TECHNICAL FIELD

This invention relates to an enzymatic process for hydrolyzing one or both acyl groups in a phospholipid.

BACKGROUND ART

Phospholipids (glycerophospholipids), such as phosphatidyl choline, consist of glycerol esterified with 2 fatty acyl groups and one phosphate or esterified phosphate group. For some applications of the phospholipid, it is desirable to hydrolyze one or both acyl groups, e.g. in order to modify the emul- sification properties or create glycerophosphoryl compounds which are building blocks for synthetic phospholipids.

Use of phospholipase A- _j and A ₂ for this hydrolysis is well known. In many cases, it is desirable to remove the residual enzyme activity, and due to the stability of some phospholipases like porcine pancreatic phospholipase A ₂ this may require special measures such as extensive heating or treatment with protease. See JP-A 63-233,750.

It is also known that hydrolysis of phospholipid can be catalyzed by lipase (EP 260,573 and JP-A 63-42,691) and by chemically modified (derivatized) lipase (JP-A 63-105,685). It is the object of this invention to provide an enzymatic hydrolysis process whereby the enzyme can be easily separated from the reaction mixture and be re-used, without the need for a large excess of water.

STATEMENT OF THE INVENTION

Surprisingly, we have found that phospholipids can be efficiently hydrolyzed by immobilized enzymes with a slight stoichiometric excess of water.

Accordingly, the invention provides an enzymatic process for hydrolyzing one or both acyl groups in a phospholipid, characterized in that the enzyme is immobil¬ ized on a particulate macroporous carrier.

Immobilization The enzyme used in the invention is immobilized on a particulate macroporous carrier. The enzyme may be simply adsorbed on the carrier, or it may be attached to the carrier by cross-linking with glutaraldehyde or other cross- linking agent known in the art.

A preferred carrier type is weakly basic anion exchange resin, e.g. of acrylic, polystyrene or phenolformaldehyde type. .Examples of commercial products are Lewatit ^® E 1999/85 (product of Bayer, West Germany) and Duolite ^® ES-568 (Rohm & Haas).

Another preferred carrier type is an adsorbent (non-ionic) carrier, e.g. of the phenol-formaldehyde type, acrylic type or polypropylene type. Examples of commercial products are Lewatit E2001/85 (acrylic, product of Bayer) and Accurel EP-100 (polypropylene, product of AKZO).

Another preferred immobilization method uses an inorganic support material, and the enzyme is preferably attached to the support by adsorption or covalent coupling. Such support materials and immobilization techniques are described in K. Mosbach (ed.): Methods in Enzymology, 44, "Immobilized

Enzymes" (Academic Press, 1976).

A preferred inorganic support material is macroporous silica or silicate carriers e.g. macroporous silica carriers from Grace Chemicals described in Biocatalyst Supports SG BC 1E/June 1987 in which more than 90% of the particles have particle sizes between 100 and 1000 p, wherein more than 80% of the pores in the particles exhibit a diameter between 5 and 45 times the diameter of the enzyme globules.

The immobilized enzymes useful for interesterification of phospholipids typically are loaded with 20,000 - 200,000 LU per g (dry weight) of catalyst (LU, Lipase Unit is defined in US 4,810,414).

Enzyme

The enzyme to be used may be a lipase of animal, plant or microbial origin. For reasons of economy, a microbially produced lipase is preferred, e.g. a bacterial or fungal lipase. Some examples of suitable enzymes are lipases derived from the following organisms:

- Positionally specific lipase from Rhizomucor (also designated Mυcoή, especially R. miehei {M. miehei), commercially available as Lipozyme™ (Novo Nordisk a/s). - Positionally specific lipase from Humicola, especially H. lanuginosa

(also designated Thermomyces lanuginosus), see US 4,810,414, EP 305,216.

- Positionally non-specific lipase from Candida rugosa (also termed C. cylindraceae, the lipase being available as Lipase OF (Meito Sangyo)).

- Positionally non-specific lipase from Pseudomonas cepacia (WO 89/01032).

Other enzymes are those indicated in JP-A 63-42,691, incorporated herein by reference, at col. 6-7.

Process conditions

The process is preferably carried out in a non-polar solvent like hexane, heptane, petroleum ether, or chlorinated hydrocarbons with a humidified immobilized enzyme.

In the process there has to be a sufficient amount of water surrounding the enzyme molecules in order to keep the enzyme molecules catalytically active. Secondly there has to be a sufficient amount of water to hydrolyze the ester bonds in the phospholipids. The necessary amount of water is generally in the range 0.5-5% by weight and may be provided simply by humidifying the immobilized enzyme, e.g. to a water content of 5-50% (w/w), especially 5-25%. The solvent may also be saturated with water, e.g. in a continuous process. The process temperature should be chosen after considering thermostability of the immobilized enzyme. In many cases 20-60°C will be suitable.

For very thermostable enzymes temperatures as high as 80°C may be used.

The process may be carried out as a batch reaction, where the ingredients are stirred gently throughout the reaction period. The amount of immobilized enzyme will typically be 1-10% (w/w), and the reaction time will generally be 10 minutes - 24 hours. After the reaction the reaction products can be separated from the immobilized enzyme simply by decanting or filtration.

Alternatively, the process may be carried out continuously by letting the phospholipid in solvent pass through a fixed bed column of immobilized enzyme. The residence time will typically be 1 - 12 hours.

Phospholipid The process of the invention may be applied to any desired kind of phospholipid containing fatty acyl ester groups and one or two phosphate groups which may be esterified. Examples of such naturally occurring phospholipids are phosphatidic acid, phosphatidyl cholϊne, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl ethanolamine and diphosphatidyl glycerol. Synthetic phospholipids with various hydroxy compounds esterified to the phosphate group may also be processed.

EXAMPLES

The following immobilized upases were prepared for use in the examples: Rhizomucor miehei lipase produced by cultivating a transformed

Aspergillus oryzae was immobilized on Duolite ^® ES-568 N. The load was 99,200 LU per g (dry weight) of catalyst.

Humicola lanuginosa lipase produced by cultivating a transformed Aspergillus oryzae was immobilized on a macroporous silica carrier (Grace 6, product of Grace Chemicals). The load was 166,500 LU per g (dry weight) of catalyst.

Candida cylindracea lipase (Lipase-OF from Meito Sangyo) was immobilized on Accurel EP-100. The load was 34,200 LU per g (dry weight) of catalyst.

EXAMPLE 1

As phospholipid was used the commercial product Epikuron 200 from Lucas Meyer GmbH. This is a fractionated soybean lecithin claimed to contain min. 95% phosphatidyl choline (PC), max. 4% lysophosphatidyl choline (LPC) and a moisture and oil content of max. 3%. 1.0 g of Epikuron 200 was mixed with 20 ml petroleum ether (b.p. 80-100°C). Each of the above immobilized R. miehei and H. lanuginosa lipases corresponding to a dry weight of 125 mg was weighed into a vial. The lipases were humidified overnight to a water content of 25% (w/w). 1.5 ml of the above mixture was added to the immobilized lipase.

Gentle stirring was then carried out at 40°C for 24 hours. Then the substrate was separated from the enzyme catalyst.

To estimate the degree of hydrolysis the following was performed: PC was separated from LPC and glycerophosphorylcholine (GPC) by thin layer chromatography on Silica gel 60 plates (Merck art. 5721) using CHCI3 : CH ₃ OH : H 0 (65:25:4, v/v/v) as solvent. After elution the plates were dried, and spots visualized by iodine vapors. The area corresponding to phosphatidyl choline and to LPC + GPC were scraped off. PC and LPC + GPC were extracted from the scraped off silica gel using in turn the following media 1.5 ml CHCI ₃ :CH ₃ OH:H ₂ 0 (65:25:4, v/v/v) 1.0 ml CHCI ₃ :CH ₃ OH:H ₂ 0 (65:25:4, v/v/v) 1.0 ml CH ₃ OH 1.0 ml CH ₃ OH:CH ₃ COOH:H ₂ 0 (94:1:5)

The extracts were pooled and adjusted to 5 ml by CHCI ₃ :CH ₃ OH:H ₂ 0 (65:25:4, v/v/v).

PC and LPC + GPC were quantitated by taking a 1 ml sample of the pooled extracts, evaporating the solvents and determining phosphorus in the residue according to Bruce N. Ames, Methods Enzymol. (8), 115-117, (1966). The degree of PC hydrolysis (DPCH) was calculated as

amount of P in LPC + GPC

DPCH = x 100% amount of P in PC + LPC + GPC

The results were:

Substrate DPCH

Untreated 5%

Treated with R. miehei lipase 85%

Treated with H. lanuginosa lipase 93%

EXAMPLE 2

Reaction was carried out as in Example 1 , but using the immobilized

C. cylindracea lipase.

After 24 hours essentially all of the phosphatidyl choline had become hydrolyzed.

EXAMPLE 3

10 mg/ml solutions or suspensions of phosphatidic acid (PA), phosphatidyl glycerol (PG), phosphatidyl ethanolamine (PE), phosphatidyl serine (PS) and lysophosphatidyl choline (LPC) in petroleum ether (b.p. 80-100°C) were made.

Samples of immobilized R. miehei lipase corresponding to a dry weight of 125 mg were weighed into vials and humidified overnight to water contents as shown in the table below. 1.5 ml of above substrates were added (at time t = 0). Gentle stirring was carried out at 40°C. 50 μ\ samples were taken at times t = 0, 2, 4, 6, and 24 hours and applied to Silica gel 60 plates. The plates were eluted with CHCI ₃ :CH ₃ OH:H ₂ 0 (65:25:4, v/v/v) and spots visualized by iodine vapors.

In all cases spots corresponding to PA, PG, PE, PS, and LPC disappeared within 24 hours. In the table below is shown the hydrolysis time T in which the phospholipid in question could no longer be discerned by TLC.

Water content (% w/w) Hydrolysis time Phos holi id of immobilized li ase T h r