WO2000056869A2

WO2000056869A2 - Surfactant-lipase complex immobilized on insoluble matrix

Info

Publication number: WO2000056869A2
Application number: PCT/IL2000/000166
Authority: WO
Inventors: Sobhi Basheer
Original assignee: Enzymotec Ltd.
Priority date: 1999-03-22
Filing date: 2000-03-16
Publication date: 2000-09-28
Also published as: KR20020010129A; JP2002539782A; IL145503A; IL129086A0; CA2368179A1; KR100774281B1; WO2000056869A3; EP1163329A2; NZ514271A

Abstract

A lipase preparation comprising an insoluble matrix and a surfactant-coated lipase complex immobilized onto said insoluble matrix. Method of preparation and the use of the novel products are disclosed.

Description

SURFACTANT-LIPASE COMPLEX IMMOBILIZED ON

INSOLUBLE MATRIX

Field of the Invention

The present invention relates to an insoluble matrix immobilized

surfactant-coated lipase complex, to a method of preparing same and to the

use of same as a biocatalyst for catalyzing, for example, inter- and/or

trans -esterification of oils and fats in hydrophobic organic media. The novel

procedures include two steps. In the first step, the enzyme is activated by

being coated with a surfactant. In the second step, the enzyme is immobilized

on the matrix of choice. These steps can be executed in any order.

Background of the Invention

Enzymatic modification of the structure and composition of oils and fats is of

great industrial and clinical interest. This process is accomplished by

exploiting regio-specific lipases in inter-esterification and/or

trans-esterification reactions utilizing fats or oils as substrates (Macrea, A.R.,

1983, J. Am. Oil Chem. Soc. 60: 291-294).

Using an enzymatic process, it is possible to incorporate a desired fatty acyl

group on a specific position of a triacylglycerol molecule, whereas conventional

chemical inter-esterification does not possess regio-specificity. Conventionally, chemical reactions are promoted by sodium metal, sodium

alkoxide or cobalt chloride that catalyze acyl migration among triglyceride

molecules, leading to the production of triglycerides possessing randomly

distributed fatty acyl residues (Erdem-Senatalar, A., Erencek, E. and Erciyes,

NT., 1995, J. Am. Oil Chem. Soc. 72: 891-894).

In recent years, a number of studies have demonstrated the potential

application of lipases as promising biocatalysts for different esterification

reactions in organic media (Wisdom, R.A., Dunhill, P., and Lilly, M.D., 1987,

Biotechnol. Bioeng. 29: 1081-1085).

Lipases with 1,3 -positional specificity principally catalyze hydrolysis of fats

and oils, to yield free fatty acids and glycerol. However, recent studies have

shown that lipases with 1,3 -positional specificity are also capable of catalyzing

two types of esterification reaction in microaqueous organic media (Quinlan,

P. & Moore, S., 1993, INFORM 4: 580-585). The first of these reactions is an

inter-esterification or acidolysis reaction in which free fatty acids react with

different triglycerides to yield new triglyceride molecules. The second type of

reaction is trans-esterification in which two different triglyceride molecules

react to give new triglyceride molecules (see Figures la-b). In both of these

enzymatic reactions, the sn-2 position of the reacting triglycerides remains

unchanged. In general, water concentration plays an important role in determining the

activity of enzymes. It also affects the equilibrium state of the reactions

performed in hydrophobic organic solvents (Naliverty, R.H., Hailing, P.J. and

Macrae, A.R., 1993, Biotechnol. Lett. 15: 1133-1138). Since water is vital for

the activity of enzymes in both hydrolysis as well as in synthesis reactions, as

a compromise between hydrolysis and synthesis of triglycerides, the

concentration of water is lowered so that the occurrence of undesirable

reactions is minimized, but the water available is sufficient for the enzyme to

remain active.

At high concentrations of water, e.g., above 5 % of solvent weight, lipases

possess preferably their natural hydrolytic activity, therefore, hydrolysis

reaction proceeds. However, at low concentrations of water, e.g., below 1 % of

solvent weight, lipases catalyze the reverse reaction, that is, synthesis.

A typical range of water concentrations needed for promotion of

inter-esterification reaction between different oils in organic media is 1-10

weight percent (wt %) of the hydrophobic organic solvent. This water

concentration can normally facilitate also the hydrolysis reaction thus

producing undesirable partial glycerides (mono- and di-glycerides) in the

range of 10-20 wt % of the initial triglycerides concentration, as byproducts. The scope for exploiting the positional specificity of lipases, especially, in the

food and oleochemical industries for the production of high-valued special fats

is enormous. For example, cocoa butter substitute, simulated human milk fat

and other structured triglycerides of specific nutritional quality can be

obtained enzymatically by employing lipases with 1,3 -positional specificity

(Vulfson, E.N., 1993, Trends Food Sci. Technol. 4: 209-215).

In view of the foregoing, it is recognized that there is a need to develop new

structured triglycerides with both medium-chain and -3 polyunsaturated

fatty acids that would be devoid of the adverse effects of the naturally

occurring α-3 polyunsaturated fatty acids, or saturated fatty acids. For

example, molecules of MCTs having one of their acyl groups substituted with

an essential long-chain fatty acid would provide the nutritional advantages of

both MCTs and LCTs. This approach is illustrated by the very useful

triglyceride that is formed by incorporating the acyl form of the

polyunsaturated fatty acids, EPA, DHA or o-linolenic acid at the Sn-2 position

of a triglyceride molecule having a medium-chain fatty acyl group at the sn-1

and sn-3 positions (Odle, J. , 1997, J. Nutr. 127: 1061).

The aforementioned polyunsaturated fatty acids incorporated into triglyceride

molecules were shown to have several health benefits with respect to

cardiovascular disease, immune disorders and inflammation, allergies,

diabetes, kidney diseases, depression, brain development and cancer.

Furthermore, medium-chain fatty acids incorporated into the same triglyceride molecule are of major importance in some clinical uses, especially,

for facilitating absorbability and solubilization of cholesterol in blood serum,

and for providing readily available energy sources for body consumption.

Many different approaches for the use of lipases in organic media have been

attempted in order to activate them and to improve their performance. These

include the use of lipase powder suspended in either microaqueous organic

solvents or in biphasic systems, and native lipases adsorbed on microporous

matrices in fixed- and fluidized-bed reactors (Malcata, et al, 1990, J. Am. Oil

Chem. Soc. 890-910). Furthermore, lipases have been hosted in reverse

micelles, and in some studies lipases were attached to polyethylene glycol or

hydrophobic residues to increase their solubility and dispersibility in organic

solvents.

None of the abovementioned approaches was found to be applicable for all

enzymatic systems. However, in many cases, when lipases were treated in

one way or another as described, their performance with respect to activity,

specificity, stability and dispersibility in hydrophobic organic systems was

improved.

In recent studies, the development of surfactant-coated lipase preparations

has been reported (e.g., Basheer. S., Mogi, K. and Nakajima, M.. 1995,

Biotechnol. Bioeng. 45: 187-195). This enzyme modification converts slightly active or completely inactive lipases, with respect to esterification of

triglycerides and fatty acids in organic media, into highly active biocatalysts.

The newly developed surfactant-lipase complexes have been further studied

and used for the inter-esterification reaction in organic solvent systems to

produce structured triglycerides of major importance in medical applications

(Tanaka, Y., Hirano, J. and Funada, T., 1994, J. Am. Oil Chem. Soc. 71:

331-334).

In another approach to the problem, various immobilized-enzyme reactor

systems were used in lipase-catalyzed reactions in microaqueous hydrophobic

organic media (e.g., Basheer, S., Mogi, K., Nakajima, M., 1995, Process.

Biochemistry 30: 531-536). These included fixed- and fluidized-bed reactors,

and a slurry reactor. In the published studies, lipase immobilized onto an

inorganic matrix was used both in a batch reactor system, and in fixed-bed

bioreactor systems. However, the lipases employed were not

surfactant-coated and therefore have the same limitations as free lipase

systems. These limitations include:

1. Difficulties in recovering the enzyme after completion of the process;

2. Rapid loss of activity of the free enzyme in the reaction medium;

3. Problems of recoverability of expensive enzymes;

4. Low synthetic activity of free lipases in organic solvents. Neither of the abovementioned strategies has satisfactorily solved the

technical problems encountered in directing trans- and inter-esterification of

fats and oils. It is therefore an object of the invention to provide a lipase

preparation that is capable of catalyzing esterification reactions in fats and

oils with a much greater efficacy than existing methods.

It is another purpose of the invention to provide a lipase preparation that

incorporates both immobilization to a matrix, and treatment by coating with

a surfactant.

It is a further object of the invention to provide such a lipase preparation that

may be used repeatedly, on an industrial scale with minimal loss of activity.

It is a further object of the invention to provide a method for preparing said

insoluble matrix-immobilized, surfactant-coated lipase complex.

Yet a further purpose of the invention is to provide a process for preparing

structured triacylglycerols, using said insoluble matrix-immobilized,

surfactant-coated lipase complexes.

Other objects and advantages of the invention will become apparent as the

description proceeds. Summary of the Invention

It has now been surprisingly found, and this is an object of the invention, that

the dual modification of crude lipase by (1) coating with a surfactant, and (2)

immobilization to an insoluble matrix; results in a synergistic improvement in

the efficiency of the enzyme to catalyze trans- and inter-esterification

reactions, when compared to either of these two treatments alone. It has been

further unexpectedly found that it is possible to enhance the catalytic stability

of said dually modified lipase for esterification reactions, by providing the

enzyme preparation in a granulated form.

The invention is primarily directed to a lipase preparation comprising an

insoluble matrix and a surfactant-coated lipase complex immobilized onto said

insoluble matrix.

The immobilization of the lipase complex onto the insoluble matrix may be

achieved by several different methods. According to a preferred embodiment

of the invention, however, the surfactant-coated lipase complex is covalently,

ionically or physically bound to the insoluble matrix.

The invention encompasses the use of many types of matrix, said matrices

being selected from the group consisting of an inorganic insoluble matrix and

an organic insoluble matrix. In a preferred embodiment of the invention, the inorganic insoluble matrix is

selected from the group consisting of alumina, diatomaceous earth, Celite,

calcium carbonate, calcium sulfate, ion-exchange resin, silica gel and charcoal.

The abovementioned ion-exchange resin may be of any suitable material, but

in a preferred embodiment is selected from the group consisting of Amberlite

and Dowex.

Although any suitable organic insoluble matrix may be use, in a preferred

embodiment of the invention, the organic insoluble matrix is selected from the

group consisting of Eupergit, ethylsulfoxycellulose and aluminium stearate.

In a preferred embodiment, the content of the lipase is 2-20 weight percent of

the surfactant-coated lipase complex. In a still more preferred embodiment,

the content of the lipase is 0.01-1.0 weight percent of the preparation.

The invention provides the above-described lipase preparation, wherein the

surfactant in the surfactant-coated lipase complex includes a fatty acid

conjugated to a hydrophilic moiety. In a preferred embodiment, the fatty acid

is selected from the group consisting of monolaurate, monomyristate,

monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate,

trilaurate, trimyristate, tripalmitate and tristearate. In a preferred

embodiment, the hydrophilic moiety is selected from the group consisting of a

sugar, a phosphate group, a carboxylic group and a hydroxylated organic residue. In a more preferred embodiment, the sugar is selected from the

group consisting of sorbitol, sucrose, glucose and lactose. Although the fatty

acid and the hydrophilic moiety may be linked by any suitable type of bond, in

a preferred embodiment, the fatty acid and the hydrophilic moiety are

conjugated via an ester bond.

Although the lipase may be derived or obtained from any convenient source,

in a preferred embodiment, the lipase is derived from a microorganism. Many

different species of both microorganisms and multicellular organisms may be

used as a source of lipase for the lipase preparation of the invention. The

invention, however, is particularly directed to the use of lipase that is derived

from a species selected from the group consisting of Burkholderia sp., Candida

antractica B, Candida rugosa, Pseudomonas sp., Candida antractica A,

Porcine pancreas lipase, Humicola sp., Mucor miehei, Rhizopus jaυan.,

Pseudomonas fluor., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae,

Mucor javanicus, Rhizopus sp., Rhizopus japonicus and Candida antractica.

In a further aspect, the invention is directed to a lipase preparation

comprising an insoluble matrix and a surfactant-coated lipase complex

immobilized onto said insoluble matrix, said lipase preparation being provided

in an organic solvent. In a preferred embodiment, the organic solvent is

selected from the group consisting of n-hexane. toluene, iso-octane. ^-octane,

benzene, cyclohexane and di-iso-propylether. The invention is further directed to the use of said lipase preparation as a catalyst for esterification,

inter-esterification and trans-esterification of oils and fats and alcoholysis of

triglycerols and fatty alcohols. In a preferred embodiment, the lipase

preparation is used as a catalyst with 1,3 -positional specificity with respect to

triacylglycerols.

In another aspect, the invention is directed to a lipase preparation as

described above, wherein said preparation is in granulated form.

The invention also provides a lipase preparation, as described hereinabove,

wherein the insoluble matrix has been modified with a fatty acid derivative.

In a further aspect the invention is directed to an enzyme preparation, as

described hereinabove, for use in a reaction environment without the need for

water addition.

The invention also encompasses a method for improving the stability of a

surfactant-coated immobilized lipase complex, comprising granulating same

prior to contacting it with the substrate to be reacted.

In a further aspect, the invention provides a method of preparing an insoluble

matrix-immobilized surfactant-coated lipase complex comprising, in any

desired order, the steps of: (a) contacting a lipase in an aqueous medium with a surfactant, at a

concentration and temperature, and for a period of time sufficient

to obtain a coating of said lipase; and

(b) contacting said lipase in an aqueous medium, with an insoluble

matrix, at a concentration, under conditions and for a period of

time sufficient to obtain immobilization of said lipase on said

matrix.

In a preferred embodiment of the abovementioned method, the lipase is first

contacted with the insoluble matrix, and thereafter with the surfactant. In

another preferred embodiment thereof, the lipase is first contacted with the

surfactant, and thereafter with the insoluble matrix.

In a preferred embodiment of the invention, the above-described method

further comprises the separation of the matrix-immobilized surfactant-coated

lipase complex from the aqueous solution in which it was formed. In a still

more preferred embodiment, this method also further comprises the step of

drying said matrix-immobilized surfactant-coated lipase complex. Although

the drying step may be accomplished by any convenient method, in a

preferred embodiment, said drying is effected by freeze drying. In another

preferred embodiment, the matrix-immobilized surfactant-coated lipase

complex is dried to a water content of less than 100 parts per million by

weight. In another preferred embodiment, the aqueous solution used in the

above-described method is a buffered-aqueous solution.

In yet another preferred embodiment of the above-described method, the

lipase and surfactant are contacted in the aqueous medium by:

(i) dissolving said surfactant in an organic solvent for obtaining a

dissolved surfactant solution; and

(ii) mixing said lipase and said dissolved surfactant solution in said

aqueous medium.

In another preferred embodiment, the method further comprises sonication of

the aqueous solution.

In yet another preferred embodiment of the method of the invention, the

insoluble matrix is selected from the group consisting of alumina,

diatomaceous earth, Celite, calcium carbonate, calcium sulfate, ion-exchange

resin, silica gel, charcoal, Eupergit, ethylsulfoxycellulose, aluminium stearate

and fatty acid derivative-treated Celite or other inorganic matrices.

In another preferred embodiment, the surfactant of the method includes a

fatty acid conjugated to a hydrophilic moiety. In a still more preferred

embodiment, said fatty acid is selected from the group consisting of monolaurate, monomyristate, monopalmitate, monostearate. dilaurate,

dimyristate, dipalmitate, distearate, trilaurate, trimyristate, tripalmitate and

tristearate.

In another preferred embodiment of the method of the invention, the

hydrophilic moiety is selected from the group consisting of a sugar and a

phosphate group and a carboxylic group and a polyhydroxylated organic

residue. In a still more preferred embodiment, the sugar is selected from the

group consisting of sorbitol, sucrose, glucose and lactose.

In another preferred embodiment of the method of the invention, the fatty

acid and the hydrophilic moiety are conjugated via an ester bond.

In a preferred embodiment of the method of the invention, the lipase is

derived from an organism. In a more preferred embodiment, the lipase is

derived from a multicellular microorganism. Although the lipase may be

derived from any suitable host, in a preferred embodiment, the lipase is

derived from a species selected from the group consisting of Burkholderia sp.,

Candida antarctica B, Candida rugosa, Pseudomonas sp., Candida antractica

A, Porcine pancreatic lipase, Humicola sp., Mucor miehei, Rhizopus javan.,

Pseudomonas fluor., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae,

Mucor javanicus. Rhizopus sp., Rhizopus japonicus and Candida antarctica. In another aspect, the invention is directed to a process for preparing

structured triacylglycerols by esterification, acidolysis, trans-esterification,

inter-esterification or alcoholysis between two substrates comprising

contacting an insoluble matrix-immobilized surfactant-coated lipase complex

with said substrates.

In a preferred embodiment of this process, the matrix-immobilized

surfactant-coated lipase complex is contacted with the substrates in the

presence of an organic solvent.

In another preferred embodiment of this process, at least one of the substrates

is selected from the group consisting of an oil, a fatty acid, a triacylglycerol

and a fatty alcohol. Although many different types of oil may be used in this

process, in a preferred embodiment, the oil is selected from the group

consisting of olive oil, soybean oil, peanut oil, fish oil, palm oil, cotton seeds

oil, sunflower oil, Nigella sativa oil, canola oil and corn oil. In another

preferred embodiment, the fatty acid is selected from the group consisting of

medium and short-chain fatty acids and their ester derivatives. In a still

more preferred embodiment, the fatty acid is selected from the group

consisting of oleic acid, palmitic acid, linolic acid, linolenic acid, stearic acid,

arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and their ester

derivatives. While the above-described process may be performed in any suitable

receptacle, said process, in a preferred embodiment, is carried out in a tank

reactor or in a fixed-bed reactor.

The invention also encompasses a triacylglycerol prepared according to the

above-described process for use as a cocoa butter substitute, human milk

fat-like triglycerides for special diets, or structured triglycerides for medical

applications.

Brief Description of the Drawings

The invention herein described, by way of example only, with reference to the

accompanying drawings, wherein:

FIG. la presents an inter-esterification acidolysis reaction catalyzed by

lipase with 1,3-positional specificity. P represents glycerol bound palmitic

acid, C represents glycerol bound capric acid. PA and CA represent free

palmitic and capric acids, respectively.

FIG. lb presents a trans-esterification reaction catalyzed by lipase with

1,3-positional specificity. P represents glycerol bound palmitic acid and C

represents glycerol bound capric acid.

FIG. 2 depicts the chemistry associated with covalent immobilization of

lipase to Eupergit C 250L followed by coating the covalently immobilized

enzyme with a surfactant. FIG. 3 presents inter-esterification reaction profiles of physically

immobilized lipases. Reaction conditions were 50 mg tripalmitin, 35 mg

capric acid and 20 mg surfactant-coated lipase (Saiken 100 - triangles, or

Lilipase A10-FG - squares) immobilized on DE in 10 ml n-hexane. The

reaction system was magnetically stirred and thermostated at 40 °C.

FIG. 4 presents an Arrhenius plot for the inter-esterification reaction of

tripalmitin and capric acid with DE -physically immobilized surfactant-coated

Lilipase A10-FG.

FIG. 5 is a bar graph showing the functional stability of Lilipase A

10FG immobilized on Celite and granulated with 2 % starch.

FIG. 6 is a bar graph showing the functional stability of powdered

Lilipase A 10FG modified with sorbitan monostearate and immobilized on

Celite.

FIG. 7 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Celite and

granulated with 2 % starch.

FIG. 8 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Celite and

granulated with ethyl cellulose.

FIG. 9 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Celite and

granulated with 2 % gum Arabic. FIG. 10 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Amberlite

IRA-900 and granulated with ethyl cellulose.

FIG. 11 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Celite and

granulated with 2 % agarose.

FIG. 12 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Celite and

granulated with 4 % starch.

FIG. 13 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan tristearate, immobilized on Celite and

granulated with 2 % starch.

FIG. 14 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on aluminium

monostearate and granulated with 2 % starch. The graph also shows

comparable results for unmodified enzyme, immobilized on aluminium

monostearate and granulated with 2 % starch.

FIG. lδ is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Amberlite

XAD-16 and granulated with 2 % starch.

FIG. 16 is a bar graph showing the functional stability of Lilipase A

10FG modified with sorbitan monostearate, immobilized on Amberlite XAD-7

and granulated with 2 % starch. Detailed Description of Preferred Embodiments

The present invention relates to a surfactant-coated lipase or phospholipase

complex immobilized on an organic or inorganic insoluble matrix (e.g.,

particulate solid support) which can be used to catalyze inter and

trans-esterification reactions, particularly of oils and fats and alcoholysis of

fatty-alcohols. The invention also makes provision for preparing the enzyme

preparation in a granulated form that demonstrates increased stability of

activity. Specifically, the present invention can be used for preparing

structured triacylglycerols possessing desired nutritional or biochemical

properties. The present invention is further directed to a method of preparing

an insoluble matrix-immobilized surfactant-coated lipase or phospholipase

complex and of a process of modifying oils and fats using an insoluble

matrix-immobilized surfactant-coated lipase or phospholipase complex.

The principles and operation of the present invention may be better

understood with reference to the drawings and accompanying descriptions.

It is to be understood that the invention is not limited in its application to the

details of construction and the arrangement of the components set forth in the

following description or illustrated in the drawings. The invention is capable

of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed

herein is for the purpose of description and should not be regarded as limiting.

According to the present invention surfactant-coated lipases or phospholipases

are immobilized onto insoluble matrices by three different methods: (i)

immobilization through hydrophobic (physical) adsorption on inorganic or

organic insoluble matrices; (ii) immobilization through ionic interactions on

various ion exchange resins (polar or apolar matrices); and (iii) immobilization

through covalent immobilization to insoluble matrix such as Eupergit (organic

matrix).

The immobilized surfactant-coated lipases prepared according to the

procedures described herein were used to catalyze inter-esterification

reactions between triglycerides and fatty acids, one-step alcoholysis reactions

between triglycerides and fatty alcohols for production of wax esters, and also

trans-esterification reactions between two different triglyceride molecules or

between two different oils.

The results indicate that coating lipases with a lipid surfactant, such as, but

not limited to, fatty acid sugar ester types, lead to activation of the lipases for

use in organic synthesis and in most cases the modification process converts

relatively inactive crude lipases to highly active biocatalysts. To develop an

efficient enzymatic inter/trans -esterification bioreactor from which the lipase enzyme can be easily recovered or used continuously, surfactant-lipase

complexes immobilized on organic and inorganic matrices, were used.

It was found that surfactant-coated lipases immobilized on an organic or

inorganic matrix showed high inter/trans-esterification activity and only

slight activity losses in five consecutive inter-esterification runs using the

same biocatalyst batch. It was further found that granulation of the

matrix-bound, surfactant-treated enzyme considerably enhanced the stability

of the enzyme, permitting its repeated use without substantial loss of activity.

The immobilized surfactant-lipase complexes prepared according to the

present invention were used for the preparation of structured triglycerides

which have potential applications in medicine and the food industry. The

triglycerides of interest that were synthesized according to the method of the

present invention were produced by inter-esterification of long-chain

triglycerides, such as the hard fraction of palm oil, with short-chain fatty acids

such as capric acid. Immobilized surfactant-coated lipase catalyzed reactions

yielded predominantly products with 1,3-positional specificity for the

triglycerides of interest. Mono- and di-glycerides were also produced in a

hydrolysis side reaction and their percentage was typically less than 7 weight

percent of the initial triglyceride concentration. The operational stability

of surfactant-lipase complexes immobilized on different solid matrices was ?9

very high, particularly following the optional granulation step, and no

significant enzyme activity losses were observed.

Inter-esterification reactions using the immobilized surfactant-lipase

complexes were also carried out to obtain fats of special characteristics with

regard to their physical properties. For example, liquid olive oil was

trans-esterified with the hard fraction of palm olein in order to obtain blends

of oil for preparation of healthier olive oil based margarines. This enzymatic

process is a useful alternative for chemical oil hydrogenation or

interesterification processes.

Thus, in accordance with the teachings of the present invention there is

provided a lipase preparation which includes an insoluble matrix and a

surfactant-coated lipase complex immobilized onto the insoluble matrix.

As used herein in the specification and in the claims section below the term

"lipase" is not limited to this specific enzyme, but is meant to embrace also

similar enzymes such as phospholipase, proteases and glycosidases. Other

suitable enzymes will be readily apparent to the skilled chemist, and are

therefore not listed herein, for the sake of brevity. According to a preferred embodiment the complex is immobilized to the

insoluble matrix via hydrophobic (physical) interaction, ionic interaction or via

covalent immobilization.

In a preferred embodiment of the invention the insoluble matrix is an

inorganic insoluble matrix, the term "insoluble" referring to its lack of

solubility in both polar (e.g., water) and non-polar (hydrophobic) solvents.

Preferably, the inorganic insoluble matrix according to the present invention

is alumina, aluminium stearate, Celite, calcium carbonate, silica gel, charcoal,

calcium sulfate, ion-exchange resin, such as, but not limited to, Amberlite and

Dowex. For physical immobilization, most preferably the inorganic insoluble

matrix employed is diatomaceous earth (DE). For ionic immobilization most

preferably the inorganic insoluble matrix employed is Amberlite and Dowex,

which are strong ion exchangers.

Suitable organic solid matrices according to the present invention include

Eupergit for covalent immobilization and ethylsolfoxycellulose for ion

interaction. Any other suitable organic solid matrix may also be used without

exceeding the scope of the invention.

In another preferred embodiment of the present invention the lipase

represents 2-20. preferably 5-11, weight percent of the surfactant-coated

lipase complex. In yet another preferred embodiment of the present invention the lipase represents 0.01-1 weight percent of the preparation. Preferably the

lipase represents about 0.7 weight percent of the preparation.

According to a preferred embodiment of the invention the surfactant employed

is a lipid, which includes a fatty acid conjugated to a hydrophilic moiety. The

fatty acid is preferably monolaurate, monomyristate, monopalmitate,

monostearate, dilaurate, dimyristate, dipalmitate, distearate, trilaurate,

trimyristate, tripalmitate or tristearate. The hydrophilic moiety is preferably

a sugar, such as, but not limited to, sorbitol, sucrose, glucose and lactose, a

phosphate group, a carboxylic group or a polyhydroxylated organic residue.

Typically, the fatty acid and the hydrophilic moiety are conjugated via an

ester bond.

According to another preferred embodiment of the invention the hpase is

derived from a microorganism or a multicellular organism. Species known to

be used for lipase extraction include Burkholderia sp., Candida antarctica B,

Candida rugosa, Pseudomonas sp., Candida antractica A, Porcine pancreas,

Humicola sp., Mucor miehei, Rhizopus javan., Pseudomonas fluor, Candida

cylindrcae, Aspergillus niger, Rhizopus oryzae, Mucor jaυanicus, Rhizopus sp.,

Rhizopus japonicus and Candida antractica.

According to a preferred embodiment of the invention the lipase preparation

maintains lipase catalytic activity in an organic solvent. Lipase catalytic activity include hydrolysis, esterification, inter-esterification,

trans-esterification, acidolysis and alcoholysis, preferably with 1,3-positional

specificity with respect to triacylglycerols. The organic solvent is typically a

hydrophobic solvent, such as, but not limited to, n-hexane, toluene, iso-octane,

n-octane, benzene, cyclohexane and di-iso-propylether.

Further according to the present invention there is provided a method of

preparing an insoluble matrix-immobilized surfactant-coated lipase complex.

The method includes the following method steps, wherein in a first step a

lipase, an insoluble matrix and a surfactant are contacted in an aqueous

solution, preferably a buffered solution. Second, conditions (e.g., sonication)

are provided for the formation of the matrix-immobilized surfactant-coated

lipase complex. Two alternative schemes are available in this respect. In the

first the lipase is first interacted with the surfactant and only thereafter the

surfactant-coated lipase is interacted with the matrix. Whereas in the second,

the lipase is first interacted with the matrix and only thereafter the matrix

immobilized lipase is interacted with the surfactant.

According to a preferred embodiment, the method further includes the step of

separating the matrix-immobilized surfactant-coated lipase complex from the

aqueous solution.

According to still another preferred embodiment of the invention the method

further includes the step of drying the matrix-immobilized surfactant-coated lipase complex. Drying is preferably effected via freeze drying, fluidization or

in an oven. Following drying, the matrix-immobilized surfactant-coated lipase

complex preferably includes less than 100, more preferably less than 50, most

preferably less than 20 parts per million water content by weight.

In a preferred embodiment of the method according to the present invention,

contacting the lipase, insoluble matrix and the surfactant within the aqueous

solution is effected by dissolving the surfactant in an organic solvent (e.g.,

ethanol) for obtaining a dissolved surfactant solution, mixing the lipase and

the dissolved surfactant solution (e.g., dropwise) in the aqueous solution;

sonicating the resulting suspension; and adding the insoluble matrix into the

aqueous solution. Alternatively, the lipase is first interacted with the

insoluble matrix and only thereafter with the surfactant.

According to a preferred embodiment, the insoluble matrix of the

above-described lipase preparation is modified with a fatty acid derivative.

This is to permit the immobilization of a hydrophobized lipase on a

hydrophobized carrier, such as aluminium stearate, fatty-acid

derivative-treated Celite and apolar or weak-polar ion-exchange resins, in

order to prepare highly active enzymes.

Further according to the present invention there is provided a process of

preparing structured triacylglycerols by esterification, trans-esterification, inter-esterification, acidolysis or alcoholysis between two substrates effected

by contacting an insoluble matrix-immobilized surfactant-coated lipase

complex with the substrates. Contacting the matrix-immobilized

surfactant-coated lipase complex with the substrates is preferably effected in

the presence of an organic solvent.

In a preferred embodiment, at least one of the substrates is an oil, a fatty acid

or a triacylglycerol. The oil may be any of the above listed oils. The fatty acid

is a medium or a short-chain fatty acid or an ester derivative thereof. A

suitable fatty acid is, for example, oleic acid, palmitic acid, linolic acid,

linolenic acid, stearic acid, arachidonic acid, eicosapentaenoic acid,

docosahexaenoic acid and their ester derivatives.

In a preferred embodiment of the invention contacting the matrix-immobilized

surfactant-coated lipase complex with the substrates is effected within a

reaction reactor, e.g., a tank reactor or a fixed-bed reactor.

Further according to the present invention there is provided a process of

changing the physical properties of oils/fats (e.g., triacylglycerols) by

trans-esterification or inter-esterification between at least two oil/fat

substrates by contacting an insoluble matrix-immobilized surfactant-coated

lipase complex with the substrates, preferably in the presence of an organic

solvent. Further according to the present invention there is provided a process of

changing the physical properties of long-chain triglycerides (LCT) and

long-chain fatty alcohols (LCFAL) to produce wax esters by alcoholysis

between at least two such substrates by contacting an insoluble

matrix-immobilized surfactant-coated lipase complex with the substrates,

preferably in the presence of an organic solvent.

According to a preferred embodiment, the matrix-immobilized

surfactant-coated lipase complex represents 2-30 weight percent of the

substrates. In another preferred embodiment the oil/fat substrates are liquid

oils and solid fats. The oil may be any of the above listed oils in a native or

hydrogenated form.

Further according to the present invention there is provided a triacylglycerol

prepared according to the above process. The triacylglycerol serves an

application such as a cocoa butter substitute, human milk fat-like,

triglycerides for special diets or structured triglycerides for medical

applications.

Yet further according to the present invention there is provided a preparation

which includes a lipase and an organic solvent. The lipase possessing both

esterification (inter- and trans-esterification), acidolysis, alcoholysis and hydrolysis catalytic activities with respect to substrates, yielding esterification

and hydrolysis products, respectively. The hydrolysis products represent less

than about 7, preferably less than about 5, more preferably less than about 3

weight percent of the products.

Examples

Reference is now made to the following examples, which together with the

above descriptions, illustrate the invention in a non-limiting fashion.

Experimental Procedures

Materials

Different crude lipase preparations were tested in this study. Table 1 below

lists commercially available lipase preparations that were employed in this

study, as well as their species source and supplier. All fatty acids and

triglycerides employed in this study were obtained from Fluka (Switzerland)

and, as reported by the supplier, were at least 99 % pure. Olive oil, sunflower

oil, palm oil, canola oil, corn oil and Nigella sativa oil were obtained from local

suppliers in the Galilee area, Israel. Fish oil,

tris(hydroxymethyl)aminomethane and the inorganic matrices used as

supports for the surfactant-coated lipase complexes, including DE, alumina

and silica gel were obtained from Sigma (USA). Analytical grade n-hexane and other solvents employed, all of analytical

grade, were from Bio Lab (Israel). Sorbitan fatty acid esters including

sorbitan monolaurate, sorbitan monomyristate, sorbitan monopalmitate and

sorbitan monostearate and sucrose fatty acid esters including mixtures of

mono-, di- and tristearate sucrose esters of variable HLB values were

obtained from Kao Pure Chemicals Ind. (Tokyo, Japan).

Tris(Hydroxymethyl)aminomethane (tris) was from Sigma (USA). Inorganic

and organic matrices used as supports for the modified lipases include

diatomaceous earth (DE), alumina and silica gel; ionic exchange resins were

purchased from Sigma, USA. Eupergit C and Eupergit C 250L

(macroporous, spherical, approximate diameter 150 and 200 μm, respectively)

were from Rhom (Germany).

Table I

Lipase modification and immobilization through physical

adsorption

Crude enzyme was first coated with lipid surfactant or other enzyme

activators e.g. gum Arabic or polyethylene glycol. A typical enzyme

modification and immobilization procedure was as follows: crude enzyme

(lipase, phospholipase, protease and glycosidases; protein content

approximately 150mg/L), was dissolved in IL phosphate or tris buffer

solution with an appropriate pH, and magnetically stirred at 10 °C for

30min. A lipid surfactant or other enzyme activator (0.5g) dissolved in

ethanol (20ml) or other solvents was added dropwise into the stirred

solution. The resulting enzyme solution was sonicated for 15min and then

vigorously stirred at 10 °C for 3 hours. An insoluble organic (20 g such as

polypropylene, aluminium stearate or chitin) or inorganic matrix (20 g such

as Celite, alumina, silica gel or ceramic support) was added into the stirred enzyme solution. The solution was magnetically stirred for a further 5 hours

at 10 °C. The precipitate was collected by centrifugation at 12000rpm

(Sorval Centrifuge, model RC-5B) or by filtration, and then was treated by

one of two different methods as follows: 1. The wet precipitate was lyophilized after freezing overnight at -20 °C. The

formed powder can be directly used for batch enzymatic reactions or

granulated for obtaining particulated modified and immobilized enzyme

with a particle size of 50-1000μm. The granulation process was performed

using various binding reagents such as starch, methyl or ethyl cellulose,

gums, agarose or other binders. For example, the granulation with starch

was conducted as follows: Starch solution (4 g starch/20ml water) was

converted to gel at 70 °C. The gel was cooled down to 60 °C and then

introduced to the modified and immobilized enzyme wet powder. The

mixture was homogenized in a high-speed mixer followed by extruding and

drying at 40-60 °C for 48 hours. The immobilized enzyme was sieved to

obtain particles in the range of 50-1000 μm. This particulated enzyme was

used mainly in packed columns.

2. The wet precipitate formed after modification and immobilization was

directly granulated with starch or other binding reagents as described

above.

Enzyme modification and immobilization through ionic adsorption

The above-described modification, immobilization and granulation

procedures were also used in conjunction with ion-exchange resins. The

types of resin used include: strong and week basic anion exchange resins,

strong and weak acidic cationic exchange resins and weak-polar and apolar

ion-exchange resins. Examples of commercially available resins used in the experiments (obtained from Sigma, USA) include: Dowex 22, Dowex

1x2-400, Dowex, 2x8-100, cellulose phosphate, Amberlite IRA-95, Amberlite

IRA-200, Amberlite IRA-900, Amberlite XAD-7, Amberlite XAD-16,

DiannonSA-lOA, Ectoela cellulose, Sephadex and sulfoxyethylcellulose. A

typical modified immobilized enzyme was prepared according to the

aforementioned procedure.

Enzyme modification and immobilization through covalent binding

Two different immobilization procedures were adopted. According to the first,

the enzyme was primarily coated with a surfactant and then the

lipase-surfactant complex was covalently linked to an Eupergit matrix, which

contains active oxirane groups. To this end, crude lipase (1 gram protein) was

dissolved in 1 liter tris or phosphate buffer pH 5.8. The enzyme solution was

vigorously stirred with a magnetic stirrer at 10 °C for 30 minutes. Sorbitan

mono-stearate (0.5 grams) dissolved in 30 ml ethanol were added dropwise to

the stirred enzyme solution. The resulting colloidal enzyme solution was

sonicated for 10 minutes and then stirred for 3 hours at 10 °C. Eupergit C or

Eupergit C 250L (125 grams) and 12 ml solution of 5 % hydrogen peroxide

were added into the enzyme solution and the resulting suspension was gently

handshaken for 1 minute, and then incubated for 48 hours at 23 °C. The

precipitate was filtered, washed with tris or phosphate buffer pH 5.8, and was

freeze-dried overnight. According to the second procedure, the lipase was first bound covalently to an

Eupergit matrix and then the bound lipase was coated with a surfactant. The

chemistry involved in this procedure is depicted in Figure 2.

To this end, crude lipase (1 gram protein) was dissolved in 1 liter tris or

phosphate buffer pH 5.8. The enzyme solution was vigorously stirred with a

magnetic stirrer at 10 °C for 30 minutes. Eupergit C or Eupergit C 250L (125

grams) and 12 ml solution of 5 % hydrogen peroxide were added into the

enzyme solution and the resulting suspension was gently handshaken for 1

minute and then incubated for 48 hours at 23 °C. Sorbitan mono-stearate (0.5

grams) dissolved in 30 ml ethanol was added dropwise to the suspension

under a gentle shake. The resulting suspension was sonicated for 10 minutes

and incubated at 10 °C for 6 hours. The precipitate was filtered, washed with

tris or phosphate buffer pH 5.8, and then freeze-dried overnight.

As a control for the activity of the covalently immobilized lipases, the same

immobilization procedures were followed however without adding surfactant

to the enzyme solution.

To this end, crude lipase (1 gram protein), was dissolved in 1 liter tris or

phosphate buffer pH 5.8. The enzyme solution was vigorously stirred with a

magnetic stirrer at 10 °C for 30 minutes. Eupergit C or Eupergit C 250L (125

grams) and 12 ml solution of 5 % hydrogen peroxide were added into the

enzyme solution and the resulting suspension was gently handshaken for 1 minute, and then incubated for 48 hours at 23 °C. The precipitate was

filtered, washed with tris or phosphate buffer pH 5.8, and then was

freeze-dried overnight.

Protein determinations according to the Bradford method indicated that all

enzyme preparations prepared according to this method contained 0.9 - 1.5 wt

% protein.

Reaction models

Three different reaction models were used to test the activity of the modified

and immobilized enzymes, compared to that of the crude enzymes and of the

immobilized enzymes without modification.

1. Esterification reaction between lauric acid and dodecyl alcohol in an

organic solvent system or in a solvent-free system.

The esterification reaction was initiated by adding 10 mg lipase preparation

to 10 ml n-hexane that contained, typically, 200 mg lauric acid and 186 mg

dodecyl alcohol. The reaction was magnetically stirred at 40° C. Samples

were periodically withdrawn (50 μl), filtered with a Millipore filter (0.45 μm)

and then mixed with a similar volume of n-hexane solution containing

n-hexadecane as an internal standard. 2. Transesterification reaction between the two triglycerides; tripalmitin

and tristearin in an organic solvent system or in solvent-free system.

The transesterification reaction was initiated by adding 10 mg lipase

preparation to 10 ml n-hexane that contained, typically, 40 mg tripalmitin

and 40 mg tristearin. The reaction was magnetically stirred at 40° C.

Samples were periodically withdrawn (50 μl), filtered with a MiUipore filter

(0.45 μm) and then mixed with a similar volume of n-hexane solution

containing n-hexadecane as an internal standard.

3. Alcoholysis reaction between Olive oil and cetyl alcohol in organic solvent

or solvent-free systems.

The alcoholysis reaction was initiated by adding 10 mg lipase preparation to

10 ml n-hexane that contained, typically, 500 mg olive oil, and 500 mg cetyl

alcohol. The reaction solution was magnetically stirred at 40 °C. Samples (50

μl) were periodically removed, filtered with MiUipore filters (0.45 μm) and

then mixed with a similar volume of n-hexane solution containing tridecanoin

as an internal standard.

Unless otherwise indicated, all experiments were conducted under the

above-described conditions. Each esterification reaction was carried out in

duplicate. In all experiments, n-hexane was dried over molecular sieves to minimize its water content down to 6 mg/liter. Thus, water concentration in

all reaction systems was less than 30 mg/liter.

Protein content

The protein content of the modified lipases, and the modified and

immobilized lipases was determined by the microkejldahl method.

Enzyme activity in a batch system

The activity of the activated modified enzyme, modified and immobilized

enzyme on insoluble matrix, crude enzymes and immobilized enzymes in

insoluble matrix, was tested using a 1ml vials containing the substrates.

The vials were shaken at 40 °C and samples were analyzed after certain

time intervals. Reaction rates were determined at substrate conversions less

than 7% per mg of protein.

Operational stability of modified and immobilized enzyme

The operational stability of the particulated modified and immobilized

enzymes was tested in a jacketed column reactor (0.5cm i.d. and 15cm long)

using the alcoholysis of olive oil and cetyl alcohol in n-hexane as a reaction

model. The enzyme particles were packed in the column and the substrate

solution was recirculated through the packed enzyme. The circulation was

stopped after one hour and the reaction solution was analyzed. After each

run the solution was discarded and the packed immobilized enzyme was washed with organic solvent (n-hexane) before charging a fresh substrate

solution. This procedure was repeated 10-20 times.

EXPERIMENTAL RESULTS

Example 1

Protein content of the lipase preparations

The protein content of the different crude lipases, lipases modified with

sorbitan monostearate (SMS), lipases immobilized on Celite and

SMS-modified lipases immobilized on Celite, was measured as described

above. The results are shown in Table II below.

The results show that there is fairly wide variation in protein concentration

between the various preparations. For the surfactant-coated,

matrix-immobilized enzymes, the protein content varied from 0.05 % to 1.12

%, by weight, according to the enzyme used in the preparation. Similar

variation was seen when one enzyme, Lilipase, was treated with different

lipid surfactants or other activating agents, and, optionally, immobilized on

Celite. The results of this investigation are shown in Table III. Table II

Lilipase A-10FG

Enzyme Protein content (%)

Lipase crude 4.6 Lipase +SMS 1.62 Lipase +SMS/Celite 0.11 Lipase /Celite 0.1

Lipase M

Enzyme Protein content (%)

Lipase crude 0.96 Lipase +SMS 1.8 Lipase +SMS/Celite 0.12 Lipase /Celite 0.1

Lipase G - Amano 50

Enzyme Protein content (%)

Lipase crude 9.65 Lipase +SMS 1.11 Lipase +SMS/Celite 0.06 Lipase /Celite 0.29

Lipase A- Amano 6

Enzyme Protein content (%)

Lipase crude 21.63

Lipase +SMS 4.85

Lipase +SMS/Celite 0.225

Lipase /Celite 0.2 Table II continued

Lipase Saiken 100

Enzyme Protein content (%)

Lipase crude 4.84

Lipase +SMS 2.87

Lipase +SMS/Celite 0.06

Lipase /Celite 0.04

Lipase F P-15

Enzyme Protein content (%)

Lipase crude 49.2

Lipase +SMS 9.31

Lipase +SMS/Celite 0.70

Lipase /Celite 0.47

Lipase F -EC

Enzyme Protein content (%)

Lipase crude 49.7

Lipase +SMS 7.2

Lipase +SMS/Celite 1.12

Lipase /Celite 0.2

Lipase EC

Enzvme Protein content (%)

Lipase crude 36.8

Lipase +SMS 6.5

Lipase +SMS/Celite 0.48

Lipase /Celite 0.39 Table II continued

Lipase PS

Enzvme Protein content (%)

Lipase crude 7.5 Lipase +SMS 0.72 Lipase +SMS/Celite 0.16 Lipase /Celite 0.08

Lipase Newlase F

Enzyme Protein content (%)

Lipase crude 25.1 Lipase +SMS 2.17 Lipase +SMS/Celite 0.24 Lipase /Celite 0.013

Lipase LP

Enzyme Protein content (%)

Lipase crude 6.16

Lipase +SMS 4.6

Lipase +SMS/Celite 0.65

Lipase /Celite 0.21

Lipase AY - Amano 30

Enzyme Protein content (%)

Lipase crude 5.14

Lipase +SMS 2.6

Lipase +SMS/Celite 0.05

Lipase /Celite 0.05 Table III

Example 2

Esterification. transesterification and alcoholysis activities of the

lipase preparations

A comparison was made of the enzymatic activities of the various lipase

preparations ( prepared as described above). For the purposes of this

comparison, the results (shown in Table TV) are presented as reaction rates

(ri).

Table IV

Lilipase A-10FG

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0.16 0.01 0

Lipase+SMS 7.43 5.2 2.1

Lipase+SMS/Celit ;e 27.4 18.1 7.1

Lipase/Celite 4.5 0.8 0.4

Lipase M

Enzyme ri π ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0.2 0 0

Lipase+SMS 6.1 4.8 2.0

Lipase+SMS/Celite 22.3 16.4 6.1

Lipase/Celite 3.2 0.1 0.3 Table PV continued

Lipase PS

Enzyme π ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0.3 0 0

Lipase+SMS 5.2 4.3 1.6

Lipase+SMS/Celite 18.3 12.5 6.5

Lipase/Celite 4.5 0.4 0.2

Lipase LP

Enzyme π π ri (alcoholysis)

(esterification) (trans e s terif icatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmo unin.mg protein)

Crude 0.3 0 0

Lipase+SMS 4.9 3.3 1.4

Lipase+SMS/Celite 15.5 9.5 2.8

Lipase/Celite 2.3 0.2 0.35

Lipase EC

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0 0 0

Lipase+SMS 0.7 1.9 1.9

Lipase+SMS/Celite 1.9 10.3 5.4

Lipase/Celite 0.1 1.1 0.3 Table TV continued

Lipase AY Amano 30

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0 0 0

Lipase+SMS 2.2 0.3 0.7

Lipase+SMS/Celite 16.3 0.8 1.3

Lipase/Celite 0.9 0.05 0.4

Lipase G

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0 0 0

Lipase+SMS 0.1 0.15 0

Lipase+SMS/Celite 1.4 0.4 0

Lipase/Celite 0.1 0 0

Lipase A

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0 0 0

Lipase+SMS 0.9 0.5 0.1

Lipase+SMS/Celite 3.0 1.2 0.6

Lipase/Celite 0.3 0.1 0 Table IV continued

Lipase F-AP15

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0.3 0 0

Lipase+SMS 6.7 4.9 1.9

Lipase+SMS/Celite 26.4 12.7 5.4

Lipase/Celite 0.8 0.93 .3

Lipase F-EC

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0.4 0 0

Lipase+SMS 7.1 3.4 1.5

Lipase+SMS/Celite 23.5 10.5 5.4

Lipase/Celite 1.9 0.6 0.6

Lipase Saiken 100

Enzyme ri n ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg

(μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0.15 0 0

Lipase+SMS 6.9 4.2 2.4

Lipase+SMS/Celite 29.3 10.3 8.2

Lipase/Celite 1.2 3.2 0.4 Table IV continued

Lipase Newlase F

Enzyme ri ri ri (alcoholysis)

(esterification) (transesterificatio (μmol/min.mg (μmol/min.mg n) protein) protein) (μmol/min.mg protein)

Crude 0 0 0 Lipase+SMS 1.3 1.3 0.4 Lipase+SMS/Celite 6.4 6.8 0.5 Lipase/Celite 02 0Λ 0

These results indicate that in their native form the crude lipases used in this

study lack measurable esterification or transesterification activity with the

low water concentrations used. In contrast, both modification with the

surfactant sorbitan monostearate and, independently, immobilization onto

Celite, resulted in detectable levels of esterification and transesterification.

When the enzyme was subjected to both of these treatments, however, the

reaction rates of both the esterification and transesterification reactions

studies increased to much greater levels. From the numerical results

presented in Table rV, it is clear that there is an unexpected synergism

between the two stages of modification, i.e., treatment with surfactant and

immobilization onto the matrix.

Figure 3 presents the conversion of tripalmitin with time when

DE-immobilized surfactant-coated Saiken- 100 (triangles) and Lilipase A10-FG

(squares) were used to catalyze the inter-esterification reaction of tripalmitin

and capric acid. The inter-esterification reaction rates thus measured were

0.096 and 0.104 mmol/min.mg biocatalyst, respectively. Example 3

Fatty acid specificity of immobilized surfactant-coated lipase complexes

The specificity of the inorganic matrix-immobilized surfactant-coated lipase

complexes of the present invention toward different fatty acid substrates was

tested by monitoring the inter-esterification of fatty acids of various chain

lengths with tripalmitin. The results are summarized in Table V below.

Reaction conditions were as follows:

Inter-esterification activity of crude lipase preparations was tested in

n-hexane using tripalmitin (50 mg) as a triacylglycerol substance and capric

acid (35 mg) as a medium-chain fatty acid substance. Inter-esterification

activities of 10 mg non-immobilized surfactant-coated hpase (5-10 % protein

content), or of 20 mg of inorganic matrix surfactant-coated lipase (0.2-2 %

protein content), in n-hexane (10 ml), were examined using the same

substrates.

Table V

Fatty acid Fatty acid chain Inter-esterification length rate

(mmol/min.mg biocat.)

Butyric acid (C4) 0.055

Hexanoic acid (C6) 0.06

Octanoic acid (C8) 0.09

Decanoic acid (CIO) 0.104

Lauric acid (C12) 0.15

Myristic acid (C14) 0.2

Palmitic acid (C16) 0.25

Stearic acid (C18) 0.26

Oleic acid (C18:l) 0.3

Arachidonic acid (C20) 0.28

Behenic acid (C22) 0.28 From Table V it can be seen that the immobilized surfactant-coated Lilipase

complexes according to the present invention predominantly catalyzed the

inter-esterification of fatty acids and tripalmitin with 1,3-positional

specificity. The concentration of hydrolysis products did not exceed 5 wt % of

the initial tripalmitin concentration.

It is further noted that the inter-esterification activity of the inorganic

matrix-immobilized surfactant-coated lipase complexes according to the

present invention was affected by the fatty acid chosen to be used as a

substrate. Thus, fatty acids having longer alkyl chains, such as palmitic and

stearic acids, are better substrates for the DE-immobilized surfactant-coated

lipase complexes than fatty acids having shorter alkyl chains.

Example 4

Influence of surfactant choice on inorganic matrix-immobilized

surfactant-coated lipase complexes

Table VI below demonstrates that the type of sorbitan fatty acid ester selected

for coating the lipase influences the inter-esterification activity of

Celite-immobilized surfactant-coated lipase complexes.

When sorbitan monostearate was used for coating, the inter-esterification

activity of the complex was the highest. However, using a shorter fatty acid chain length in the sorbitan ester led to decrease in the activity of the

complex.

Table VI

Lilipase A- 10FG

Enzyme π ri π

(esterification) (transester (alcoholysi

(mol/min.mg s) ification) ((mol/min. protein) (mol/min. mg mg protein) protein)

Lilipase crude 0.16 0.01 0 Lilipase/Celite 4.5 0.1 0.4

Lilipase+sorbitan monostearate 7.43 5.75 1.2 Lilipase+sorbitan monostearate/Cehte 27.4 15.5 2.3

Lilipase+sucrose ester HLB=5 23.6 4.5 3.5 Lilipase+sucrose ester HLB=5/Celite 22.4 7.5 5.5

Lilipase+sucrose ester HLB=11 31.1 12.5 6.2 Lilipase+sucrose ester HLB=11/Celite 25.3 30 8.2

Lilipase+sucrose ester HLB=16 8.5 2 1.1 Lilipase+sucrose ester HLB=16/Celite 16.2 12.5 4.2

Lilipase+sorbitan monolaurate 6.2 4 0.6 Lilipase+sorbitan monolaurate/Celite 7.1 11.5 2.1

Lilipase+sorbitan tristearate 10.1 4 1.9 Lilipase+sorbitan tristearate/Celite 22.5 31 7.2

Lilipase+sorbitan trioleate 18.3 7.5 2.6 Lilipase+sorbitan trioleate/Celite 27.3 20 9.3

Lilipase+ sorbitan monooleate 6.2 2.5 1.4 Lilipase+monooleate/Celite 8.5 10 4.2

Lilipase+lecithin 10.2 3.5 1.15 Lilipase+lecithin/Celite 16.2 10 3.2

Lilipase+stearic acid 6.3 3.75 1.6 Lilipase+stearic acid/Celite 13.4 14 5.3

Lilipase+Octadecanoic acid 6.2 3 0.81 Lilipase+Octadecanoic acid/Celite 14.6 11.5 3.4

Lilipase polyoxyethylene-8-stearate 6.5 4.4 2.1

Lilipase 13.5 16 6.2 polyoxvethylene-8-stearate/Celite

Lilipase +polyethylenglycol 5.4 0 0 Lilipase +polvethvlenglycol Celite 9.2 0.4 0.4

Lilipase gum Arabic 9.3 2 0.5 Lilipase gum Arabic/Celite 11.1 5 1.1 Example 5

Physical binding of modified lipase to insoluble matrices

The esterification, transesterification and alcoholysis activity for Lilipase

A-IOFG immobilized on various insoluble matrices was measured, and

compared with the activities of Lilipase A-IOFG modified with sorbitan

monostearate and immobilized on different inorganic matrices. The results

are shown in Table VII.

Table VII

Enzyme/Insoluble ri ri (transri matrix* (esterification) esterification) (alcoholysis) (μmolVmin.mg (μmol/min.mg (μmol/min.m protein) protein) g protein)

Lilipase A10-FG crude 0.16 0.01 0 Lilipase + SMS 7.43 5.2 2.1

Lilipase + SMS/Celite 27.4 18.1 7.1

(acid-washed) Lilipase/Celite 4.5 0.8 0.4

(acid-washed)

Lilipase + SMS/Celite 25.4 13.5 6.0 (acid-nonwashed)

Lilipase/Celite 3.5 0.5 0.1 (acid-nonwashed)

Lilipase + SMS/fatty^* 41.3 27.4 17.3 acid-treated Celite Lilipase/fatty acid-treated 19.7 9.5 4.6 Celite

Lilipase+ SMS /Alumina 23.2 11.1 5.4 Lilip as e/ Alumina 1.6 0.3 0.4

Lilipase + SMS 45.3 29.1 19.2 /Aluminium monostearate Table VII continued

Lilipase/ Aluminium 19.2 9.8 6.1 monostearate

Lilipase + SMS/Silica 21.3 10.8 5.7 Lilipase/Silica 3.2 0.4 0.2

Lilipase + SMS/Calcium 18.6 12.3 6.1 carbonate Lilipase/Calcium 2.4 0.4 0.2 carbonate

Lilipase + SMS/Calcium 16.6 11.3 5.6 sulfate Lilipase/Calcium sulfate 0.3 0.2 0.3

* Fatty acid-(or its derivative) treated Celite (hydrophobized Celite) was prepared as follows: Celite (100 g) was suspended in phosphate buffer solution (100 ml) of pH=5.7. A solution of free fatty acid or its derivative (stearic acid or fatty acid sugar ester) dissolved in ethanol (5g/30ml) was added dropwise to the vigorously stirred suspension at 70°C. The suspension was stirred for 2 h, filtered, washed with water and then lyophilized.

The most suitable carriers for the immobilized enzymes used in this study

are aluminum monostearate and fatty acid-treated Celite. As can be seen in

Table VII, nonmodified Lilipase immobilized on Aluminum monostearate or

fatty acid treated- Celite gave relatively good activity in all of the three

reaction models. The results presented in this Table prove that the enzyme

modification with a surfactant in a first step and then immobilizing the modified enzyme on a fatty acid-treated insoluble matrix leads to a further

increment in the activity of the enzyme. As can be seen in the above Table,

the activity of the fatty acid derivative -modified and immobilized lipase on a

fatty acid derivative-treated insoluble matrix (Aluminum monostearate, fatty acid derivative-treated Celite) is much greater than the activity of

lipase immobilized on a fatty acid derivative-treated insoluble matrix

Example 6

The effect of the choice of ion-exchange resin on enzyme activity

The esterification, transesterification and alcoholysis activities of Lilipase

A-IOFG immobilized on various ion-exchange resins, and of the same enzyme

modified with sorbitan monostearate prior to immobilization were compared.

The results of this comparison (expressed as reaction rate, ri) are shown in

Table VIII.

Table VIII

Lilipase/Ion-exchange ri ri (transri resin* (esterification) esterification) (alcoholysis) (μmol/min.mg (μmol/min.mg (μmol/min.m protein) protein) g protein)

Dowex 22 4.9 4.1 1.9 Dowex 22 + SMS 32.2 23.1 6.3

Dowex 1x2-400 6.9 3.5 1.1 Dowex 1x2-400 + SMS 29.6 25.4 6.1

Dowex2x8-100 5.5 4.7 2.2 Dowex2x8-100 + SMS 21.3 16.2 7.3

Cellulose phosphate 3.5 0.2 0.1 Cellulose phosphate + 6.3 1.5 1.1

SMS

Amberlite IRA-95 7.6 0.8 0.9 Amberlite IRA-95 + SMS 26.2 12.5 2.3

Amberhte IRA-200 9.2 3.1 1.4 Amberlite IRA-200 + 28.6 18.9 5.3

SMS

Amberlite IRA-900 6.1 4.2 2.3 Amberlite IRA-900 + 37 28.3 8.6 SMS

DiannonSA-lOA 6.1 2.8 1.2 DiannonSA-lOA + SMS 31 19.5 11.3

Ectoela cellulose 5.4 2.3 1.7 Ectoela cellulose + SMS 29.5 12.2 5.1

Sephadex 1.5 0.2 0.1 Sephadex + SMS 2.9 0.9 0.8

Sulfoxyethylcellulose 5.6 3.2 2.5 Sulfoxyethylcellulose + 32.3 17.0 12.6 SMS

Amberlite XAD-7 8.3 4.5 3.8 (Weak-polar) Amberlite XAD-7 + SMS 37.3 29.5 17.5

Amberlite XAD-16 6.1 6.5 4.3 (apolar)

Amberlite XAD-16 + SMS 33.4 24.4 14.3

* All ion-exchange resins were purchased from Sigma, USA.

These results demonstrate the dramatic increase in esterification and

trans-esterification activity of the modified enzymes upon immobilization.

Furthermore, it can be seen from Table VIII that ion-exchange resins containing hydrophobic groups in their structure behave much better as

carriers for the surfactant-modified enzymes.

Example 7

Biosynthesis of structured triglycerides from different oils

using inorganic matrix-immobilized surfactant-coated lipase

complexes

Table IX below demonstrates the rates of inter-esterification reactions

between different oils and capric acid in n-hexane system and in a solvent free

system at 60 °C. The inter-esterification reactions were catalyzed by

DE-immobilized surfactant-Lilipase A10-FG complexes.

DE-immobilized surfactant-Lilipase complexes predominantly catalyzed the

inter-esterification of various oils with capric acid in n-hexane and solvent

free systems. The highest inter-esterification reaction was obtained when

olive oil was employed. Similar reaction rates where obtained when the

inter-esterification reactions were carried-out in a solvent-free system.

Table LX

Source of oil R.R. (mmol min-mg biocat.) R.R. (mmol min-mg biocat.) in n-hexane in solvent free system

Olive oil 0.52 0.73

Fish oil 0.4 0.53

Sun flower oil 0.3 0.42

Palm oil 0.2 0.31

Canola oil 0.32 0.45

Corn oil 0.24 0.41

Nigella satiυa oil 0.2 0.46 Example 8

Operational stability of modified and immobilized enzymes

The stability of the enzyme preparations over repeated cycles of use was

determined as described in the methods section above. For the purposes of

this investigation, Lilipase A-IOFG was used to catalyze the conversion of

olive oil to a wax ester. Activity was measured in a 10-run experiment, each

run requiring one hour for completion. The level of activity was measured as

percentage conversion of the olive oil. The reaction conditions used were as

follows:

100 ml of n-Hexane containing 20g olive oil and 20g cetyl alcohol recirculated

over an immobilized enzyme packed-bed at a circulation rate of 2.3 ml/min at

40° C. The column used to pack the granulated enzyme was 12 cm long with

an internal diameter of 0.75 cm. The results of this investigation are shown

in figures 5 — 12.

Fig. 5 shows that the activity of Lilipase A-10FG immobilized on Celite

(without modification) and granulated with 2% starch was low, and that the

activity decreased with each re-use. Fig. 6 shows that the activity of the

same lipase powder after modification with sorbitan monostearate and then

immobilization on Celite (without granulation) was 9-fold higher than the

activity of the same non-modified lipase. It can be seen from Fig. 2 that

there was sharp activity loss after the first, second and the third runs,

which can be attributed to washing out of the immobilized enzyme. Figs. 7-16 show that the enzyme activity in organic solvent can be essentially

retained by granulation with different binding reagents such as starch,

ethyl cellulose, gums, starch, etc. The granulation process also facilitates the

flow in the packed column with enzyme. The binders used in these figures

are as follows:

Fig. 7 Starch 2%

Fig. 8 Ethyl cellulose 2 %

Fig. 9 Gum Arabic 2 %

Fig. 10 Ethyl cellulose 2 %

Fig. 11 Agarose 2%

Fig. 12 Starch 4%

Figs. 13 - 16 Starch 2 %

Immobilization was performed on Celite in the experiments shown in figures

5 - 9 and 12 - 13, on Amberlite IRA-900 in the experiments shown in figures

10 and 11, on aluminium monostearate in figure 14, on Amberlite XAD-16 in

figure 15 and on Amberlite XAD-7 in figure 16. In all cases where lipase was

modified by surfactant, the surfactant used for this treatment was sorbitan

monostearate, except for the experiment shown in figure 13, where the

surfactant was sorbitan tristearate. Example 9

Covalent binding of modified and non-modified lipases to Eupergit

Lipases were covalently bound to Eupergit according to the manufacturer's

specification. The covalent binding was carried out the two procedures

described above.

Table X

Lilipase AlO-FG

Enzyme form Conversion (%)

Crude Lilipase AlO-FG 2

Lilipase on Eupergit 4

Lilipase on Eupergit + SMS 19

Lilipase on Eupergit + SMS after 48h 26

Lipase LP

Enzyme form Conversion (%)

Crude lipase LP 1

Lipase LP on Eupergit 2

Lipase LP on Eupergit + SMS 14

Lipase LP on Eupergit + SMS after 48h 18

Lipase PS

Enzyme form Conversion (%)

Crude lipase PS 1

Lipase PS on Eupergit 2

Lipase PS on Eupergit + SMS 8

Lipase PS on Eupergit + SMS after 48h 12

It can be seen from Table X that different lipases show different

inter-esterification activity when are treated similarly. This result is

ascribed to the different sources of the lipases used. All crude lipases showed

very low inter-esterification activity under the described conditions while

their activity has slightly increased when they were covalently immobilized on

Eupergit. It is interesting to notice that when lipases were coated with a

surfactant their inter-esterification activity has significantly increased. The highest conversion of tripalmitin to its inter-esterification products with

1,3-positional specificity that was achieved after 2 h reaction, was when the

lipases were first covalently immobilized on Eupergit and then coated with

surfactant. Lilipase AlO-FG coated with the surfactant and immobilized on

Eupergit yielded the highest inter-esterification activity within the three

lipases tested in this respect (Table X).

Example 10

Effect of binders on the enzyme activity.

As previously mentioned, different binders have been used for the

granulation of the modified-immobilized lipases. Table XI shows the average

conversion of olive oil to its wax esters in the first and second runs, using

different binders for granulating Lilipase AlO-FG modified with sorbitan

monostearate and immobilized on Celite. The percentage of all binders was

2% dry weight of the granules. In these experiments, the granulated

enzyme was packed in a column and used in 10 runs.

The average conversion of olive oil to its wax esters in the first and second

runs using Lilipase A 10FG modified with sorbitan monostearate and

immobilized on Celite and then granulated with different binders (2%).

Reaction conditions: 100ml of n-Hexane containing 2g olive oil and 2g cetyl

alcohol recirculated over an immobilized enzyme packed-bed at a circulation

rate of 2.5 ml/min and at 40°C. Column dimensions: 12 cm long, 0.75 cm i.d. Table XI

Binder Conversion (%)

Starch 92

Ethyl cellulose 44.6

Methyl cellulose 38.1

Agarose 16.8

Gelatin Inactive

Polyvinylpyrrollodone Inactive

Gum Arabic 49.1

Gum Xan 33.2

Gum Karaya 49.5

Gum Tragacanth 20.1

Gum Locas 33.6

From the above table it may be concluded that starch, Gum Arabic and Gum

Karaya are the most effective binders of those tested in this study, that

yielded active biocatalysts.

Although the invention has been described in conjunction with specific

embodiments thereof, it is evident that many alternatives, modifications and

variations will be apparent to those skilled in the art. Accordingly, it is

intended to embrace all such alternatives, modifications and variations that

fall within the spirit and broad scope of the appended claims.

Claims

1. A lipase preparation comprising an insoluble matrix and a

surfactant-coated lipase complex immobilized onto said insoluble matrix.

2. The lipase preparation of claim 1, wherein the surfactant-coated

lipase complex is covalently, ionically or physically bound to the insoluble

matrix.

3. The lipase preparation of claim 1, wherein the insoluble matrix is

selected from the group consisting of an inorganic insoluble matrix and an

organic insoluble matrix.

4. The lipase preparation of claim 3, wherein the inorganic

insoluble matrix is selected from the group consisting of alumina,

diatomaceous earth, Celite, calcium carbonate, calcium sulfate, ion-exchange

resin, silica gel and charcoal.

5. The lipase preparation of claim 4, wherein the ion-exchange resin

is selected from the group consisting of Amberlite and Dowex.

6. The lipase preparation of claim 3, wherein the organic insoluble

matrix is selected from the group consisting of Eupergit, ethylsulfoxycellulose

and aluminium stearate.

7. The lipase preparation of claim 1, wherein the content of the

lipase is 2-20 weight percent of the surfactant-coated hpase complex.

8. The lipase preparation of claim 1, wherein the content of the

lipase is 0.01-1.0 weight percent of the preparation.

9. The lipase preparation of claim 1, wherein the surfactant in the

surfactant-coated lipase complex includes a fatty acid conjugated to a

hydrophilic moiety.

10. The lipase preparation of claim 9, wherein the fatty acid is

selected from the group consisting of monolaurate, monomyristate,

monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate,

trilaurate, trimyristate, tripalmitate and tristearate.

11. The lipase preparation of claim 9, wherein the hydrophilic moiety

is selected from the group consisting of a sugar, a phosphate group, a

carboxylic group and a hydroxylated organic residue.

12. The lipase preparation of claim 11, wherein the sugar is selected

from the group consisting of sorbitol, sucrose, glucose and lactose.

13. The lipase preparation of claim 9, wherein the fatty acid and the

hydrophilic moiety are conjugated via an ester bond.

14. The lipase preparation of claim 1, wherein the lipase is derived

from a microorganism.

15. The lipase preparation of claim 1, wherein the lipase is derived

from a species selected from the group consisting of Burkholderia sp., Candida

antractica B, Candida rugosa, Pseudomonas sp., Candida antractica A,

Porcine pancreas lipase, Humicola sp., Mucor miehei, Rhizopus javan.,

Pseudomonas fluor., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae,

Mucor jaυanicus, Rhizopus sp., Rhizopus japonicus and Candida antractica.

16. The lipase preparation of claim 14, wherein the lipase is derived

from a multicellular organism.

17. A lipase preparation comprising an insoluble matrix and a

surfactant-coated lipase complex immobilized onto said insoluble matrix, said

lipase preparation being provided in an organic solvent.

18. The lipase preparation of claim 17, wherein the organic solvent

is selected from the group consisting of n-hexane, toluene, iso-octane,

n-octane, benzene, cyclohexane and di-iso-propylether.

19. The lipase preparation of claim 17, for use as a catalyst for

esterification, inter-esterification and trans-esterification of oils and fats and

alcoholysis of trigiycerols and fatty alcohols.

20. The lipase preparation of claim 19 for use as a catalyst with

1,3-positional specificity with respect to triacylglycerols.

21. The lipase preparation of claim 1, wherein said preparation is in

granulated form.

22. The lipase preparation of claim 1, wherein the insoluble matrix

has been modified with a fatty acid derivative.

23. An enzyme preparation according to claim 1, for use in a reaction

environment without the need for water addition.

24. A method for improving the stability of a surfactant-coated

immobilized lipase complex, comprising granulating same prior to contacting

it with the substrate to be reacted.

25. A method of preparing an insoluble matrix-immobilized

surfactant-coated lipase complex comprising, in any desired order, the steps

of:

(a) contacting a lipase in an aqueous medium with a surfactant, at a

concentration and temperature, and for a period of time sufficient to

obtain a coating of said lipase; and

(b) contacting said lipase in an aqueous medium, with an insoluble

matrix, at a concentration, under conditions and for a period of

time sufficient to obtain immobilization of said lipase on said

matrix.

26. The method of claim 25, wherein said lipase is first contacted

with the insoluble matrix, and thereafter with the surfactant.

27. The method of claim 25, wherein the lipase is first contacted with

the surfactant, and thereafter with the insoluble matrix.

28. The method of claim 25, further comprising the step of:

(c) separating the matrix-immobilized surfactant-coated lipase

complex from the aqueous solution in which it was formed.

29. The method of claim 28, further comprismg the step of: (d) drying the matrix-immobilized surfactant-coated lipase complex.

30. The method of claim 29, wherein drying is effected by freeze

drying.

31. The method of claim 29, wherein the matrix-immobilized

surfactant-coated lipase complex is dried to a water content of less than 100

parts per million by weight.

32. The method of claim 25, wherein the aqueous solution is a

buffered aqueous solution.

33. The method of claim 25, wherein the lipase and surfactant are

contacted in the aqueous medium by:

(i) dissolving said surfactant in an organic solvent for obtaining a

dissolved surfactant solution; and

(ii) mixing said lipase and said dissolved surfactant solution in said

aqueous medium.

34. The method of claim 25, further comprising sonicating the

aqueous solution.

35. The method of claim 25, wherein the insoluble matrix is selected

from the group consisting of alumina, diatomaceous earth, Celite, calcium

carbonate, calcium sulfate, ion-exchange resin, silica gel, charcoal, Eupergit,

ethylsulfoxycellulose, aluminium stearate and fatty acid derivative-treated

Celite or other inorganic matrices.

36. The method of claim 25, wherein the surfactant includes a fatty

acid conjugated to a hydrophilic moiety.

37. The method of claim 36, wherein the fatty acid is selected from

the group consisting of monolaurate, monomyristate, monopalmitate,

monostearate, dilaurate, dimyristate, dipalmitate, distearate, trilaurate,

trimyristate, tripalmitate and tristearate.

38. The method of claim 36, wherein the hydrophilic moiety is

selected from the group consisting of a sugar and a phosphate group and a

carboxylic group and a polyhydroxylated organic residue.

39. The method of claim 38, wherein the sugar is selected from the

group consisting of sorbitol, sucrose, glucose and lactose.

40. The method of claim 36, wherein the fatty acid and the

hydrophilic moiety are conjugated via an ester bond.

41. The method of claim 25, wherein the lipase is derived from an

organism.

42. The method of claim 41, wherein the lipase is derived from a

multicellular microorganism.

43. The method of claim 41, wherein the lipase is derived from a

species selected from the group consisting of Burkholderia sp., Candida

antarctica B, Candida rugosa, Pseudomonas sp., Candida antractica A,

Porcine pancreatic lipase, Humicola sp., Mucor miehei, Rhizopus jaυan.,

Pseudomonas fluor., Candida cylindrcae, Aspergillus niger, Rhizopus oryzae,

Mucor jaυanicus, Rhizopus sp., Rhizopus japonicus and Candida antarctica.

44. A process for preparing structured triacylglycerols by

esterification, acidolysis, trans-esterification, inter-esterification or alcoholysis

between two substrates comprising contacting an insoluble

matrix-immobilized surfactant-coated lipase complex with said substrates.

45. The process of claim 44, wherein the matrix-immobilized

surfactant-coated lipase complex is contacted with the substrates in the

presence of an organic solvent.

46. The process of claim 44, wherein at least one of the substrates is

selected from the group consisting of an oil, a fatty acid, a triacylglycerol and

a fatty alcohol.

47. The process of claim 46, wherein the oil is selected from the

group consisting of olive oil, soybean oil, peanut oil, fish oil, palm oil, cotton

seeds oil, sunflower oil, Nigella sativa oil, canola oil and corn oil.

48. The process of claim 46, wherein the fatty acid is selected from

the group consisting of medium and short-chain fatty acids and their ester

derivatives.

49. The process of claim 46, wherein the fatty acid is selected from

the group consisting of oleic acid, palmitic acid, linolic acid, linolenic acid,

stearic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and

their ester derivatives.

50. The process of claim 44 which is carried out in a tank reactor or

in a fixed-bed reactor.

51. A triacylglycerol prepared according to the process of claim 44,

for use as a cocoa butter substitute, human milk fat-like triglycerides for

special diets, or structured triglycerides for medical applications.