totally

18 hours. The product is filtered and rinsed with 100 ml of deionized water. The product is vacuum dried at 40°C.

Immobilization yield = 100% (calculated from LU determinations) Load = 48,200 LU/g of dry matter BlU/g approx. 0; PLU/g = 370

A similar experiment, in which Duolite was used instead of Accurel, showed a much lower immobilization yield and a much lower ester synthesis activity.

EXAMPLE 4

Comparison

A suspension of 4.0 g (dry matter) of Duolite ES-568N was adjusted to pH 7, and 1.87 g of L(A+B) (85,600 LU/g) dissolved in deionized water was added (total weight of the L(A+ B) solution was 12 g). pH was readjusted to 7, and the suspension was stirred overnight, totally 17 hours. The product was filtered and rinsed with 60 ml of deionized water. The product was vacuum dried at 40°C.

Immobilization yield = 13% (calculated on LU) Load = 5,400 LU/g of dry matter BlU/g = 8; PLU/g = 0.2

EXAMPLE 5

According to the invention

1 g (dry matter) of Accurel EP 100 is mixed with 10 ml of 96% ethanol. Stirring is carried out for 10 minutes, followed by vacuum filtration. 0.4 g of L(A+ B) (142,000 LU/g) dissolved in 5 ml deionized water was added. pH

was adjusted to 6.5, and the suspension was stirred overnight. The product was filtered and rinsed with 50 ml of deionized water. The product is air dried at room temperature.

Immobilization yield = 98% (calculated from LU determinations) Load = 55,900 LU/g dry matter BlU/g = 61 ; PLU/g = 346

It appears from Examples 4 and 5, that the immobilized lipase preparation according to the invention is highly superior to the corresponding prior art preparation, both in regard to the immobilization yield, the load and to the ester synthesis activity and the interesterification activity.

EXAMPLE 6

According to the invention

This example describes the use of LA and LB for interesterification. Procedures are as in Example 3, except for the following modifications and results:

4.43 g of LB (112,900 LU/g) to 4.0 g Accurel EP 100 Immobilization yield = 100% Load = 45,200 LU/g of dry matter PLU/g = 370 (same as in Example 3)

25 g Accurel EP 100 5.61 g of LA (222,800 LU/g) Immobilization yield = 99% Load = 44,700 LU/g of dry matter BlU/g = 59

The interesterification activity was measured at catalyst hydrations from 0-10%:

Hvdration (% HoHO) QJ _. 2 5 10

Interesterification activity (BlU/g):

Immobilized LB 14 10 0-1 0-1

Immobilized LA 12 59

It is remarkable that interesterification with LB is possible only at very low hydration levels.

EXAMPLE 7

According to the invention

This example shows immobilization of LB on polyethylene powder, Accurel EP 400, with ethanol and isopropanol as wetting agents.

Variations from Example 3 are:

0.338 g of LB (360,000 LU/g) on 4.1 g Accurel EP 400 / ethanol Immobilization yield = 30% Load = 9,200 LU/g of dry matter PLU/g = 369

0.341 g of LB (360,000 LU/g) on 4.1 g Accurel EP 400 / iso-PrOH Immobilization yield = 22%

Load = 6,800 LU/g of dry matter PLU/g = 294