WO2004035773A1

WO2004035773A1 - Immobilization of compounds on polymeric matrix

Info

Publication number: WO2004035773A1
Application number: PCT/IL2003/000822
Authority: WO
Inventors: Sobhi Basheer; Muhammad Kaiyal; Aviv Boltanski
Original assignee: Enzymotec Ltd.
Priority date: 2002-10-14
Filing date: 2003-10-12
Publication date: 2004-04-29
Also published as: AU2003272056A1; IL152290A0

Abstract

The present invention relates to a process for immobilizing a compound containing a functional group selected from the group consisting of amine, carboxyl, hydroxyl, thiol and/or other nucleophilic groups, onto an insoluble polymeric matrix containing epoxy groups, wherein said process comprises covalently binding said compound onto said insoluble polymeric matrix in a medium containing an organic solvent, preferably in the presence of water and/or a surface-active material. The process of the invention is particularly intended for the immobilization of proteins, preferably enzymes and specifically enzymes intended for industrial applications. The immobilized enzymes obtained by the process of the invention have enhanced activity and stability and therefore can be used either in cycles in batch reactors or continuously in fixed-bed and fluidized-bed reactors of an industrial process.

Description

IMMOBILIZATION OF COMPOUNDS ON POLYMERIC MATRIX

Field and Background of the Invention

A vast number of research works describe various methods for the immobilization of biomolecules, in general, and enzymes in particular. The main goals of enzyme immobilization remain to greatly restrict the freedom of movement of an enzyme, reduce the cost contribution of an enzyme in the -whole process, to allow heterogeneous catalysis of enzymatic reactions and operation under continuous processes, and also to avoid the necessity to remove the enzyme from the end product. Enzymes and other biomolecules have been immobilized according to many procedures. These procedures include:

1. Physical adsorption of enzymes to solid insoluble carrier matrices, such as silica, Celite, glass beads, and polymers.

2. Adsorption on ion-exchange resins.

3. Adsorption to a solid carrier and with subsequent cross-linking with bi-functional molecules, such as gluta aldehyde.

4. Covalent binding to a solid support material, such as activated silica.

5. Entrapment of enzymes in a growing polymer, such as acrylic-based polymers.

6. Encapsulation of enzymes in semi-permeable gels or by confinement in a membrane reactor.

7. Cross-linked enzyme crystals (CLEC) or aggregates (CLEA).

All the above mentioned enzyme immobiHzation procedures have been carried out by adding an insoluble carrier support into an aqueous enzyme solution under certain conditions with respect to pH, ionic strength, type of buffer and, in general, at room temperature, whereas in some procedures the temperature had to be lowered to 10°C. At the end of the process, immobilized enzymes were washed with a buffer solution or with water, to remove loosely bound biomolecules. Immobilized enzymes were used wet (following filtration and washing) or were first dried by lyophilization, or dried under reduced pressure (over molecular sieve and dried air/nitrogen stream). It has been reported that in most cases there were substantial .enzyme activity losses following the above procedures.. Some enzymes . retained their full activity following immobilization, in particular, those which were immobihzed by covalent binding. Nery few cases have been reported where enzymes increased their specific activity after immobilization. However, it has been reported in many studies that enzymes improved their operational stability after immobilization.

Immobilized enzymes according to the aforementioned procedures have been used as wet or dried catalysts in enzymatic reactions in aqueous media, in aqueous/organic media (bi-phasic reactions) and in organic media (organic synthesis).

Within the aforementioned procedures, immobilization via adsorption is the simplest for large-scale production. The procedure in general consists of mixing and incubating together an enzyme and an adsorptive support, under suitable conditions of pH, ionic strength, and so on, for a predefined period of time, followed by separating the immobihzed enzyme and extensive washings for removing unbound or loosely bound enzyme. This procedure is based on surface interaction between the enzyme and the adsorptive matrix that involves electrostatic forces, such as Nan der Waals forces, ionic forces, hydrophobic interactions and hydrogen bonding. In most cases these forces are weak and inadequate for retaining an immobihzed enzyme adsorbed on the support, especially under operation conditions, resulting in washing out the enzyme.

Covalent binding of enzymes on supporting carriers has been considered a promising potential for industrial application. When an enzyme is covalently bound to an insoluble matrix, it shows higher operational stability. This method of immobihzation involves the formation of a covalent bond between the enzyme and the (activated) support material. Such a covalent bond is normally formed between certain reactive (or functional) group(s) present on the surface of the support and reactive (or functional) groups present in the amino acid residues and exposed on the surface of the biological components. Typically, amino, carboxyl, thiol and hydroxyl groups in the amino acids of the biomolecules are the main groups involved in the formation of the covalent bond with an activated support. Epoxy supports are potentially the optimal industrial supports for enzyme immobihzation. The epoxy group-containing polymeric resins, such as Eupergit of Roehm (Germany), Sepabeads of (Residion-Mitzubishi, Italy) and Polymer Carrier NA-epoxy Biosynth of Riedel deHaen have been identified as the most suitable supports for covalent immobilization of enzymes intended for industrial applications.

A conventional protocol for immobilization of enzymes (and other biomolecules) on common epoxy supports, such as Eupergit and Sepabeads, involves two steps:

1. Hydrophobic-hydrophilic adsorption of an enzyme that is dissolved in an aqueous buffer solution on the external surface (or in the pores) of an epoxy support.

2. A slow chemical reaction between the adsorbed enzyme and epoxy groups of the support via intermolecular reactions between the nucleophilic groups (such as amino or thiol groups) on the protein surface and the epoxy groups on the support surface. The reaction results in a covalent bond between the nitrogen of an amino group (of the enzyme) and the carbon of- methylenic group (of the epoxy- support):

Support-CH (OH)-CH₂-NH-Enzyme.

Similarly, a covalent bond may be formed between the epoxy-support and enzymes containing carboxyl group (s)

Support-CH (OH)-CH₂-OC(O)-Enzyme; thiol group (s):

Support-CH (OH)-CH₂-S-Enzyme; and hydroxyl group (s):

Support-CH (OH)-CH₂-O-Enzyme.

Different strategies have been adopted for stabilization of immobilized enzymes on epoxy-supports. These methods include:

1. Immobilization of enzymes dissolved in aqueous solutions on conventional mono-functional epoxy supports.

2. Immobihzation of_enzym.es dissolved in aqueous solutions on hetero-functional glyoxyl-epoxy supports.

3. Immobilization of enzymes dissolved in aqueous solutions on a number of hetero-functional epoxy supports.

4. Immobilization of genetically altered (mutated) enzymes dissolved in aqueous solutions on (thiol or chelate) epoxy-supports.

Excess of epoxy groups on the support normally facihtates the reaction with different nucleophiles (such as, for example, thiol and amine) of nucleophile(s)-containing biomolecules. Binding of biomolecules on epoxy- supports is affected by different parameters, including pH, ionic strength, type of buffer and temperature. Effective and efficient immobilization of enzymes on Eupergit, for example, involves contacting an aqueous enzyme solution, preferably at pH above 7 and 1M ionic strength of phosphate buffer at room temperature for 72 hours. Binding of enzymes on epoxy- supports, such as Eupergit can also occur at lower pH values, however at a slower rate. It has been reported that epoxy-support can serve as an excellent support, for the immobilization of most, if not all, classes of enzymes, including hydrolases, oxidoreductases, transferases, and others. Many research reports have documented the favorable use of immobilized enzymes, in particular lipases, on epoxy-supports, especially, Eupergit C in aqueous systems (for hydrolysis). For example, it has been reported that immobihzed lipases on Eupergit C have shown high operational stability in enantioselective hydrolysis of esters, hydrolysis of oils and fats, and in deacylation reactions, all in biphasic or in emulsion systems.

Immobilized enzymes on epoxy-supports, especially on Eupergit C, for organic synthesis, have been extensively studied during the last decade. A systematic study conducted by Ivanov and Schneider (J. Mol. Catal. B: Enzym. 3, 303-309 (1997)) for the comparison of synthetic activity in organic solvents and operational stability of immobilized lipases (following various immobilization procedures) has concluded the following:

1. Adsorption of lipase on Celite yielded the most active enzyme preparation for ester synthesis; however, the synthetic activity was completely lost after 10 cycles.

2. Adsorption of lipase on octyl silica yielded reasonably active enzyme preparation for ester synthesis; however, activity was lost after 5 cycles. 3. Covalent binding of lipase to aminopropylsilica and glutaraldehyde- activated silica yielded hpases of low synthetic activity.

4. Covalent binding of lipase on Eupergit C yielded 25% less active lipase preparation compared to octyl silica-hpase preparation, and 20 times less active than that of hpase adsorbed on Celite.

5. Immobilized lipase on Eupergit C retained 30% of its initial activity from cycle No. 6 through cycle No. 10; consequently, it was the most stable lipase preparation prepared in this study.

A recent work by M. Ferrer et al. (BioTrans 2001 Conference Proceedings p. 186, Darmstadt, Germany) showed that immobihzed lipase on Eupergit C exhibited low synthetic activity compared to the one adsorbed on silica or porous polypropylene (Accurel).

Reviewing the data obtained so far regarding covalently-immobilized enzymes reveals an important need for defining improved conditions for covalent-immobilization of an enzyme onto a solid matrix, for providing an enzyme complex preparation possessing high activity and stability for as many operation cycles as possible.

Consequently, it is an object of present invention to provide an improved immobilization process for covalently binding a compound, in particular a proteinous biocatalyst, such as an enzyme or catalytic antibody onto a solid polymeric matrix, without affecting, or whilst minimally affecting the activity of the biocatalyst in either synthetic and/or hydrolytic reactions. It is an additional object of present invention to provide such an immobihzation process for providing a covalently-bound immobihzed enzyme complex possessing high activity and stability for as many as possible synthetic and/or hydrolytic reaction cycles. It is yet a further object of present invention to provide such an immobilization process in which the enzyme is dissolved in a recyclable organic solvent instead of water.

These, and other objects of the invention, will become apparent as the description proceeds.

Summary of the Invention

The present invention relates to a process for immobilizing a compound containing a functional group selected from the group consisting of amine, carboxyl, hydroxyl, thiol and/or other nucleophilic groups onto an insoluble polymeric matrix containing epoxy groups wherein said process comprises covalently binding said compound onto said insoluble polymeric matrix in a medium containing an organic solvent, preferably in the presence of water and/or a surface-active material.

The compound to be immobilized by the process of the present invention is preferably proteinous biocatalyst. The biocatalyst may be in any one of solid form, suspended in a solution, particularly an aqueous solution, or confined in a reverse micellar system which is a water in oil (W/O) system, or in micellar system which is oil in water (O/W).

The insoluble polymeric matrix is preferably an acrylic polymer containing epoxy groups, particularly any one of Eupergit C 250L, containing at least 200μmol of epoxy groups per gram polymer and Eupergit C, containing at least 600μmol of epoxy groups per gram polymer. The organic solvent used in the immobihzation process is preferably selected from the group consisting of n-hexane, 2,2-dimethoxypropane, acetone, diethyl ether, iso-propanol, toluene, iso-octane and any mixture thereof. ^{' "}

The biocatalyst is preferably, but not limited to an enzyme, particularly a lipase or a phospholipase.

Specific lipases are obtained from a microorganism selected from the group consisting of Thermomyces lanuginosus, Candida antarctica B, Rhizomucor miehei, Rhizopus strains, Rhizopus oryzae and Candida rugosa.

In another embodiment, the enzyme may be a saccharidase, particularly an invertase.

The covalent-immobilization process of the invention may be carried out in a medium comprising the said proteinous biocatalyst, the said insoluble polymeric matrix containing epoxy groups and the said organic solvent, wherein said organic solvent is mixed with water and a surface active ingredient to form an emulsion. The amount of said organic solvent in said emulsion is particularly in the range of from 10wt% to 99.9wt%, preferably from 30 wt% to 99wt% and most preferably from 90wt% to 95wt%.

The covalent-immobilization process of the invention can also be carried out in a medium comprising the said proteinous biocatalyst, the said insoluble polymeric matrix containing epoxy groups and the said an organic solvent, wherein said organic solvent is water-insoluble and is mixed with water to form a bi-phase system. The amount of said organic solvent in said bi-phase system is particularly in the range of from 10wt% to 99.9wt%, preferably from 30wt% to 99wt% and most preferably from 90wt% to 95wt%.

In another embodiment, the said proteinous biocatalyst may be in solid form and the covalent-immobilization process is carried out in a medium comprising the said insoluble polymeric matrix containing epoxy groups and the said organic solvent, preferably in the presence of water and/or surface -active ingredient.

In a further embodiment, the biocatalyst may be suspended in a solution and the covalent-immobilization process is carried out in a medium comprising the said insoluble polymeric matrix containing epoxy groups and the said organic solvent, preferably in the presence of water and/or surface -active ingredient.

The covalent-immobilization process of the invention may be carried out in a medium containing a surface-active material, for example a polyol ester surfactant, and particularly a polyol fatty acid ester, such as sorbitan monooleate (SMO) and sorbitan monostearate (SMS).

In specific embodiments of the process of the invention, the biocatalyst may be coated with said surface-active material before, during or after the covalent-immobilization process.

In a particular embodiment, the invention relates to a process for immobilizing a compound containing a functional group selected from the group consisting of amine, carboxyl, hydroxyl, thiol and/or other nucleophilic groups, onto an insoluble polymeric matrix containing epoxy groups, comprising:

(i) contacting said compound with a medium comprising an insoluble polymeric matrix containing epoxy groups suspended in an organic solvent, preferably in the presence of water and/or a surface-active material;

(ii) incubating the mixture at a temperature, ranging preferably from 25°C to 70°C, until the binding reaction is completed;

(iii) separating the complex consisting of said compound covalently- immobilized on said insoluble polymeric matrix; and optionally

(iv) washing the separated complex with water followed by washing with acetone.

Also in this embodiment, the compound to be immobilized is a proteinous biocatalyst, preferably an enzyme.

The organic solvent employed in said step (i) is preferably water-insoluble and is mixed with water and a surface active ingredient to form an emulsion. The amount of said organic solvent in said emulsion is particularly in the range of from 10wt% to 99.9wt%, preferably from 30 wt% to 99wt% and most preferably from 50wt% to 95wt%.

In another embodiment, the said organic solvent employed in step (i) is water-insoluble and is mixed with water to form a bi-phase system. The amount of said organic solvent in said bi-phase system is particularly in the range of from 10wt% to 99.9wt%, preferably from 30wt% to. 99wt% and most preferably from 50wt% to 95wt%. The said step (ii) may be carried out in the presence of glutaraldehyde.

In a further aspect, the invention relates to a process for reactivating an exhausted biocatalyst covalentl -bound onto an insoluble polymeric matrix containing epoxy groups, referred to as "covalently-immobihzed biocatalyst complex", prepared according to claim 1, comprising the steps:

(i) resuspending said exhausted covalently-immobihzed biocatalyst complex in a phosphate buffer solution;

(ii) separating said covalently-immobihzed biocatalyst complex; and optionally

(iii) washing the separated complex with an organic solvent, preferably acetone or n-hexane followed by removing the organic solvent and drying the replenished covalently-immobihzed biocatalyst complex for re-use.

The various processes of the invention are particularly intended for the preparation of a hpase or phospholipase covalently-immobihzed onto an insoluble polymeric matrix which contains epoxy groups, said immobilized hpase or phospholipase being intended for catalyzing either synthetic reactions, in which a new ester group is formed and/or hydrolytic reactions, in which an ester group is cleaved. The immobilized lipase or phospholipase can be particularly intended for catalyzing an interesterification or transesterification reaction, an esterification reaction or a hydrolytic reaction.

The invention further relates to immobilized enzymes obtained by any of the embodiments of the process of the invention, which have enhanced activity and stability and therefore can be used either in cycles in batch reactors or continuously in fixed-bed and

The invention will be described in more detail on hand of the attached drawings.

Brief Description of the Figures

Figure 1 shows the operational stability of different lipase (Lipozyme TL 100L) preparations immobilized on Eupergit C in (a) n-hexane; (b) n- hexane + SMO; (c) phosphate buffer solution (PBS); and (d) PBS + SMO in consecutive batch experiments.

Figure 2 shows the operational stability of different hpase (Lipozyme TL 100L) preparations immobilized on Eupergit C 250L in (a) n-hexane; (b) n- hexane + SMO; (c) phosphate buffer solution (PBS); and (d) PBS + SMO in consecutive batch experiments.

Figure 3 shows the operational stability of different lipase (Lipozyme TL 100L) preparations immobilized on Eupergit C 250L in (a) acetone; and (b) acetone + SMO, in consecutive batch experiments.

Figure 4 shows the interesterification activity of hpozyme TL 100L immobilized on Eupergit C 250L through 45 cycles.

Figure 5 shows the operational stability of immobilized hpase (Lipozyme TL 100L) on Eupergit C and on Eupergit C 250L in the presence of water. Figure 6 shows the operational stability of different lipase preparations (Lipozyme TL 100L) immobilized on Eupergit C 250L in the presence of toluene (with and without SMO) at 70°C, in 10 successive cycles.

Figure 7 shows the hydrolytic reaction profile using immobilized Lipase OF (Candida rugosa) on Eupergit C 250L in n-hexane in the presence of SMO with and without cross-linking with glutaraldehyde, compared with the same lipase when immobilized on the same matrix in 1M phosphate buffer solution (pH 7) in the presence of SMO with and without cross- hnking with glutaraldehyde, and crude enzyme preparation.

Description of the Invention

The present invention relates to the preparation of immobilized biocatalysts, preferably an enzyme, on an epoxy polymer matrix, preferably epoxy-support acrylic beads (such as, Eupergit C and Eupergit C 250L) so that the immobihzed-enzyme preparation exhibits high activity (either synthetic or hydrolytic) in aqueous or organic media. In contrast to well familiar, conventional procedures for covalent-immobihzation of enzymes onto epoxy-supports. in aqueous media, the present invention provides a new procedure in which the contact between the enzyme and epoxy-support is carried out in an organic solvent, or in organic solvent- containing media, such as bi-phase systems, emulsion or reverse micellar systems, preferably at room temperature and up to 70°C.

The covalent binding between the enzyme and the epoxy resin can be carried either with an enzyme in a sohd form or with an enzyme in a liquid form. Most commercial liquid enzyme preparations contain 1-10% active protein (enzyme), which is dissolved in a mixture of water and a polyol, such as glycerol and propanediol, which serve as preservants and suspenders. Other commercial enzyme preparations are in solid form, and also contain 1-10% protein (active enzyme) dispersed in a sohd polyol, typically a disaccharide such as lactose, or a polysaccharide, such as starch. For covalently immobilizing the enzyme, according to the present invention, both enzyme preparations (sohd, as well as liquid) are suspended in a medium containing organic solvent. The immobilized enzyme is collected by simple filtration where the organic solvent may be recycled for use in, another immobilization process.

Some crude enzymes, including lipases and proteases, were reported to increase their synthetic activity upon increasing the concentration of buffer salts (such as sodium chloride) or polyols (such as sorbitol) in their reaction medium. Other reports have shown that incubating enzymes in organic media for a certain period of time resulted in increasing the synthetic activity of some lipases and proteases. Typical organic solvents used in those studies are in particular hydrophobic organic solvents, such as iso-octane, n-hexane, toluene and cyclohexane. Other organic solvents, such as acetone, iso-propanol and ethyl acetate, caused deterioration of the synthetic activity of enzymes incubated in them. In view of possible deterioration of enzyme activity in an organic solvent during the immobilization binding, certain polyesters (such as sugar fatty acid esters) and polyol compounds (such as polyethylene glycol and saccharides) have been added into the organic solvents for protecting the enzymes from the harsh reaction conditions

The present invention thus provides a new process for immobilizing a compound containing a functional group selected from the group consisting of amine, carboxyl, hydroxyl, thiol and/or other nucleophilic groups onto an insoluble polymeric matrix containing epoxy groups, wherein said process comprises covalently binding said compound onto said insoluble polymeric matrix in a medium containing an organic solvent, preferably in the presence of water and/or a surface-active material.

The present invention further provides a process for covalently bindly and immobilizing a proteinous biocatalyst, such as an enzyme or a catalytic antibody onto an insoluble polymeric matrix containing epoxy groups.

The present invention further provides a process for reactivating an exhausted biocatalyst, such as an enzyme or a catalytic antibody, covalently-bound onto an insoluble polymeric matrix containing epoxy groups.

Thus, the present invention provides a new process for the preparation of highly active covalently-immobihzed biocatalyst molecules, especially enzymes, on epoxy groups-containing sohd supports, such as Eupergit matrix (copolymerisates of methacrylamide, N,N'-methylene-bis(methyl- acrylamide) and monomers containing oxirane groups) produced by Roehm, Germany; polymer carrier NA-epoxy and Biosynth (spherically macroporous beads of a copolymer based on vinyl acetate and divinyl ethyleneurea with a surface area modifier with oxirane groups after hydrolysis of the acetate groups) of Riedel de-Haen, Germany, and the epoxy polymer Sepabeads EC-EP (highly porous spherical polymer beads based on poly methacrylate that contains oxirane groups) of Resindion- Mitsubishi, Japan.

In a preferred embodiment, the immobihzation procedure of the present invention is carried out by mixing an enzyme preparation with an organic solvent comprising a chemically reactive insoluble epoxy group -containing polymeric matrix, such as Eupergit C and Eupergit C 250L. The term "enzyme preparation" includes, inter alia, an enzyme in a sohd form, an aqueous solution of an enzyme, optionally containing polyol compound(s); enzyme that is confined in a micellar (in an emulsion or in a dispersion) or reverse micellar system. Micellar system (emulsion) is oil in water (o/w), while reverse micellar system is water in oil (w/o), both systems have a defined micelle structure. In contrast, a dispersion represents a suspension of particles with no defined structure in the solution. The enzyme preparation may optionally contain a stabilizer or modifier, such as a surfactant. The organic solvent that contains the polymeric matrix for immobihzation may be, for example, n-hexane, acetone, 2,2- dimethoxypropane, diethyl ether, iso-propanol, toluene, iso-octane and a mixture of any or all of them.

The term "organic solvent" as used herein includes mixtures of at least one organic solvent and water, defined as "an emulsion" or '^bi-phase system" in which the amount of said organic solvent in said mixture is in the range of from lwt% to 99wt%, particularly from 10wt% to 99.9wt%, preferably from 30 wt% to 99wt% and most preferably from 50wt% to 95wt%.

The enzyme preparation, optionally containing a modifier, and the polymeric matrix in an organic solvent are mixed gently at room temperature or in a heated incubator, preferably at a temperature in the range of 25 to 70°C. Following incubation, the mixture is filtered off, washed with the same solvent used in the immobihzation procedure, and optionally washed with water and then with acetone. Residues of organic solvents and water retained on the enzyme-support particles following the immobihzation process can be removed by drying under reduced pressure (over silica gel in a desiccator) or by lyopbilization in a lyophilizer. The organic solvent in the filtrate can be recycled and used for a consecutive enzyme immobihzation process. In the process of present invention, the nucleophile group (s) on the biocatalyst or enzyme surface, such as amino, hydroxyl, thiol, imidazole, carboxyl, or other groups, react with the epoxy groups of the polymeric support to produce a covalently-bound enzyme- support complex. Also, unlike conventional immobilization techniques carried out in aqueous solutions, the process of present invention can be conducted at high temperatures, such as 70°C and higher, resulting in reducing the immobilization time as well as in facihtating multi-point covalent bindings between the enzyme molecules and the polymeric support, which, has a significant effect on major increment of the operational enzyme stability.

Without being bound by theory, it may be suggested that the process of present invention provides an interface layer formed by mixing an organic solvent with an aqueous buffer system containing the enzyme (with or without surface active ingredient). Such an interface layer would urge "interfaciaUy active" enzymes to adopt a unique conformation which facilitates both the specific catalytic activity and the covalent binding with a chemically reactive functional group, such as an epoxy group. This conformation that simultaneously facilitates catalytic activity and capability of the enzyme molecule to react with a chemically reactive group on a polymer would produce an active enzyme covalently-bound on an insoluble matrix. For other types of enzymes that do not need an interface for expressing their activity, it is most likely that in the presence of an organic solvent some changes in the position of the functional groups on the surface of the enzyme molecule occur that lead to a preferred molecule conformation that allow the reaction with the epoxy polymeric resin. Such covalently-bound immobilized enzyme preparations may be used either in a stirred-tank reactor or fixed in a column reactor. The use of both alternatives ensures full-recovery of the immobilized enzyme following operation in a series of cycles.

All pubhcations mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It is to be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Abbreviations

SMO: Sorbitan Monooleate

SMS: Sorbitan Monostearate

PBS: Phosphate Buffer Solution

GC: Gas Chromatography

HPLC: High Pressure Liquid Chromatography

FID: Flame Ionization Detector

RI: Reaction rate

Examples

Materials and methods

Different enzyme preparations were used; the protein in each enzyme preparation was determined according to Bradford method [Bradford, M. M. A Rapid and Sensitive for the Quantitation of Microgram Quantitites of Protein Utihzing the Principle of Protein-Dye Binding, Analytical Biochemistry 72: 248-254. 1976].

The enzyme preparations used in demonstrating the present invention include, but are not limited to: Lipozyme TL 100L (Thermomyces lanuginosus ): an aqueous solution of the enzyme containing protein content 3.5% by weight mixed with a polyol, typically, glycerol or propane diol);

Novozym 525L (Candida antarctica B): an aqueous solution of the enzyme containing protein content 4.5% by weight mixed with a polyol, typically, glycerol or propane diol;

Novozym 388 (Rhizomucor miehei): an aqueous solution of the enzyme containing protein content 9.2% by weight mixed with a polyol, typically, glycerol or propane diol all from Novozymes, Denmark;

Lipase Saiken 100 (Rhizopus strain):¹ a dried powdery preparation containing protein content 13% by weight from Nagase, Japan;

Lipase F-AP 15 (Rhizopus oryzae): a dried powdery preparation containing protein content 16.5% by weight from Amano, Japan;

Lipase OF (Candida rugosa): a dried powdery preparation containing protein content 14% by weight from Meito Sangyo, Japan;

Lecitase novo (a microbial phospholipase Al): an aqueous preparation containing protein content 5% by weight obtained from Novozymes- Denmark; and

Bioinvert L: an aqueous invertase preparation containing protein content 3.5% by weight from Quest, England.

Supports used in the present invention, include, but are not hmited to:

Eupergit C, a macroporous, spherical acrylic polymer carrier having a diameter of 150μm, contains >600 μmol of epoxy groups/ (dry) (Roehm- Germany).

Eupergit C 250L, a highly macroporous, spherical acrylic polymer carrier having a diameter of 200μm, contains >200 μmol of epoxy groups/g (dry) (Roehm-Germany). Polymer Carrier NA-epoxy and BIOSYΝTH (spherically macroporous beads of a copolymer based on vinyl acetate and divinyl ethyleheurea with a surface area modifier with oxirane groups after hydrolysis of the acetate groups) of Riedel de-Haen (Germany)

The epoxy polymer SEPABEADS EC-EP (highly porous spherical polymer beads based on polymethacrylate that contains oxirane groups) of Resindion-Mitsubishi (Japan).

All chemicals and solvents were purchased from Sigma.

Triglycerides, fatty acid esters, fatty alcohols and fatty acids were analyzed by a GC equipped with an FID. Sucrose, glucose and fructose were analyzed by an HPLC, equipped with an RI detector.

Example 1; Enzyme Immobilization on Epoxy -Supports in Organic Solvents.

Enzyme preparation (lg or 1ml preparation contains about 2-10% by wt of active enzymatic protein) was added into a screw-cap vial that contains the selected organic solvent (10ml). The mixture was shaken vigorously for lOmin. Eupergit C or Eupergit C 250L (lg) was added into the mixture and then the screw-cap vial was tightly closed and gently shaken for 72h at room temperature. The mixture was filtered and washed several times with the same organic solvent. The cake was divided into two portions: the first portion was washed with water and then with acetone, while the second portion did not undergo any further washing. The rationale behind the selection of these two alternatives was derived from the fact that washing with water would remove loosely, non-covalently bound enzymes from the matrix surface and then washing with acetone would remove excess water from the matrix. Both portions were either lyophilized in a freeze-dryer or dried over sihca gel in a desiccator for 24 hours at room temperature.

Example 2: Immobilization of a Modified-Enzyme on Epoxy- Supports in Organic Solvents.

To immobihze modified-lipase enzymes, the procedure of Example 1 was followed. However, in such case, the organic solvent contained a surfactant modifier (lOOmg), such as sorbitan monooleate (SMO) and sorbitan monostearate (SMS).

It should be noted that adding the surfactant could be carried out before, during or following the covalent binding of the enzyme onto the polymeric matrix containing epoxy groups.

Control experiments were carried out using the same procedure as described above, substituting the organic solvent(s) with 1M PBS (as in the conventional procedures reported by the manufacturer Roehm, Germany).

Example 3: Enzyme activity assay.

The synthetic and hydrolytic activities of lipases and phosphohpases were determined by using the following assay procedures:

A. Synthetic activity:

The synthetic activity of the prepared immobihzed enzyme-support complex was tested using two reaction models: (1) an esterification reaction of free fatty acid with fatty alcohol in n-hexane system; and (2) an interesterification reaction of ohve oil triglycerides with lauric acid in a solvent-free system.

(a) Esterification reaction: Immobilized lipase (typically, lOOmg), prepared as described in Example 1, was added into n-hexane solution (1ml) containing lOOmg lauric acid and lOOmg lauryl alcohol. The reaction was carried out while shaking the mixture for a period of time (typically 2-4 hours, depending on the particular lipase used) at 60°C. Samples were taken at the same time for all the esterification reactions, as described hereinafter, diluted with n-hexane and injected into a gas chromatograph (GC). The percent peak area ratio of formed ester is calculated as: peak area of formed lauryl ester/total peak area of lauric acid, lauric alcohol and lauryl ester X 100. The percent peak area value was chosen as a measure for the activity of the immobihzed lipase.

(b) Interesterification reaction: Immobilized lipase preparation (lOOmg), prepared as described in Example 1, was added into a mixture containing olive oil (0.9g) and lauric acid (0.6g). The reaction was carried out at 60°C for a period of time (typically 2-4 hours, depending on the lipase used). The sum peak area for the newly formed triglycerides is calculated as: peak area of new formed lauryl-containing triglycerides/total peak area of lauric acid and triglycerides X 100. The sum peak area value was determined as a measure for enzymatic interesterification activity.

All lipases used for interesterifcation reactions were 1,3 -positional specific lipases.

(c) Alcoholysis reaction of de-oiled phospholipids: Immobilized Lecitase novo on Eupergit C (lOOmg), prepared as described in Example 1, was added into a mixture containing de-oiled phosphohpids (200mg) and ethanol (20μl) dissolved in 2ml n-hexane that contains 20μl water. The reaction was carried out at 40°C for 16h. The conversion of phosphohpids was determined based on the peak area ratio obtained by HPLC analysis for phospholipids before and after reaction.

B. Hydrolytic activity:

The hydrolytic activity of hpase s was determined applying the most common routine assay that is performed in a thermostated reaction vessel containing the triglyceride tributyrin in phosphate buffer solution in the presence of bile acid salt (0.05%) while keeping the pH constant (by a pH stat). After addition of the enzyme the fatty acid (butyric acid) released with time is automatically titrated with NaOH solution (0.1M).

Example 4: The Esterification Activity of Covalently -Immobilized Lipase.

Tables 1 to 3 show the esterification activities of different immobilized lipases, prepared in the processes described in Examples 1 and 2:

Table 1: The esterification activity of Lipozyme TL 100L (Thermomyces Lanuginosa lanuginosus; Novozy es-Denmark) immobihzed on Eupergit C in different organic solvents with and without surfactant (SMO or SMS), before washing and after washing with water followed by acetone.

Esterification reaction conditions: Immobilized hpase (lOOmg), prepared as described in Examples 1 and 2, was added into n-hexane solution (1ml) containing lOOmg lauric acid and lOOmg lauryl alcohol. The reaction was carried out while shaking for a period of 2 hours, at 60°C.

Table 1 shows that Lipozyme TL 100L covalently-immobihzed on Eupergit C using buffer system in the immobihzation procedure, as recommended by the manufacturer, exhibits a very low activity. Covalently-immobihzed modified Lipozyme TL 100L (using SMO or SMS) under the same conventional immobilization procedure, did not result in a significant improvement in the esterification activity of the enzyme. Washing the formed immobilized enzyme particles with water and acetone did not affect the enzymatic activity, either. Of the various organic solvents used in the covalent-binding procedure, according to present invention, n- hexane and 2,2-dimethoxypropane were the most effective. More specifically, highly active immobihzed hpases were obtained when the immobihzation on Eupergit C was carried out in n-hexane. It can be seen that there is no positive effect when SMO or SMS are added during the immobilization procedure. Washing the immobilized enzyme preparation with water and then with acetone resulted in losing about half of the enzymatic activity compared to the non-washed immobilized enzyme preparation. This loss of activity can be attributed to removing the loosely, non-covalently, bound enzyme from the surface of the matrix and/or due to enzyme inactivation caused by treatment with acetone. The data demonstrates that immobihzation of the enzyme in the presence of acetone resulted in results comparable to those obtained in the conventional immobilization procedure, using buffer as a solvent.

The addition of SMO and SMS into the immobilization medium (acetone) led to no improvement in the esterification activity of the immobihzed enzyme. These results support the hypothesis that acetone might deactivate the covalently-bound Lipozyme TL 100L. Immobihzation of Lipozyme TL 100L on Eupergit C using 2,2-dimethoxypropane as a solvent resulted in good esterification activity. In this particular case, the addition of SMO or SMS into the solvent medium caused a significant increment-m the esterification activity of the immobilized enzyme. The data further demonstrates that washing the immobilized enzyme preparation (following immobilization process in 2,2-dimethoxypropane) with water and then with acetone led to a significant loss of the enzyme synthetic activity. In summary, the results in Table 1 reveal that covalent immobihzation of Lipozyme TL 100L in water-insoluble organic solvents, such as n-hexane and 2,2-dimethoxypropane, according to the present invention, provides a highly active immobihzed enzyme preparation, applicable in esterification reactions, whereas immobilization of the same hpase in water-soluble solvents such as, water (the conventional procedure), acetone and iso-propanol provides an immobilized enzyme of very low esterification activity. Using the surfactants SMO and SMS resulted in increasing enzymatic activity in some cases (2,2- dimethoxypropane and acetone) and decreasing enzymatic activity in others (n-hexane, and iso-propanol). In all cases, when organic solvent was used in the process of immobilization, washing of immobihzed enzyme preparation resulted in significant decrease in esterification activity. This negative effect of the washing procedure could be ascribed to either washing out of loosely bound enzymes from the surface of the matrix or to enzyme inactivation due to enzyme exposure to water-soluble solvents.

Table 2: The esterification activity of Lipase Saiken (Rhizopus, Nagase- Japan) immobilized on Eupergit C, in different organic solvents with and without surfactant (SMO or SMS), before washing and after washing with water followed by acetone.

Esterification reaction conditions: Immobihzed lipase (lOOmg), prepared as described in Examples 1 and 2, was added into n-hexane solution (1ml) containing lOOmg lauric acid and lOOmg lauryl alcohol. The reaction was carried out while shaking for a period of 2 hours, at 60°C.

Table 2 summarizes the results obtained with covalently immobilized hpase Saiken 100, (Rhizopus sp.) on Eupergit C using during the immobihzation procedure aqueous and various organic solvents, as far as an esterification reaction of lauryl alcohol and lauric acid in n-hexane system, is concerned. For covalent immobihzation of said hpase, a powdery form of crude Saiken 100 was added to the different solvent media. Following completion of the immobilization procedure the enzyme immobihzed on Eupergit C was sieved to remove the enzyme suspender, namely, the saccharide (lactose or starch), from the immobilized enzyme. The results indicate that most immobilized lipase preparations when applying a procedure that does not contain washing with water and acetone, possess low esterification activity. However, the same preparations show a dramatic increase in esterification activity when washed with water and acetone, in particular when the covalent immobilization was carried out in n-hexane, acetone, and in 2,2- dimethoxypropane. Immobilization of the same hpase on Eupergit C in buffer medium provides low esterification activity and this activity is not affected by washing with, acetone and water. Furthermore, Table 2 demonstrates that the type of organic solvent used in the immobilization of the enzyme on Eupergit is very important for yielding a biocatalyst possessing satisfying synthetic activity. Covalent immobihzation of lipase Saiken 100 in n-hexane yielded the most active biocatalyst. The results further demonstrate the positive role that the modifier (SMO and SMS) plays in facilitating the synthetic activity of the immobihzed enzymes. This is in particular true for n-hexane + SMS and, 2,2-dimothoxypropane + SMS. -

Table 3: The esterification activity of Novozym 525L (Candida Antarctica B, Novozymes-Denmark) immobilized on Eupergit C in different organic solvents with and without surfactant (SMO or SMS), before washing and after washing with water followed by acetone. Esterification reaction conditions: Immobilized lipase (lOOmg) prepared as described in Examples 1 and 2, was added into n-hexane solution (1ml) containing lOOmg Lauric acid and lOOmg Lauryl alcohol. The reaction was carried out while shaking for a period of 2 hours, at 60°C.

Table 3 shows that covalently-bound Novozym 525L on Eupergit C maintains high esterification activity when immobihzed in a conventional aqueous medium or in organic solvent, according to present invention. However, enzyme preparations immobilized in organic solvents, according to present invention, possess higher esterification^' activity compared to the ones immobihzed using the conventional technology. The data reveals that washing the immobilized enzyme preparation with water and acetone reduces its esterification activity only when the enzyme is covalently- immobihzed in the presence of iso-propanol. There is yet no clear explanation to this phenomenon.

Example 5: The Interesterification Activity of Covalently -Immobilized Lipase.

Tables 4 to 6 show the interesterification activities of different immobihzed lipases, prepared in the processes described in Examples 1 and 2:

Table 4: The interesterification activity of Lipozyme TL 100L (Thermomyces lanuginosus, Novozymes-Denmark) immobihzed on Eupergit C in different organic solvents with and without surfactant (SMO or SMS), before washing and after washing with water followed by acetone.

Interesterification reaction conditions: Immobilized lipase preparation (lOOmg), prepared as described in Examples 1 and 2, was added into a mixture containing ohve oil (0.9g) and lauric acid (0.6g). The reaction was carried out at 60°C for a period of 4 hours.

Table 4 shows that Lipozyme TL 100L, when covalently immobilized on Eupergit C using a buffer solution, according to the recommended conventional procedure, exhibits low intersterifcation activity. The addition of sugar fatty acid ester surfactant such as SMO or SMS improved significantly the interesterification activity of the immobilized enzyme. Washing the immobilized enzyme with water and acetone reduced the activity of both, surfactant-coated and non-surfactant coated lipase. Immobilization of Lipozyme TL 100L on Eupergit C in iso-propanol resulted in very low interesterification activity and the covalently-bound enzyme completely lost its activity when it was washed with water and then with acetone. Immobilization of Lipozyme TL 100L on Eupergit C in n-hexane + SMO resulted in a significant increase in interesterification activity compared to the same enzyme when immobihzed on Eupergit C in water + SMO. Table 4 further shows that addition of SMO or SMS to the medium during the immobilization process resulted in increasing the interesterification activity of the enzyme.

Table 5: The interesterification activity of Lipase Saiken (Rhizopus, Nagase-Japan) immobihzed on Eupergit C in different organic solvents with and without surfactant (SMO or SMS), before washing and after washing with water followed by acetone.

In contrast to covalently-bound Lipozyme TL 1001 (Table 4), the hpase Saiken 100 covalently-immobihzed on Eupergit C (Table 5) exhibits a significant increase in interesterification activity when washed with water and acetone. It is speculated that the powdery lipase Saiken does lack the adequate amount of water that other commercial lipases may have, which is probably required during the covalent-immobilization process on Eupergit C. The results show that washing immobihzed lipase Saiken on Eupergit C with water and acetone resulted in a significant increase in the interesterification, activity. These results indicate again the significance of water for this type of enzyme, in order to express its synthetic activity. The results further show that addition of SMO or SMS into the medium during lipase Saiken immobihzation improved significantly the interesterification activity of same enzyme especially when n-hexane, acetone and 2,2 dimethoxypropane were used as solvent in the immobihzation process.

Table 6: The interesterification activity of Lipase Novozym 525L (Candida Antarctica B, Novozymes-Denmark) immobihzed on Eupergit C in different organic solvents with and without surfactant (SMO or SMS), before washing and after washing with water followed by acetone. Interesterification reaction conditions: Immobilized lipase preparation (lOOmg), prepared as described in Examples 1 and 2, was added into a mixture containing ohve oil (0.9g) and lauric acid (0.6g). The reaction was carried out at 60°C for a period of 4 hours.

Table 6 shows that hpase Novozym-525L exhibits low interesterification activity when immobihzed on Eupergit C in n-hexane, acetone and 2,2- dimothoxypropane compared to no detectable inter-esterification activity when it is covalently-immobihzed in the conventional procedure using aqueous medium. Furthermore, washing with water and acetone resulted in loss of any detectable interesterification activity. The immobihzed Novozyme 525L prepared in buffer and in iso-propanol showed no detectable interesterification activity with and without surfactant modifier. The low interesterification activity for this type of lipase is expected since it is well known to catalyze more efficiently esterification reactions.

Example 6: The Operational Activity of Covalently -Immobilized Lipase in interesterification reactions .

Figures 1 to 3 show the operational stability of a covalently-immobihzed lipase throughout successive cycles of operation in interesterification of olive oil and lauric acid in solvent-free system.

Figure 1 presents the operational stability of different lipase (Lipozyme TL 100L) preparations immobilized on Eupergit C in (a) n-hexane; (b) n- hexane + SMO; (c) phosphate buffer solution (PBS); and (d) PBS + SMO in consecutive batch experiments.

Interesterification reaction conditions: Immobilized lipase preparation (lOOmg), prepared as described in Examples 1 and 2, was added into a mixture containing ohve oil (0.9g) and lauric acid (0.6g). The reaction, in each cycle, was carried out at 60°C for 4 hours.

Fig. 1 demonstrates the operational stability of different preparations of Lipozyme TL 100L covalently-immobihzed on Eupergit C, with and without SMO, in n-hexane and in buffer solution (PBS) throughout, up to 50 consecutive cycles using the same batch of enzyme. The data presented in Fig. 1 demonstrates that Lipozyme TL 100L immobilized on Eupergit C using n-hexane as a solvent has much higher interesterification activity compared to the same hpases when immobilized under the same conditions but in buffer solution. The enzyme immobilized in n-hexane was used without washing with water and acetone. Fig. 1 further demonstrates that immobilized Lipozyme TL 100L on Eupergit C in n- hexane + SMO medium exhibits the highest interesterification activity as well as the best operational stabihty. In addition, immobilized Lipozyme TL 100L on Eupergit C in n-hexane + SMO medium shows three times as much higher interesterification activity compared to the same enzyme when immobihzed in buffer solution + SMO. Immobilized Lipozyme TL 100L on Eupergit according to the recommended conventional protocol (using PBS) yielded low interesterification activity. Fig. 1 shows that all immobilized lipase preparations, excluding the one that is immobihzed in buffer solution without SMO, showed high interesterification activity in the first cycle, which sharply dropped in the second cycle. This phenomenon is attributed to washing out the loosely bound enzyme from the surface of the matrix during the reaction of the first cycle. The results further indicate that there is a steady loss in the enzymatic interesterification activity by all enzyme preparations throughout the cycles. This loss of activity might be attributable to the exhaustion of enzyme resulted from stripping of essential water molecules bound to the enzyme molecules.

Fig. 2 presents the operational stability of different lipase (Lipozyme TL 100L) preparations immobilized on Eupergit C 250L in (a) n-hexane; (b) n- hexane + SMO; (c) phosphate buffer solution (PBS); and (d) PBS + SMO in consecutive batch experiments.

Interesterification reaction conditions: Immobilized hpase preparation (lOOmg), prepared as described in Examples 1 and 2, wherein Eupergit C 250L substitutes Eupergit C, was added into a mixture containing olive oil (0.9g) and lauric acid (0.6g). The reaction was carried out at 60°C for 4 hours. Fig. 2 demonstrates the operational stabihty of covalently-immobihzed Lipozyme TL 100L on the highly porous epoxy resin, Eupergit C 250L in n-hexane compared to the same enzyme when immobilized on same matrix in buffer with and without SMO. All hpase preparations were used with no wash with water and acetone after immobihzation. Lipozyme TL 100L immobilized on Eupergit C 250L, with or without SMO, yielded high interesterification activity. The higher interesterification activity of lipases immobihzed on Eupergit C 250L compared to same lipases immobihzed on Eμpergit C is attributable to the high porosity of Eupergit C 250L. Both enzyme preparations showed a first order decrease in enzyme interesterification activity throughout the 45 cycles. As expected, immobihzed Lipozyme TL 100L on Eupergit C 250L in buffer medium + SMO exhibits substantial higher interesterification activity compared to the same hpase when immobilized under the same conditions, but without adding SMO. Fig. 2 further shows that the interesterification activity of both immobihzed lipases in buffer medium have sharply decreased after the first few cycles.

Fig. 3 presents the operational stabihty of different hpase (Lipozyme TL 100L) preparations immobilized on Eupergit C 250L in (a) acetone; and (b) acetone + SMO, in consecutive batch experiments.

Interesterification reaction conditions: Immobilized lipase preparation (lOOmg), prepared as described in Examples 1 and 2, with Eupergit C 250L instead of Eupergit C, was added into a mixture containing olive oil (0.9g) and lauric acid (0.6g). The reaction was carried out at 60°C for 4 hours. Fig. 3 shows the operational stability of immobilized Lipozyme TL 100L on Eupergit C 250L in acetone, with and without SMO. Immobilized enzyme preparations were used with no wash with water and acetone. It can be seen that Lipozyme TL 100L exhibits higher activity as well as higher stability when it is immobihzed in n-hexane in the presence of SMO (cf. Fig. 2). These results may indicate that SMO has a protecting role on enzyme activity once it is exposed to acetone or other enzyme-deactivating solvents. The interesterification activity of the immobihzed hpase in the absence of SMO dropped sharply after the first cycle, most likely because of washing out the unbound enzyme molecules adsorbed on the surface of the matrix. This may suggest that SMO enhances the covalent binding of enzymes ©nto the epoxy-support.

Example 7: Reactivation of the Interesterification Activity of Exhausted Covalently-Immobilized Lipase.

A well-known phenomenon for lipases is that they become exhausted after several cycles of use in operation. This phenomenon is in general attributed to the loss of crucial water molecules bound to the immobilized enzyme. Many research studies have failed to regenerate the activity of exhausted (deactivated) immobihzed lipases. In an effort to regenerate the interesterification activity of exhausted covalently-immobihzed lipase on Eupergit C 250L (as demonstrated in Fig. 2), following 46 cycles and loss of around 80% of the initial activity the exhausted immobilized lipase in the reaction system was treated with equal reaction volume (1.5ml) of phosphate buffer solution (PBS) of 0.1M (pH 7) and then the mixture was shaken for 12h at 60°C.

The covalently-immobihzed lipase-support complex was filtered, resuspended twice, each time with 1.5ml of phosphate buffer solution (pH 7), and optionally resuspended three times, each time with 1.5ml of acetone or n-hexane and then the immobilized enzyme was filtered, dried in a desiccator and reused.

Fig. 4 presents the interesterification activity of lipozyme TL 100L immobihzed on Eupergit C 250L through 45 cycles (see Fig. 2: n- hexane+SMO). At the 46^th cycle the exhausted immobilized enzyme was washed with buffer solution and then with n-hexane. Reaction conditions are as described for Fig. 2.

Fig. 4 describes the reactivation of interesterification activity of exhausted lipase (Lipozyme TL 100L) covalently-immobihzed on Eupergit C 250L. It demonstrates the interesterification activity of the exhausted lipase from Fig. 2 after the above described treatment procedure. The activity of the exhausted immobilized lipase that is covalently bound onto Eupergit C 250L is regenerated almost to its full initial activity by a simple procedure including washing with phosphate buffer solution and then filtering the immobilized enzyme and reusing it. These results indicate to the possibility that the loss of enzyme interesterification activity is most likely because of stripping some essential water from the enzyme molecules. This suggests that a certain water amount should be kept in the reaction system to avoid loss of enzyme activity.

Example 8: The effect of water on the operational stability of the covalently bound enzyme used in interesterification reactions.

Fig. 5 presents the operational stability of hpase (Lipozyme TL 100L) immobihzed on Eupergit C and on Eupergit C 250L in the presence of water. Reaction conditions: olive oil (0.9g), lauric acid (0.6g) and lOμl of water were incubated at 60°C for 2h using either Lipozyme TL 100L immobilized on Eupergit C in n-hexane + SMO or Lipozyme TL 100L immobilized on Eupergit C 250L in n-hexane + SMO (50mg each) using the same batch of biocatalyst for 18 consecutive cycles.

Fig. 5 shows the effect of water on the operational stabihty of Lipozyme TL 100L immobilized on Eupergit C and Eupergit C 250L. The results indicate that the presence of supplemented water in the reaction system plays a major role in determining the operational stabihty of the hpases immobihzed onto epoxy matrices. Fig. 5 reveals that addition of 0.6% water into the reaction system resulted in retaining the hpase interesterification activity throughout at least 18 successive cycles.

Example 9: Covalent-immobilization of a Lipase in the Presence of an Organic Solvent at a High Temperature (> 60°C).

Fig. 6 presents the operational stability of different lipase preparations (Lipozyme TL 100L) immobihzed on Eupergit C 250L in the presence of toluene (with and without SMO) at 70°C, in 10 successive cycles.

Interesterification reaction conditions: Immobilized hpase preparation (150mg), prepared as described in Examples 1 and 2, but applying 70°C instead of 60°C (without washing with water and acetone) was added into a mixture containing olive oil (0.9g) and lauric acid (0.6g). The reaction was carried out at 60°C for 4 hours.

Fig. 6 relates to covalent-immobilization of Liposyme TL 100L on an epoxy-support (Eupergit C 250L) in toluene at high temperature (70°C). Unexpectedly, the results show that covalent-binding (immobilization) of this particular enzyme (and may be other lipases, as well) according to the process of present invention can be carried out at high temperatures, such as 70°C. This finding is in contradiction "to other known enzyme covalent- immobihzation procedures that have been documented as being carried out at, or below, room temperature. The data further indicates the major effect which SMO exhibits in providing an immobihzed lipase possessing high interesterification activity. Fig. 6 demonstrates that highly active immobihzed lipas.e preparations were obtained when SMO was present during the immobilization of the enzyme, whereas the absence of SMO resulted in production of immobilized enzyme that exhibits very low interesterification activity. It may be suggested that the increment in the activity in the second cycle was due to structural changes in the pores of the matrix.

Tables 7 and 8 show the interesterification activity in three consecutive cycles using Lipozyme TL 100L immobilized on Eupergit C at different temperatures in iso-octane-containing medium as well as in aqueous medium as a control both in the presence of SMO. The enzyme was immobihzed as described in Example 1, but the enzyme incubation was carried out at different temperatures in the range of 25-70°C. Table 7 shows that the lipase Lipozyme TL 100L was successfully covalently immobihzed at different temperatures in the range of 25-70 to give immobilized hpase preparations equally active for interesterification reactions. It can be also seen that the lipase preparations retained their approximately full activity in three consecutive cycles, in particular that which was prepared at 70 °C. The results presented in Table 8 show that the same hpase, however immobihzed in aqueous medium, gave lower interesterification activity and there was a significant loss of activity in the third cycle. Further more, the results show also that when the hpase was immobilized at 70 °C in aqueous medium its interesterification activity dropped sharply, apparently due to the thermal inactivation of the enzyme during the immobihzation procedure.

Table 7: The interesterification activity of Lipozyme TL 100L immobihzed on Eupergit C in iso-octane-containing medium in the presence of SMO at different temperatures in the range of 25-70 °C.

Reaction conditions: A mixture of ohve oil (0.9g) and lauric acid (0.6g) was mixed with Lipozyme TL 100L immobilized on Eupergit C (lOOmg) at 60°C for 2h.

Table 8: The interesterification activity of Lipozyme TL 100L immobihzed on Eupergit C in aqueous medium in the presence of SMO at different temperatures in the range of 25-70 °C. Reaction conditions: A mixture of olive oil (0.9g) and lauric acid (0.6g) was mixed with Lipozyme TL 100L immobilized on Eupergit C (lOOmg) at 60°C for 2h.

Example 10: Covalent immobilization of enzymes in bi-phase, micellar and in reverse micellar systems.

An important embodiment of the present invention relates to covalent immobilization of enzymes confined in a micellar or reverse micellar systems. More particularly, the process of present invention also provides a new process for covalently-binding of enzymes, in particular interfacial enzymes, such as lipases in dispersion, in emulsion system, micellar system or in reverse micellar system (Table 7). The amount of said organic solvent in said bi-phase and emulsion systems is in the range of from 10wt% to 99.9wt%, preferably from 30 wt% to 99wt% and most preferably from 50wt% to 95wt%. The immobilization procedure is carried out by adding the epoxy-support in an organic solvent into an enzyme confined in a reverse micellar system, micellar system or in a dispersed system; followed by shaking for 48h at room temperature or higher, as described above. The suspension is filtered and the cake is washed either with the organic phase or with water and optionally with acetone and then dried in a desiccator.

(i) Lipase immobilization in a reverse micellar system (water/oil): An aqueous solution of commercial Novozym 388 (2ml) was added to a solution of iso-octane (10ml) that contained the surfactant bis(2- ethylhexyl) sulfosuccinate sodium salt (lOOmg, AOT, Fluka). The solution was vigorously stirred to produce reverse micellar (water in oil) lipase system. Epoxy-matrix (lg Eupergit C 250L) was added into the reverse micellar hpase solution and the system was shaken for 48h at room temperature. The suspension was filtered and the cake was washed with iso-octane. The covalently-immobihzed lipase was dried over night in a desiccator.

(ii) Lipase immobilization in a micellar system (oil in water): An emulsion system of a commercial lipase Novozym 388 was prepared by adding the enzyme (2ml) into a mixture of 10ml phosphate buffer of 0.1M (pH 6) and 2ml iso-octane that contains lOOmg SMO. For enzyme immobihzation an amount of lg Eupergit C250L was added into the emulsion system. The system was shaken for 48h at room temperature and then filtered. The cake was washed with iso-octane and dried in a desiccator.

(iii) Immobilization of lipases in a bi-phase system: The same Novozym 388 preparation was prepared as described in (ii) above, however, in this case the immobilization process was carried out in a bi-phase system (a surfactant was not added) that consisted of iso-octane (10ml) Novozym 388 (2ml) and Eupergit C 250L (lg) to serve as a control (as far as activity of the surface-active ingredient, is concerned) to the above two experiments. The mixture was shaken for 48h, filtered and the cake was washed with iso-octane and then dried in a desiccator.

(iv) Immobihzation of lipase in aqueous system: The same Novozym 388 was covalently-immobihzed on Eupergit C 250L applying 1M phosphate buffer solution of pH 6, as recommended by the matrix manufacturer. This conventionally-immobilized enzyme preparation served as a second control system.

Table 9 shows the interesterification activity of covalently-immobihzed Novozyme 388 on Eupergit C 250L preparations, as described in (i) to (iv) above.

Table 9: The interesterification activity of immobilized lipase Novozym 388 on Eupergit C 250L, wherein the immobilization process was carried out in a biphase, reverse micellar, emulsion and in buffer systems.

Interesterification reaction conditions: Immobilized hpase preparation (lOOmg), prepared as described in (i) to (iv) above, was added to a mixture containing olive oil (0.9g) and lauric acid (0.6g). The reaction was carried out at 60°C for a period of 1 hour.

Table 9 shows that the enzyme lipase Novozym 388 when immobilized according to the recommended conventional procedure (cf. (iv) hereinabove) yielded, as expected, low interesterification activity. However, the same hpase when immobilized onto Eupergit C 250L in a biphase system without the addition of any surface-active ingredient (cf. (iii) hereinabove), yielded an immobihzed enzyme possessing about sevenfold higher interesterification activity. This data supports the assumption that providing a surface -active ingredient during immobilization of a lipase significantly facilitates the yield of an active immobilized biocatalyst. It is speculated that during the covalent-immobilization process of the lipase the surface-active material plays a role in adapting the lipase desired conformation (open-lid conformation) that could be coupled covalently to the epoxy-resin to yield an active biocatalyst. The data presented in Table 9 regarding immobihzation of the same lipase in an emulsion system, in the presence of SMO as a surfactant (cf. (ii) hereinabove), or in a reverse micellar system (cf. (i) hereinabove) support the above hypothesis. In both cases, a surfactant was present in the immobihzation procedure, resulting in increasing the interface between the organic and the aqueous phases, and consequently, a biocatalyst of higher interesterification activity was obtained. Example 11: The Hydrolytic Activity of a Covalently -Immobilized Lipase.

The hydrolytic activity of covalently-immobihzed lipases on epoxy- supports in an emulsion system has been also tested following the immobihzation as follows:

Lipase OF dissolved in 0.05M phosphate buffer solution of pH 7 (1ml buffer contains 67mg lipase OF; Mieto Sangyo, Japan) was added to a solution of n-hexane (15ml) containing SMO (150mg). The mixture was shaken for 70 h with Eupergit C 250L (lg) and then was optionally treated with 25% aqueous solution of glutaraldehyde (0.5ml) for 6h. The medium was filtered and the Eupergit particles were washed with n-hexane.

A control procedure was carried out to immobilize the same hpase on Eupergit C 250L using 1M phosphate buffer solution as recommended by the manufacturer of the matrix with and without cross-linking with glutaraldehyde. The glutaraldehyde was added for cross-linking the enzyme in order to minimize enzyme leakage in case it was not covalently bound to the epoxy resin matrix

Fig. 7 presents the hydrolytic reaction profile using immobilized Lipase OF (Candida rugosa) on Eupergit C 250L in n-hexane in the presence of SMO with and without cross-linking with glutaraldehyde, compared with the same lipase when immobihzed on the same matrix in 1M phosphate buffer solution (pH 7) in the presence of SMO with and without cross- linking with glutaraldehyde, and crude enzyme preparation. Hydrolytic reaction conditions: Phosphate buffer solution of 0.05M (15ml) containing bile acid salts (0.05%) and tributyrin (0.25ml) at pH 7.3 was added to an appropriate amount of immobilized lipase preparation. The mixture was stirred while keeping the pH constant at 7.3 by a pH stat at room temperature.

The data indicates that lipase OF covalently-immobihzed on Eupergit C 250L in the presence of organic solvent, such as n-hexane, exhibited higher hydrolytic activity compared to the same crude non-immobihzed hpase OF. The results further demonstrate that cross-linking the immobilized hpase in organic solvent with glutaraldehyde resulted in reducing the hydrolytic activity. Fig. 7 further reveals that hpase immobilized on Eupergit C 250L in buffer system according to the recommended conventional procedure, in the presence of SMO, yielded a biocatalyst having much lower hydrolytic activity compared to the same lipase when immobihzed in n-hexane system. Consequently, the data indicates that lipases when covalently-immobihzed according to the process of present invention would exhibit higher hydrolytic activity compared to the hydrolytic activity of the same lipases when immobihzed in the conventional procedure, using a buffer system.

It should be further mentioned that lipase OF covalently-immobihzed on Eupergit C 250L in n-hexane + SMO was used in 6 consecutive cycles with a minor loss of hydrolytic activity.

Example 12: Covalent immobilization of phospholipases onto epoxy- containing polymeric matrix.

Lecitase novo, a microbial phospholipase Al, was immobihzed on Eupergit C according to the procedure described in Example 1, however using acetone as an organic solvent. A control preparation for Lecitase novo immobihzed on Eupergit C in aqueous solution in the presence of SMO was also prepared. Table 10 shows the alcoholysis results of de-oiled phospholipids with ethanol both dissolved in n-hexane in the presence of covalently-immobihzed Lecitase novo. The alcoholysis activity of both enzyme preparations was tested in three consecutive cycles using the same batch of enzyme. The Lecitase immobihzed in an organic medium according to the process of present invention demonstrated higher alcoholic activity. Furthermore, this high activity remained unchanged following three consecutive cycles. On the other hand, Lecitase immobihzed according to the conventional procedure in an aqueous system yielded immobilized enzyme preparation demonstrating a reasonable enzyme activity. However, this activity was totally lost after one batch (or cycle) of operation. It is speculated that this loss of activity resulted from detachment of enzyme molecules which are not covalently-firmly bound to the matrix.

Table 10: The alcoholysis activity in three consecutive cycles of Lecitase novo immobihzed on Eupergit C in acetone medium compared to the same enzyme when immobilized in aqueous medium. .

Reaction conditions: De-oiled phosphohpids (200mg), ethanol (20μl), water (20μl) and Lecitase novo immobilized on Eupergit C (200mg). The reaction was carried out for 16h under stirring at 40°C.

Example 13: Covalent-immobilization of Enzymes in Organic Solvents for Applications in Aqueous Medium.

The novel covalent-immobihzation process according to the present invention is applicable for covalent-binding of other types of enzymes, including ones that require aqueous medium or a biphase system for catalytic operation. As an example, the enzyme invertase which catalyzes the inversion of sucrose to glucose and fructose in an aqueous medium is demonstrated. For covalent-binding of invertase onto Eupergit C the same procedure as described in Example 1 was used. Several different immobihzed invertase preparations (prepared in various organic solvents), were obtained. Following the immobihzation in organic solvents cross- linking with glutaraldehyde was optionally carried out in the same solvent in order to reduce leakage of loosely bound enzyme and also for stabihzing the immobihzed enzymes of high molecular weight.

Invertase (1ml of Bioinvert L, Quest, England) was added into a mixture of an organic solvent (15ml; see Table 11) that contains Eupergit C (lg). The mixture was shaken for 72h at room temperature, and then filtered. The immobilized enzyme- Eupergit particles were washed with same solvent.

Table 11 demonstrates the inversion activity of different preparations of the invertase immobilized on Eupergit C. The data shows that covalent immobihzation of invertase onto Eupergit C in an organic solvent, such as diethyl ether and n-hexane, according to the process of present invention, resulted in obtaining an immobilized invertase possessing 1.5- to 2.5-fold higher inversion activity compared to the activity of invertase when immobihzed on the same ' epoxy matrix applying the recommended conventional covalent binding procedure using buffer solutions. These results indicate that covalent-immobilization of an enzyme on epoxy matrices applying the process of present invention yields a biocatalyst possessing higher enzymatic activity in either an aqueous or non-aqueous systems, compared to the same enzyme when covalently bound to same epoxy-matrix applying the conventional immobilization procedure using a buffer solution (as recommended by the producer of the epoxy-matrix).

Table 11: The inversion activity of different preparations of immobihzed invertase on Eupergit C (lOOmg) in different media, using 2g of aqueous sucrose solution (60 wt%) at 60°C for a period of 15min.

Claims

1. A process for immobihzing a compound containing a functional group selected from the group consisting of amine, carboxyl, hydroxyl, thiol and/or other nucleophilic groups onto an insoluble polymeric matrix containing epoxy groups wherein said process comprises covalently binding said compound onto said insoluble polymeric matrix in a medium containing an organic solvent, preferably in the presence of water and/or a surface -active material.

2. A process for immobihzing a compound according to claim 1, wherein the compound is a proteinous biocatalyst.

3. A process according to claim 2, wherein the biocatalyst is in a solid form.

4. A process according to claim 2, wherein the biocatalyst is suspended in a solution.

5. A process according to claim 4, wherein the solution is an aqueous solution.

6. A process according to claim 2, wherein the biocatalyst is confined in a reverse micellar system which is a water in oil (W/O) system, or in micellar system which is oil in water (O/W).

7. A process according to any one of claims 2 to 6, wherein the biocatalyst is an enzyme.

8. A process according to any one of claims 1 to 7, wherein the insoluble polymeric matrix is an acryhc polymer containing epoxy groups.

9. A process according to claim 8, wherein the acrylic polymer is any one of Eupergit G 250L, containing at least 200μmol of epoxy groups per gram polymer and Eupergit C, containing at least βOOμmol of epoxy groups per gram polymer.

10. A process according to claim 1, wherein said organic solvent is selected from the group consisting of n-hexane, 2,2-dimethoxypropane, acetone, diethyl ether, iso-propanol, toluene, iso-octane and mixture thereof.

11. A process according to claim 7, wherein the enzyme is a lipase or phospholipase.

12. A process according to claim 11, wherein the lipase is obtained from a microorganism selected from the group consisting of Thermomyces lanuginosus, Candida antarctica B, Rhizomucor miehei, Rhizopus strains, Rhizopus oryzae and Candida rugosa.

13. A process according to claim 7, wherein the enzyme is a saccharidase.

14. A process according to claim 13, wherein the saccharidase is an invertase.

15. A process according to any one of claims 2 to 14, wherein the covalent-immobihzation process is carried out in a medium comprising the said proteinous biocatalyst, the said insoluble polymeric matrix containing epoxy groups and the said organic solvent, wherein said organic solvent is mixed with water and a surface active ingredient to form an emulsion.

16. A process according to claim 15, wherein the amount of said organic solvent in said emulsion is in the range of from 10wt% to 99.9wt%, preferably from 30 wt% to 99wt% and most preferably from 90wt% to 95wt%.

17. A process according to any one of claims 2 to 14, wherein the covalent-immobilization process is carried out in a medium comprising the said proteinous biocatalyst, the said insoluble polymeric matrix containing epoxy groups and the said an organic solvent, wherein said organic solvent is water-insoluble and is mixed with water to form a bi-phase system.

18. A process according to claim 17, wherein the amount of said organic solvent in said bi-phase system is in the range of from 10wt% to 99.9wt%, preferably from 30wt% to 99wt% and most preferably from 90wt% to 95wt%.

19. A process according to claim 3, wherein the said proteinous biocatalyst is in solid form and the covalent-immobilization process is carried out in a medium comprising the said insoluble polymeric matrix containing epoxy groups and the said organic solvent, preferably in the presence of water and/or surface -active ingredient.

20. A process according to claim 3, wherein the biocatalyst is suspended in a solution and the covalent-immobihzation process is carried out in a medium comprising the said insoluble polymeric matrix containing epoxy groups and the said organic solvent, preferably in the presence of water and/or surface -active ingredient.

21. A process according to claim 1, wherein the covalent-immobilization process is carried out in a medium containing a surface-active material.

22. A process according to claim 21, wherein the surface-active material is a polyol ester surfactant.

23. A process according to claim 22, wherein the surface-active material is selected from the group consisting of polyol fatty acid esters, such as sorbitan monooleate (SMO) and sorbitan monostearate (SMS).

24. A process according to claim 1 and any one of claims 21 to 23, wherein the biocatalyst is coated with said surface-active material before, during or after the covalent-immobilization process.

25. A process for immobihzing a compound containing a functional group selected from the group consisting of amine, carboxyl, hydroxyl, thiol and/or other nucleophilic groups, onto an insoluble polymeric matrix containing epoxy groups, according to claim 1, comprising: (i) contacting said compound with a medium comprising an insoluble polymeric matrix containing epoxy groups suspended in an organic solvent, preferably in the presence of water and/or a surface-active material;

(iii) separating the complex consisting of said compound covalently- immobihzed on said insoluble polymeric matrix; and optionally

(iv) washing the separated complex with water followed by washing with acetone.

26. A process according to claim 25, wherein the compound is a proteinous biocatalyst.

27. A process according to claim 26, wherein the proteinous biocatalyst is an enzyme.

28. A process according to any one of claims 25 to 27, wherein said organic solvent employed in step (i) is water-insoluble and is mixed with water and a surface active ingredient to form an emulsion.

29. A process according to claim 28, wherein the amount of said organic solvent in said emulsion is in the range of from 10wt% to 99.9wt%, preferably from 30 wt% to 99wt% and most preferably from 50wt% to 95wt%.

30. A process according to any one of claims 25 to 27, wherein said organic solvent employed in step (i) is water-insoluble and is mixed with water to form a bi-phase system.

31. A process according to claim 30, wherein the amount of said organic solvent in said bi-phase system is in the range of from 10wt% to 99.9wt%, preferably from 30wt% to 99wt% and most preferably from 50wt% to 95wt%.

32. A process according to any one of claims 25 to 31, wherein the biocatalyst in step (i) is in sohd form.

33. A process according to any one of claims 25 to 31, wherein the biocatalyst in step (i) is suspended in a solution.

34. A process according to claim 33, wherein the biocatalyst in step (i) is suspended or dissolved in an aqueous solution.

35. A process according to any one of claims 25 to 31, wherein the biocatalyst in step (i) is confined in a reverse micellar system.

36. A process according to any one of claims 25 to 35, wherein the medium of step (i) contains a surface -active material.

37. A process according to claim 36, wherein the surface -active material is a polyol ester surfactant.

38. A process according claim 37, wherein the surfactant is selected from the group consisting of sorbitan fatty acid esters, typically, sorbitan monooleate (SMO) and sorbitan monostearate (SMS).

39. A process according to any one of claims 36 to 38, wherein the biocatalyst is coated with said surface-active material before, during or following step (i).

40. A process according to any one of claims 25 to 39, wherein step (ii) is carried out in the presence of glutaraldehyde.

41. A process according to any one of claims 27 to 40, wherein the enzyme is a lipase or a phosphohpase.

42. A process according to claim 41, wherein the lipase is obtained from a microorganism selected from the group consisting of Thermomyces lanuginosus, Candida antarctica B, Rhizomucor miehei, Rhizopus strains, Rhizopus oryzae and Candida rugosa.

43. A process according to any one of claims 27 to 40, wherein the enzyme is a saccharidase.

44. A process according to claim 43, wherein the saccharidase is an invertase.

45. A process for reactivating an exhausted biocatalyst covalently- bound onto an insoluble polymeric matrix containing epoxy groups, referred to as "covalently-immobihzed biocatalyst complex", prepared according to claim 1, comprising the steps:

(ii) separating said covalently-immobihzed biocatalyst complex; and optionally

46. A process according to any one of the preceding claims, for preparing a lipase or phospholipase covalently-immobihzed onto an insoluble polymeric matrix which contains epoxy groups, said immobihzed hpase or phospholipase being intended for catalyzing either synthetic reactions, in which a new ester group is formed and/or hydrolytic reactions, in which an ester group is cleaved.

47. A process according to claim 46 for preparing a lipase or phospholipase covalently-immobihzed onto an insoluble polymeric matrix, said immobihzed lipase or phospholipase being intended for catalyzing an interesterification or transesterification reaction.

48. A process according to claim 46 for preparing a hpase or phosphohpase covalently-immobihzed onto an insoluble polymeric matrix, said immobihzed lipase or phospholipase being intended for catalyzing an esterification reaction.

49. A process according to claim 46 for preparing a lipase or phospholipase covalently-immobihzed onto an insoluble polymeric matrix, said immobilized lipase or phospholipase being intended for catalyzing a hydrolytic reaction.

50. A stabilized and activated immobilized enzyme preparation prepared by the process of any one of claims 1 to 10 and 15 to 40.

51. A stabihzed and activated immobilized lipase or phosphohpase prepared by the process of any one of claims 46 to 49.