JP2002539782A - Surfactant-lipase complex immobilized on an insoluble matrix - Google Patents

Abstract

  (57) [Summary] A lipase preparation comprising an insoluble matrix and a surfactant-coated lipase complex immobilized on the insoluble matrix. Methods for the preparation and use of this new product are disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an insoluble matrix-immobilized surfactant-coated lipase complex, a method for preparing the same, and a biocatalyst for catalyzing the transesterification and / or transesterification of oils and fats in a hydrophobic organic medium. The use of the above conjugates as This new procedure involves two steps. In a first step, the enzyme is activated by coating it with a detergent. In a second step, the enzyme is immobilized on a special matrix. These steps can be performed in any order.

[0002]

[Prior art]

Enzymatic modification of the structure and composition of fats and oils is of great industrial and clinical interest. This method is achieved by utilizing a site-specific lipase in a transesterification and / or transesterification reaction using an oil or fat as a substrate (
Macrea, AR, 1983, J. Am. Oil Chem. Soc. 60: 291-294).

[0003] Whereas enzymatic methods allow the incorporation of a desired fatty acyl group at a specific position on a triacylglycerol molecule, conventional chemical transesterification has no site specificity. Heretofore, sodium metal, sodium alkoxy, or cobalt chloride, which catalyzes the acyl transfer between triglyceride molecules, has facilitated the chemical reaction, resulting in the formation of triglycerides with randomly distributed fatty acyl residues (Erdem-Senatalar, A., Erencek). , E. and Erciyes, AT, 1995, J. Am. Oi
l Chem. Soc. 72: 891-894).

[0004] Recently, a number of studies have demonstrated the potential application of lipases as potential biocatalysts for various esterification reactions in organic media (Wisdom, RA, Duhill. P.,
And Lilly, MD, 1987, Biotechnol. Bioeng. 29; 1081-1085).

[0005] 1,3-position specific lipase mainly catalyzes the hydrolysis of fats and oils to produce free fatty acids and glycerol. However, recent studies have also shown that 1.3-position specific lipases can catalyze two types of esterification reactions in trace aqueous organic media (Quinlan, P. & Moore, S., 1993). , INFORM 4: 580-585). The first of these reactions is a transesterification or acidolysis reaction, where free fatty acids react with various triglycerides to produce new triglyceride molecules. The second type of reaction is an ester displacement reaction, where two heterologous triglyceride molecules react to give a new triglyceride molecule (see FIGS. 1a-b). In both of these enzymatic reactions, the sn-2 position of the reacting triglyceride remains unchanged.

In general, water concentration plays an important role in determining enzyme activity. Water concentration also affects the parallel state of the reactions performed in hydrophobic organic solvent media (Valiverty, R
.H., Halling, PJ and Macrae, AR, 1993, Biotechnol. Lett.l 15: 113
8-1138). Since water is essential for enzymatic activity in both hydrolysis and synthesis reactions, a compromise between triglyceride hydrolysis and synthesis is to reduce water concentrations to minimize unwanted reactions, while effectively using water. Is sufficient to maintain enzyme activity.

At high water concentrations, for example above 5% by weight of the solvent weight, the lipases preferably retain their natural hydrolytic activity and the hydrolysis reaction proceeds. However, at low water concentrations, eg, less than 1% of the weight of the solvent, the lipase catalyzes a reverse or synthetic reaction.

[0008] A typical water concentration range required to promote the transesterification reaction between different fatty oils in an organic medium is 1 to 10% by weight of the hydrophobic organic solvent. Generally, this water concentration can also facilitate the hydrolysis reaction, so that undesired partial glycerides (mono and diglycerides) are formed as by-products in the range of 10-20% by weight of the initial triglyceride concentration.

[0009] The range of utilizing the positional specificity of lipase is particularly large in the food industry and the oleochemical industry that produce high value-added special fats. For example, cocoa butter replacer, pseudo-human milk fat and other structured triglycerides of specific nutritional quality are
, 3-enzyme can be obtained enzymatically using regiospecific lipase (Vulfson, EN, 1993,
Trends Food Sci. Technol. 4: 209-215).

For the foregoing, there is a need to develop novel structured triglycerides having both medium chain and α-3 polyunsaturated fatty acids without the adverse effects of naturally occurring α-3 polyunsaturated fatty acids or saturated fatty acids. There is. For example, an MCT molecule having one of the acyl groups substituted with an essentially long chain fatty acid would provide the nutritional benefits of both MCT and LCT. This approach is illustrated by the very useful triglycerides, which contain acyl forms of the polyunsaturated fatty acids EPA, DHA or α-linolenic acid with medium chain fatty acyl groups at the sn-1 and sn-3 positions. Formed by incorporation into the sn-2 position of a triglyceride molecule having
, 1997, J. Nutr. 127: 1061].

[0011] The polyunsaturated fatty acids incorporated into the triglyceride molecule may be used for cardiovascular diseases,
It has been found that it has several health benefits with respect to immunodeficiency and inflammation, allergy, diabetes, kidney disease, depression, disability and cancer. Moreover, medium-chain fatty acids incorporated into the same triglyceride molecule are even more important in certain clinical applications, especially in applications to facilitate serum cholesterol absorption and dissolution, and for body consumption. It is even more important in applications for providing energy sources that can be provided to the public.

A number of different approaches to using lipase in organic media have been attempted for lipase activation and performance improvement. These include the use of lipase powder suspended in either a trace aqueous organic solvent or a two-phase system, and the use of natural lipase adsorbed on a porous matrix in fixed and fluidized bed reactors. (Malcata et al. 1990, J. Am. Oil Chem. Soc. 890-910). Moreover, lipases are accepted in reverse micelles, and in some studies, lipases have been attached to polyethylene glycol or hydrophilic residues in order to enhance the solubility and dispersibility of the lipase in organic solvents.

[0013] None of the above approaches has been found to be applicable to all enzyme systems. However, in many cases, some treatment of the enzyme as described above improved the lipase's performance with respect to lipase activity, specificity, stability and dispersibility in hydrophobic organic systems.

[0014] Recent studies have reported the development of surfactant-coated lipase preparations (eg, Basheer, S., Mogi, K. and Nakajima. M., 1995, Biotechnol. Bioeng.
45: 187-195). This enzymatic modification converts a slightly active or completely inactive lipase to a highly active biocatalyst for the esterification of triglycerides with fatty acids in organic media. This newly developed surfactant-lipase complex has been further studied,
It has been used in organic solvent-based transesterification reactions to produce more important structured triglycerides for pharmaceutical applications (Tanaka. Y., J. and Funada, T., 1994, J.
Am. Oil Chem. Soc. 71: 331-334).

In another approach to this problem, various immobilized enzyme reactors have been used for lipase-catalyzed reactions in trace aqueous hydrophobic organic media (eg, Basheer. S., Mogi,
k., Nakajima, M., 1995, Process Biochemistry 30: 531-536). These devices include fluidized bed reactors and slurry reactors. In the disclosed study,
Lipase immobilized on an inorganic matrix was used for both batch reaction systems and fixed bed bioreactor systems. However, the lipase used was not coated with detergent and therefore had the same limitations as the lipase-free system. These limitations include: 1. Difficulty in enzyme recovery after process completion; 2. a rapid decrease in the activity of free lipase in the reaction medium; 3. The problem of recovery of expensive enzymes; Low synthetic activity of free lipase in organic solvents;

[0016]

[Problems to be solved by the invention]

None of the above strategies has satisfactorily solved the technical problems encountered in managing transesterification and transesterification reactions of fats and oils. Accordingly, an object of the present invention is to provide a lipase preparation which can catalyze the esterification reaction of fats and oils with higher efficiency than the conventional method.

It is another object of the present invention to provide lipase preparations that incorporate both immobilization to a matrix and treatment by coating with a surfactant.

It is a further object of the present invention to provide a lipase preparation that can withstand repeated use on an industrial scale with minimal loss of activity.

A further object of the present invention is to provide a method for preparing the insoluble matrix-immobilized surfactant-coated lipase complex.

It is a further object of the present invention to provide a method for preparing a structured triacylglycerol using the surfactant-coated lipase complex immobilized on the insoluble matrix.

[0021] Other objects and advantages of the invention will become apparent as the description proceeds.

[0022]

Overview SUMMARY OF THE INVENTION The invention this time (1) a surfactant coating, and (2) Immobilization of insoluble matrix; is by double modification of crude lipase catalyzes the ester-substituted and transesterification It has unexpectedly been found to improve the efficiency of the enzyme in a synergistic manner compared to either of these two treatments alone, and this is one object of the present invention. Furthermore, it has been unexpectedly found that providing the enzyme preparation in particulate form can enhance the catalyst stability for the esterification reaction of the double-modified lipase.

The present invention is primarily directed to a lipase preparation comprising an insoluble matrix and a surfactant-coated lipase complex immobilized on the insoluble matrix.

[0024] Immobilization of the lipase complex on the insoluble matrix can be accomplished in several different ways. However, according to a preferred embodiment of the present invention, the detergent-coated lipase complex is covalently, ionically or physically bound on an insoluble matrix.

The present invention encompasses a number of matrix types, these matrices being selected from the group consisting of inorganic and organic insoluble matrices.

In a preferred embodiment of the present 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 ion exchange resin can be of any suitable material, but in a preferred embodiment is selected from the group consisting of Amberlite and Dowex.

While any suitable organic insoluble matrix can be used, in a preferred embodiment, the organic insoluble matrix is selected from the group consisting of Eupergit, ethylsulfoxylulose and aluminum stearate.

In a preferred embodiment, the lipase content is between 2 and 20% by weight of the detergent-coated lipase complex. In a more preferred embodiment, the lipase content is between 0.01 and 1.0% by weight of the preparation.

The present invention provides the above lipase preparation, wherein the surfactant in the surfactant-coated lipase conjugate comprises a fatty acid attached 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, trimiristate, tripalmitate and tristearate. You. In a preferred embodiment, the hydrophilic moiety is
It is selected from the group consisting of sugars, phosphate groups, carboxyl groups and hydroxylated organic residues. In a more preferred embodiment, the sugar is selected from the group consisting of sorbitol, sucrose, glucose and lactose. The fatty acid and the hydrophilic moiety can be linked by any suitable type of bond, but in this case, the fatty acid and the hydrophilic moiety are linked via an ester bond.

Although the lipase can 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 can be used as lipase sources for the lipase preparations of the present invention. However, the present invention relates to Burkholderia sp., Candida antractica B, Candida ru.
gosa, Pseudomonas sp., Candida antractica A, Porcine pancreas lipas
e, Humicola sp., Mucor miehei, Rhizopus javan., Pseudomnas fluor., C
andida cylindrcae, Aspergillus niger, Phizopus oryzae, Mucor javanic
The invention is particularly directed to the use of a lipase derived from a species selected from the group consisting of Rhizopus sp., Rhizopus japonicus and Candida antractica.

In a further aspect, the present invention is directed to a lipase preparation, which comprises an insoluble matrix and a surfactant-coated lipase complex immobilized on the insoluble matrix, wherein the lipase complex is dissolved in an organic solvent. Provided to In a preferred embodiment, the organic solvent is selected from the group consisting of n-hexane, toluene, isooctane, n-octane, benzene, cyclohexane and diisopropyl ether. The present invention is further directed to the use of the above lipase preparations as catalysts for the esterification, transesterification and transesterification of fats and oils and alcoholysis of triglycerol and fatty alcohols. In a preferred embodiment, the lipase preparation is used as a catalyst with 1,3-position specificity for triacylglycerol.

In another aspect, the invention is directed to a lipase preparation as described above, wherein the preparation is in particulate form.

The present invention is directed to a lipase preparation as described above, wherein the insoluble matrix has been modified with a fatty acid derivative.

In a further aspect, the present invention is directed to an enzyme preparation as described above, which preparation is for use in a reaction environment without the need for the addition of water.

The present invention also encompasses a method of improving the stability of a surfactant-coated immobilized lipase conjugate, which includes granulating the conjugate prior to contacting with a treatment substrate.

In a further aspect, the present invention provides a method for preparing an insoluble matrix-immobilized surfactant-coated lipase complex, the method comprising the following steps in any order: (a) lipase in an aqueous medium Contacting the surfactant with the surfactant field at a constant concentration and at a constant temperature for a period of time sufficient to obtain a coating of the lipase; and (b) contacting the lipase in an aqueous medium at a constant concentration with the matrix. Contacting with an insoluble matrix under conditions and over a time period sufficient to achieve immobilization of the lipase thereon.

In a preferred embodiment of the above method, the lipase is first contacted with an insoluble matrix and then with a detergent. In another preferred embodiment of the invention, the lipase is first contacted with a detergent and then with an insoluble matrix.

In a preferred embodiment of the invention, the method further comprises separating the matrix-immobilized surfactant-coated lipase complex from the aqueous solution in which it was formed. In an even more preferred embodiment, the method further comprises the step of drying the matrix-immobilized surfactant-coated lipase complex. This drying step can be accomplished in any conventional manner, but in a preferred embodiment the drying is performed by lyophilization. In another preferred embodiment, the matrix-immobilized surfactant-coated lipase complex is dried to a water content of less than 100 ppm by weight.

[0039] In another preferred embodiment, the aqueous solution used in the above method is a buffered aqueous solution.

In yet another preferred embodiment of the above method, the lipase and the surfactant are contacted in an aqueous medium by the following method: (i) dissolving the surfactant in an organic solvent and dissolving the surfactant Obtaining a solution, and further (ii) mixing the lipase and the dissolved surfactant solution in the aqueous medium.

In another preferred embodiment, the method further comprises sonication of the aqueous solution.

In yet another preferred embodiment of the method according to the invention, the insoluble matrix is alumina, diatomaceous earth, celite, calcium carbonate, calcium sulfate, ion exchange resins, silica gel and charcoal, Eupergit, ethylsulfoxycellulose, It is selected from the group consisting of aluminum stearate, and celite or other inorganic matrices treated with fatty acid derivatives.

In another preferred embodiment, the surfactant of the method of the invention comprises a fatty acid attached to a hydrophilic site. In a more preferred embodiment, the fatty acid is monolaurate, monomyristate, monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate, trilaurate,
Selected from the group consisting of trimiristate, tripalmitate and tristearate

In another preferred embodiment, the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxyl 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.

In another preferred embodiment of the present invention, the fatty acid and the hydrophilic moiety are linked via an ester bond.

In a preferred embodiment of the method according to 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 can be derived from any suitable host, in a preferred embodiment the lipase is Burkholderia sp., Candida antarctica B, Candida rugosa, Pseud
omonas sp., Candida antractica A, Porcine pancreatic lipase, Humico
la sp., Mucor miehei, Rhizopus javan., Pseudomnas fluor., Candida cy
lindrcae, Aspergillus niger, Phizopus oryzae, Mucor javanicus, Rhiz
opus sp., derived from a species selected from the group consisting of Rhizopus japonicus and Candida antarctica.

In another aspect, the invention is directed to a method of preparing a structured triacylglycerol by esterification, acidolysis, transesterification, transesterification, or alcoholysis between two substrates, the method comprising an insoluble matrix. Contacting the immobilized surfactant-coated lipase complex with the substrate.

[0048] In a preferred embodiment of the method, the matrix-immobilized surfactant-coated lipase conjugate is contacted with the substrate in the presence of an organic solvent.

In another preferred embodiment of the method, at least one of the substrates is selected from the group consisting of oils, fatty acids, triacylglycerols and fatty alcohols. Although a number of different types of oils can be used in this method, in a preferred embodiment the oils are olive oil, soybean oil, peanut oil, fish oil, palm oil, cottonseed oil, sunflower oil, Ni
selected from the group consisting of gellas sativa oil, canola oil and corn oil. In another preferred embodiment, the fatty acids are selected from the group consisting of medium and short chain fatty acids and their ester derivatives. In a more preferred embodiment, the fatty acid is selected from the group consisting of oleic acid, palmitic acid, linoleic acid, linolenic acid, stearic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and ester derivatives thereof.

Although the above process may be performed in any suitable vessel, in a preferred embodiment the process is performed in a tank reactor also in a fixed bed reactor.

The method also includes a triacylglycerol prepared according to the above method, which is used as a cocoa butter replacer, a special dietary human milk fat-like triglyceride, or a structured triglyceride for medical use. The present invention will now be described by way of example with reference to the accompanying drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to surfactant-coated lipase or phospholipase conjugates immobilized on an organic or inorganic insoluble matrix (eg, a particulate solid support), wherein the conjugates are In particular, it is used to catalyze transesterification and transesterification of fats and oils and alcoholysis of fatty alcohols. The invention also provides for the preparation of a granular enzyme preparation, which preparation exhibits enhanced activity stability. In particular, the present invention can be used to prepare structured triacylglycerols having desired nutritional or biochemical properties. The present invention is further directed to a method for preparing an insoluble matrix-immobilized surfactant-coated lipase or phospholipase complex and a method for modifying an oil or fat using the insoluble matrix-immobilized surfactant-coated lipase or phospholipase complex.

The principles and operation of the present invention will be described with reference to the drawings and the accompanying drawings.
You should understand better.

It is to be understood that in its application, the invention is not limited to the specific arrangements and arrangements of 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, the expressions and predicates employed herein are for description purposes and should not be considered limiting.

According to the invention, the surfactant-coated lipase or phospholipase is immobilized on the insoluble matrix in three different ways: (i) hydrophobic (physical) on inorganic or organic insoluble matrices Immobilization through adsorption; (ii) immobilization through ionic interaction on various ion exchange resins (polar or non-polar matrices); and (iii) covalent bonding to insoluble matrices such as Eupergit (organic matrix). Immobilization through immobilization.

The immobilized surfactant-coated lipase prepared by the procedure described herein can be used for transesterification between triglycerides and fatty acids, one-stage alcoholysis between triglycerides and fatty alcohols for the production of wax esters. And also used to catalyze an ester substitution reaction between two different triglycerides or between two different fatty oils.

The results show that lipases coated with lipid surfactants such as, but not limited to, fatty acid sugar esters cause lipase activation that can be used for organic synthesis, and in most cases,
We show that this modification process converts relatively inactive crude lipase to a highly active biocatalyst. In order to develop a reactor in which lipase enzyme can be easily recovered from the reactor, or a bioreactor for efficient enzyme transesterification / substitution reaction that can be used continuously,
Detergent-lipase conjugates immobilized on organic and inorganic matrices were used.

The surfactant-coated lipase immobilized on an organic or inorganic matrix shows high transesterification / substitution activity in five consecutive transesterification reactions using the same biocatalyst batch, with only a small loss of activity. Indicated. It has further been found that granulation of the matrix-bound surfactant-treated enzyme significantly enhances enzyme stability and can be used repeatedly without substantial loss of activity.

The immobilized surfactant-lipase conjugate prepared according to the invention was used for the preparation of structured triglycerides with potential use in the pharmaceutical and food industries. The subject triglycerides synthesized according to the method of the present invention were produced by transesterification of long chain triglycerides, such as the hard fraction of palm oil, with short chain fatty acids, such as capric acid. Reactions catalyzed by immobilized surfactant-coated lipase resulted in products with 1,3-regiospecificity for the triglycerides of interest. Mono and diglycerides also formed in the hydrolysis side reactions, but their percentages were usually less than 7% by weight of the initial triglyceride concentration. The operational stability of the detergent-lipase complex immobilized on different solid matrices was very high, especially after any granulation step, and no significant loss of enzyme activity was observed.

The transesterification reaction using the immobilized surfactant field-lipase complex was also carried out for the purpose of obtaining fats having special characteristics with respect to physical properties. For example, liquid olive oil was ester-substituted with a hard palm oil fraction in order to obtain a mixed oil for preparing margarine based on healthier olive oil. This enzymatic method is a useful alternative to chemical oil hydrogenation or transesterification methods.

Thus, according to the teachings of the present invention, there is provided a lipase preparation comprising an insoluble matrix and a surfactant-coated lipase complex immobilized on the insoluble matrix.

The term “lipase” as used in this specification and in the following claims is not limited to this particular enzyme, but also encompasses similar enzymes such as phospholipases, proteases and glycosidases. That means. Other suitable enzymes will be apparent to the skilled chemist.

According to a preferred embodiment, the conjugate is formed via a hydrophobic (physical) interaction
It is immobilized on an insoluble matrix via ionic interactions or via covalent immobilization.

The insoluble matrix in a preferred embodiment of the present invention is an inorganic insoluble matrix, and the term “insoluble” refers to a lack of solubility in polar (eg, water) and non-polar (hydrophobic) solvents. Inorganic insoluble matrices according to the present invention include alumina, aluminum stearate, celite, calcium carbonate, silica gel, charcoal, calcium sulfate, and, but not limited to, Amberlite (Ambe).
ion exchange resins such as rlite) and Dowex. For physical immobilization, the most preferably employed inorganic insoluble matrix is diatomaceous earth (DE). For ionic immobilization, the most preferably employed inorganic insoluble matrices are Amberlite and Dowex, which are strong ion exchangers.

Suitable organic solid matrices according to the invention are eupergit for covalent immobilization and ethylsulfoxycellulose for ionic interactions. Any other suitable organic solid matrix can be used without departing from the scope of the present invention.

In another preferred embodiment of the invention, the lipase makes up from 2 to 20%, preferably from 5 to 11% by weight of the detergent-coated lipase complex. Furthermore, in a preferred embodiment of the invention, the lipase makes up from 0.01 to 1% by weight of the preparation.
Preferably, the lipase makes up about 0.7% by weight of the preparation.

According to a preferred embodiment of the present invention, the surfactant used is a lipid, which comprises a fatty acid bound to a hydrophilic moiety. This fatty acid is preferably
Monolaurate, monomyristate, monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate, trilaurate, trimiristate, tripalmitate or tristearate. The hydrophilic moiety is preferably, but not limited to, a sugar such as sorbitol, sucrose, glucose and lacrose, a phosphate group, a carboxyl group or a polyhydroxylated organic residue. Usually, the fatty acid and the hydrophilic moiety are linked via an ester group.

According to a preferred embodiment of the present invention, the lipase is derived from a microorganism or a multicellular organism. Known species used for lipase extraction include Burkholderia sp., Candi
da antarctica B, Candida rugosa, Pseudomonas sp., Candida antractica
A, Porcine pancreas, Humicola sp., Mucor miehei, Rhizopus javan.
, Pseudomnas fluor, Candida cylindrcae, Aspergillus niger, Phizopus
oryzae, Mucor javanicus, Rhizopus sp., Rhizopus japonicus and Cand
ida antractica.

According to a preferred embodiment of the present invention, the lipase preparation maintains lipase catalytic activity in an organic solvent. Among the lipase catalytic activities include hydrolysis, esterification, transesterification, ester substitution, acidolysis and alcoholysis, preferably acidolysis and alcoholysis with 1,3-position specificity with respect to triacylglycerol. Organic solvents are hydrophobic solvents such as, but not limited to, n-hexane, toluene, isooctane, n-octane, benzene, cyclohexane, and diisopropyl ether.

Further, according to the present invention, there is provided a method for preparing a surfactant-coated lipase complex immobilized on an insoluble matrix. The method comprises the following method steps, in which the first step involves contacting the lipase, insoluble matrix and surfactant field in an aqueous solution, preferably a buffer solution. In the second step, conditioning (eg, sonication) is provided for matrix-immobilized surfactant-coated lipase complex formation. In this regard, two alternatives are available. First, the lipase is first allowed to interact with the detergent world, and only thereafter with the detergent-coated lipase with the matrix. On the other hand, the second is to allow the lipase to first react with the matrix and then only after that to react the matrix-immobilized lipase with the detergent field.

According to a preferred embodiment of the present invention, the method further comprises the step of separating the matrix-immobilized surfactant-bound lipase complex from the aqueous solution.

According to another preferred embodiment of the invention, the method further comprises the step of drying the matrix-immobilized surfactant-coated lipase complex. Drying is preferably effected by freeze drying, fluidized bed drying or drying in an oven. After drying, the matrix-immobilized surfactant-coated lipase conjugate exhibits a water content of less than 100 ppm by weight, preferably less than 50 ppm by weight, and most preferably less than 20 ppm by weight.

In a preferred embodiment of the method according to the invention, lipase in aqueous solution,
Contacting the insoluble matrix with the surfactant field involves dissolving the surfactant field in an organic solvent (eg, ethanol) to obtain a surfactant-dissolving solution; The resulting suspension is sonicated; this is done by adding an insoluble matrix to the aqueous solution. Alternatively, the lipase is first allowed to interact with the insoluble matrix and then only afterwards in contact with the detergent field.

According to a preferred embodiment, the insoluble matrix of the lipase preparation is modified with a fatty acid derivative. This makes it possible to immobilize the hydrophobic lipase on a hydrophobic carrier such as aluminum stearate, celite treated with a fatty acid derivative, and a nonpolar or weakly polar ion exchange resin for the purpose of preparing a highly active enzyme.

The invention further provides, in accordance with the present invention, a method of preparing a structured triacylglycerol, comprising the steps of esterifying, transesterifying, transesterifying, acidilyzing, or insoluble matrix-immobilized surfactant-coated lipase conjugate. By alcoholysis between the two substrates consisting of contacting the substrates. The contact between the matrix-immobilized surfactant-coated lipase complex and the substrate is performed in the presence of an organic solvent.

In a preferred embodiment, at least one of the substrates is a fatty oil, fatty acid or triacylglycerol. The fatty oil may be any of the above fatty oils. Fatty acids are medium or short chain fatty acids or their ester derivatives. Suitable fatty acids are for example oleic acid, palmitic acid, linoleic acid, linolenic acid, stearic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and their ester derivatives.

In a preferred embodiment of the invention, the contacting of the matrix-immobilized surfactant-coated lipase complex with the substrate is carried out in a reactor, for example a tank reactor or a fixed-bed reactor.

Further in accordance with the present invention, there is provided a method of altering the physical properties of fats and oils (eg, triacylglycerols), the method comprising the steps of: removing an insoluble matrix-immobilized surfactant-coated lipase complex; By transesterification or transesterification between at least two oleaginous substrates, which comprises contacting the substrate in the presence of a solvent.

Further according to the present invention, long chain triglycerides (L
CT) and a method for altering the physical properties of long chain fatty alcohols (LCFAL), comprising contacting an insoluble matrix-immobilized surfactant-coated lipase complex with a substrate, preferably in the presence of an organic solvent. At least by alcoholysis between two substrates of this kind.

According to a preferred embodiment, the matrix-immobilized surfactant-coated lipase complex accounts for 2 to 30% by weight of the substrate. According to another preferred embodiment, the oil / fat substrates are liquid oils and solid fats. The oils are any of the above oils, natural or hydrogenated.

Further according to the present invention there is provided a triacylglycerol prepared by the above method. The triacylglycerols are useful for the use of cocoa butter substitutes, human milk fat-like substances, special dietary or medical structured triglycerides, and the like.

Further according to the present invention there is provided a preparation comprising a lipase and an organic solvent.
This lipase undergoes both esterification (transesterification and substitution reactions) with respect to the substrate,
It has acidolysis, alcoholysis and hydrolysis catalytic activity and produces both esterification and hydrolysis products. The hydrolysis products make up less than about 7%, preferably less than about 5%, more preferably less than about 3% by weight of the total product.

[0083]

【Example】

Reference will now be made to the examples, which together with the above description, illustrate the invention in an unrestricted manner.

Experimental Procedure Materials A variety of crude lipase preparations were tested in this study. The commercial lipase preparations used in the study and their seed sources and suppliers are listed in Table 1 below. All fatty acids and triglycerides used in this study were obtained from Fluka (Switzerland) and were at least 99% pure, as reported by the supplier. olive oil,
Sunflower oil, palm oil, canola oil, corn oil and Nigella sativa oil were obtained from local suppliers (Galilee Province, Israel). Inorganic matrices, alumina and silica gel containing fish oil, tris (hydroxymethyl) aminomethane and DE used as a support for the surfactant-coated lipase conjugate were obtained from Sigma (USA).

[0085] Analytical grade n-hexane and other solvents employed were all of analytical grade and were obtained from Bio Lab (Israel). Mixtures of sorbitan monolaurate, sorbitan monomyristate, sorbitan monopalmitate and sorbitan fatty acid esters, including sorbitan monostearate, and mono-, di-, and sucrose tristearate esters exhibiting various HLB values A sucrose fatty acid ester containing was obtained from Kao Pure Chemicals Ind. (Tokyo, Japan). Tris (hydroxymethyl) aminomethane (Tris) was obtained from Sigma (USA). The inorganic and organic matrices used as supports for the modified lipase include:
Includes diatomaceous earth (DE), alumina and silica gel; ion exchange resin is Sigma
(USA). Eupergit C and Eupergit C2
50L "EupergitC 250L" (porous sphere, diameter about 150 to 200μm each)
) Was obtained from Rhom (Germany).

[Table 1]

The immobilized crude enzyme via lipase modification and physical adsorption was first coated with a lipid surfactant or other enzyme activator such as gum arabic or polyethylene glycol. A typical enzyme modification and immobilization procedure is as follows: dissolve the crude enzyme (lipase, phospholipase, protease and glycosidase; protein content about 150 mg / L) in 1 L phosphate solution or Tris buffer at about pH And stirred at 10 ° C. for 30 minutes. A lipid surfactant or other enzyme activator (0.5 g) dissolved in ethanol (20 ml) or another solvent was added dropwise to this stirred solution. The resulting enzyme solution was sonicated for 15 minutes and then vigorously stirred at 10 ° C. for 3 hours. Insoluble organic matrix (polypropylene, aluminum stearate or chitin (20 g) or Celite "C
elite, alumina, silica gel or a ceramic support, 20 g) was added to the stirred enzyme solution. The solution was magnetically stirred at 10 ° C. for another 5 hours. The precipitate was collected by centrifugation (12000 rpm) (Sorval Centrifuge, model RC-5B) or by filtration and processed by one of the following two methods: The wet precipitate was frozen at -20 ° C for 24 hours and then made lipophilic. The powder formed can be used directly for batch enzymatic reactions or modified in granular form and has a particle size of 50-1000 μm.
m can be granulated to obtain a granular immobilized enzyme. Granulation was accomplished using starch, methyl or ethyl cellulose, gum, agarose or other binders. For example, granulation with starch is carried out as follows: A starch solution (4 g starch / 20 ml water) is gelled at 20 ° C. The gel is cooled to 60 ° C. and introduced into the modified and immobilized enzyme wet powder. The mixture is homogenized in a high speed mixer, then extruded and dried at 40-60 ° C for 48 hours. The immobilized enzyme is sieved to obtain particles in the range of 50 to 1000 μm. This granular enzyme is mainly used in packed columns.

[0087] 2. The wet precipitate formed after modification and immobilization was granulated directly with starch or other binding reagents as described above.

Enzyme Modification and Immobilization via Ion Adsorption The above modification, immobilization and granulation procedures are also used in conjunction with ion exchange resins. The types of resins employed include the following: strong and weakly basic ion exchange resins, strong and weakly acidic ion exchange resins, and weakly polar and nonpolar ion exchange resins. Examples of commercially available resins (obtained from Sigma, USA) used in the experiments 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-10A", "Ectoela" cellulose, "Sephdex
And sulfoxyethylcellulose. A typical modified immobilized enzyme was prepared according to the procedure described above.

Enzymatic Modification via Immobilization and Immobilization Two different procedures were employed. According to a first procedure, the enzyme was first coated with a detergent and then the lipase-detergent complex was covalently bound to an Eupergit "Eupergit" matrix, which contains active oxirane groups. To achieve the goal, crude lipase (1 g protein) was dissolved in 1 liter of Tris or phosphate buffer pH 5.8. This enzyme solution is added at 30 ° C.
Vigorous magnetic stirring for 0 minutes. Sorbitan monostearate (0.5 g) dissolved in 30 ml of ethanol was added dropwise to this stirred enzyme solution. The resulting colloidal enzyme solution was sonicated for 10 minutes and then stirred at 10 ° C. for 3 hours. "Eupergit C"
Alternatively, “Eupergit C250L” (125 g) and 12 ml of a 5% hydrogen peroxide solution were added to the enzyme solution, and the resulting suspension was shaken by hand for 1 minute, and then incubated at 23 ° C. for 48 hours. The precipitate was filtered, washed with Tris or phosphate buffer pH 5.8, and lyophilized overnight.

According to a second procedure, the lipase was first covalently bound to an “Eupergit” matrix, and then the bound lipase was coated with a detergent. The chemistry involved in this procedure is shown in FIG.

For the purpose, the crude lipase (1 g protein) was dissolved in 1 liter of Tris or phosphate buffer pH 5.8. The enzyme solution was vigorously magnetically stirred at 30 ° C. for 10 minutes. "Eupergit C" or "Eupergit C250L" (125 g) and 12 ml of a 5% hydrogen peroxide solution were added to the enzyme solution, the resulting suspension was shaken by hand for 1 minute and then incubated at 23 ° C. for 48 hours. Then, sorbitan monostearate (0.5 g) dissolved in 30 ml of ethanol was added dropwise to the suspension while mildly stirring. The resulting suspension was sonicated for 10 minutes and then incubated at 10 ° C. for 6 hours. The precipitate was filtered, washed with Tris or phosphate buffer pH 5.8, and lyophilized overnight.

As a control for the covalently immobilized lipase, the same immobilization procedure was performed without the addition of a detergent to the enzyme solution.

To achieve the objective, the crude lipase (1 g protein) was dissolved in 1 liter of Tris or phosphate buffer pH 5.8. The enzyme solution was vigorously magnetically stirred at 30 ° C. for 10 minutes. "Eupergit C" or "Eupergit C250L" (125 g) and 12 ml of a 5% hydrogen peroxide solution were added to the enzyme solution, the resulting suspension was shaken by hand for 1 minute and then incubated at 23 ° C. for 48 hours. The precipitate was filtered, washed with Tris or phosphate buffer pH 5.8, and lyophilized overnight.

According to the protein measurement according to the Bradford method, the enzyme preparation prepared by this method contained 0.9-1.5% by weight of protein.

Reaction Models Three different reaction models were used for testing modified and immobilized enzyme activity and compared with the corresponding activity of the crude enzyme and the corresponding activity of the unmodified immobilized enzyme.

1. Esterification between lauric acid and dodecyl alcohol in organic and solvent-free systems.

The esterification reaction was started by adding 10 mg of the lipase preparation to 10 ml of n-hexane containing usually 200 mg of lauric acid and 186 mg of dodecyl alcohol. The reaction was magnetically stirred at 40 ° C. Collect samples periodically (50μl)
And filtered through a Millipore filter (0.45 μm) followed by n
-Mixed with a similar volume of n-hexane solution containing hexadecane.

2. Ester substitution reaction between two triglycerides; tripalmitin and tristearin in organic or solvent-free system.

The ester displacement reaction was started by adding 10 mg of the lipase preparation to 10 ml of n-hexane, usually containing 40 mg of tripalmitin and 40 mg of tristearin. The reaction was magnetically stirred at 40 ° C. Samples are taken periodically (50 μl), filtered through a Millipore filter (0.45 μm) and then n-
It was mixed with a similar volume of n-hexane solution containing hexadecane.

[0100] 3. An alcoholysis reaction between olive oil and cetyl alcohol in an organic or solvent-free system.

The alcoholysis reaction was started by adding 10 mg of the lipase preparation to 10 ml of n-hexane, usually containing 500 mg of olive oil and 500 mg of cetyl alcohol. The reaction was magnetically stirred at 40 ° C. Samples are collected periodically (50 μl
) And filtered through a Millipore filter (0.45 μm), then mixed with a similar volume of n-hexane solution containing tridecanoin as an internal standard.

Unless otherwise specified, all experiments were performed under superficial conditions. Each esterification reaction was performed in duplicate. In all experiments, n-hexane was dried over molecular sieves to minimize water content to less than 6 mg / liter.

Protein content The protein content of the modified and immobilized lipase was measured by the microkejldahl method.

Enzyme-activated activating modification enzyme in a batch system , modification and immobilized enzyme immobilized on an insoluble matrix,
The activity of the crude enzyme and the enzyme immobilized in the insoluble matrix was measured using a 1 m
Tested using 1 glass bottle. The vials were shaken at 40 ° C. and analyzed after a certain period of time. Reaction rates were measured at substrate conversions of less than 7% / mg protein.

Operational Stability of Modified and Immobilized Enzymes The operation stability of the granulated modified and immobilized enzymes was determined using a jacketed column reactor (alcolysis of olive oil and cetyl alcohol in n-hexane) as a reaction model. (0.5 cm inner diameter and 15 cm length). The enzyme particles were packed into the column and the substrate solution was circulated through the packed enzyme. After one hour, the circulation was stopped and the reaction solution was analyzed. After each experimental work, the solution was discarded, and the immobilized enzyme was washed with an organic solvent (n-hexane) before loading the new substrate solution. This procedure is 1
Repeated 0-20 times.

Experimental results Example 1 Protein content of lipase preparation Heterologous crude lipase, lipase modified with sorbitan monostearate (SMS),
The protein content of the lipase immobilized on celite and the SMS modified lipase immobilized on celite were measured as described above. The results are shown in Table II.

These results indicate that there is fairly wide variation in protein concentration between the various preparations. In the case of the surfactant-coated matrix-immobilized enzyme, the protein content is 0
. It varies from 05% to 1.12% by weight depending on the enzyme used in the preparation. A similar variation was observed when the enzyme Lilipase was treated with a heterologous lipid surfactant or other activator and optionally immobilized on celite. The results of this study are shown in Table III.

[0108]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

Example 2 Esterification, ester substitution and alcoholysis activity of lipase preparation Preparation of lipase The enzyme activities of various lipase preparations (prepared as described above) were compared. For comparison purposes, the results (shown in Table IV) are expressed as reaction rates (ri).

[0110]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

These results indicate that the crude lipase used in this study lacks measurable esterification or ester displacement activity at low water concentrations in its native form. In contrast, both modification with the surfactant sorbitan monostearate and, independently, immobilization on celite resulted in a detectable degree of esterification and ester substitution. However, when the enzyme was subjected to both of these treatments, the kinetics of both the esterification and ester displacement reactions increased to a greater extent. From the numerical results shown in Table IV, it is clear that there is an unexpected synergy between the two stages of modification, surfactant treatment and immobilization on the matrix.

In FIG. 3, DE-immobilized surfactant-coated “Saiken-100” (triangles) and “Lipase A10
"-FG" (squares) shows the conversion of palmitin over time when used in the transesterification of tripalmitin and capric acid. The transesterification rates thus determined were 0.098 and 0.104 mmol / min-mg biocatalyst, respectively.

Example 3 Fatty Acid Specificity of Immobilized Surfactant-Coated Lipase Complex The specificity of the inorganic matrix-immobilized surfactant-coated lipase complex of the present invention with respect to various kinds of different fatty acid substrates was evaluated using various chain length fatty acids and tripalmitin. Tested by monitoring transesterification with The results are shown in Table V. The reaction conditions are as follows: The transesterification activity of the crude lipase preparation was determined using tripalmitin (50 mg) as a triacylglycerol substrate and capric acid (35 m2) as a medium chain fatty acid substrate.
g) in n-hexane. Non-immobilized surfactant-coated lipase (5
-10% protein-containing), or inorganic matrix surfactant-coated lipase (
(0.2-2% protein content) was tested in n-hexane using the same substrate.

[0114]

[Table 11]

From Table V, it can be seen that the immobilized surfactant-coated “Lilipase” complex of the present invention significantly catalyzes the transesterification of fatty acids with tripalmitin in a 1,3-position specific manner. The hydrolysis product concentration did not exceed 5% by weight of the initial tripalmitin concentration.

Further, it can be seen that the surfactant-coated lipase complex immobilized on the inorganic matrix in the present invention is affected by the fatty acid selected as the substrate. Thus, fatty acids with longer alkyl chains, such as palmitic acid and stearic acid, are better substrates for DE-immobilized surfactant-coated lipase complexes than fatty acids with shorter chains.

[0117] EXAMPLE 4 Effect Table VI inorganic matrix immobilized surfactant coated lipase on complex selected surfactant, type of sorbitan fatty acid ester used in the coating of lipase, celite immobilized surfactant-coated lipase It shows that it affects the transesterification activity of the complex.

When sorbitan monostearate was used for coating, the conjugate had the highest transesterification activity. However, the use of shorter chain fatty acids in sorbitan esters results in reduced complex activity.

[0119]

[Table 12]

Example 5 Physical Binding of Modified Lipase on Insoluble Matrix Esterification, ester substitution, and alcoholysis activity of “Lilipase A-10FG” immobilized on various insoluble matrices were measured, and sorbitan monostearin was measured. The activity was compared with that of “Lilipase A-10FG” modified with an acid salt and immobilized on various inorganic matrices. The results are shown in Table VII.

[Table 13]

The most suitable carriers for the immobilized enzymes used in this study are aluminum monostearate and fatty acid treated celite. As can be seen from Table VII, the unmodified "Lilipase" immobilized on aluminum monostearate and fatty acid-treated celite gave relatively good activity in the three reaction models. The results shown in this table demonstrate that modification of the enzyme with a surfactant in the first step and subsequent immobilization of the modified enzyme on a fatty acid treated insoluble matrix results in a further improvement in enzyme activity. As can be seen from the table above, the activity of the lipase immobilized on the fatty acid derivative-modified and fatty acid derivative-treated insoluble matrix (aluminum monostearate, fatty acid derivative-treated celite) was confirmed by the lipase immobilized on the fatty acid derivative-treated insoluble matrix. Much greater than the activity.

Example 6 Effect of Selection of Ion Exchange Resin on Enzyme Activity Esterification of "Lilipase-10FG" immobilized on various ion exchange resins and the same enzyme modified with sorbitan monostearate prior to immobilization, Ester substitution and alcoholysis activity were compared. The results of the comparison (expressed as reaction rates) are shown in Table VIII.

[0123]

[Table 14]

[0124] These studies illustrate that immobilization dramatically increases the esterification and ester displacement activity of the modifying enzyme. In addition, Table VII shows that ion exchange resins having hydrophilic groups in their structure behave better as carriers for surfactant-modified enzymes.

Example 7 Biosynthesis of structured triglycerides from various fatty oils using surfactant-coated lipase complex immobilized on an insoluble matrix Table IX shows n-hexane and solvent-free between various fatty oils and capric acid. 6 in the system
The rate of transesterification at 0 ° C. is shown. This transesterification is carried out according to DE-
Catalyzed by immobilized surfactant-coated “Lilipase A10-FG” complex.

The DE-immobilized surfactant-coated “Lilipase” conjugate significantly catalyzed the transesterification of various fatty oils with capric acid in n-hexane. The best transesterification was obtained with olive oil. Similar reaction rates were obtained when the transesterification was performed in a solventless system.

[0127]

[Table 15]

Example 8 Operating Stability of Modified and Immobilized Enzyme The stability of the enzyme preparation used over repeated cycles was determined as described in the Methods section above. For this purpose, "Lilipase A10-FG" was used as a catalyst for the conversion of olive oil to wax esters. The activity was measured in ten experimental runs, each of which took one hour to complete. Activity levels were measured as olive oil conversion%. The reaction conditions are as follows: 100 g of n-hexane containing 20 g of olive oil and 20 g of cetyl alcohol.
was circulated through the immobilized enzyme packed bed at 40 ° C. at a rate of 2.3 ml / min. The column used for granular enzyme packing was 12 cm long and 0.75 cm inside diameter. The results of this study are shown in FIGS.

FIG. 5 shows “Lilipas” fixed on (unmodified) celite and granulated with 2% starch.
This shows that the activity of "eA10-FG" is low, and the activity decreases with each reuse. FIG.
Shows that the activity of the same lipase after modification with sorbitan monostearate and then immobilized on celite (no granulation) was 9 times higher than that of the same unmodified lipase. From FIG. 2, it can be seen that there is a sudden loss of activity after the first, second and third experimental work, which may be due to the efflux of the immobilized enzyme. 7-1
No. 6 shows that the enzymatic activity in the organic solvent can be essentially maintained by granulation using various binding reagents such as starch, ethylcellulose, gum, starch and the like. Granulation is
It also facilitates flow through the enzyme packed column. The binders used are as follows.
Figure 7 Starch 2% Figure 8 Ethyl cellulose 2% Figure 9 Gum arabic 2% Figure 10 Ethyl cellulose 2% Figure 11 Agarose 2% Figure 12 Starch 4% Figure 12-16 Starch 2%

Immobilization was performed on celite in the experiments shown in FIGS. 5-9 and 12-13, on “Amberlite IRA-900” in FIGS. 10 and 11, on aluminum monostearate in FIG. 14, and on “Amberlite XAD” in FIG. -16 "above, and in Figure 16" Amberlite XAD-7
Went on. In all cases where the lipase was modified with a surfactant, the surfactant used for the treatment was sorbitan monostearate except for the experiment of FIG. 13, and in the case of FIG. 13, it was sorbitan tristearate.

Example 9 Covalent attachment of modified and unmodified lipase to "Eupergit"

[0132]

[Table 16]

From Table X it can be seen that different lipases show different transesterification activities when treated similarly. This result can be attributed to the different sources of lipase used. All crude lipases showed very low transesterification activity under the conditions described, while their covalent immobilization on Eupergit resulted in a slight increase in activity. Interestingly, when lipases are coated with detergents, their transesterification activity is significantly increased. The highest conversion of palmitin to transesterification products with 1,3-specificity is achieved after a reaction time of 2 hours, the lipase is first covalently immobilized on "Eupergit" and then coated with detergent. Is achieved if "Lilipase A10-FG" coated with surfactant produced the highest transesterification activity of the three lipases tested in this regard.

Example 10 Effect of Binders on Enzyme Activity Different binders have been used for granulation of modified immobilized lipase as described above. Table XI shows the average conversion when olive oil is converted to its wax ester in the first and second experimental runs, where "Lilipase A10-FG" immobilized on celite modified with sorbitan monostearate. Different binders were used for the granulation of. The conversion of all binders was 2% based on the dry weight of the particles. In these experiments, the granulated enzyme was packed in a column and the experiment was performed 10 times.

Modified with sorbitan monostearate and immobilized on Celite,
Then the average conversion of olive oil to wax ester in the first and second experiments using "Lilipase A10-FG" granulated with different binders (2%) Reaction conditions: including 2 g of olive oil and 2 g of cetyl alcohol 100 ml of n-hexane was circulated through the immobilized enzyme packed bed at a rate of 2.5 ml / min at 40 ° C. Column dimensions: length 12 cm, inner diameter 0.75 cm.

[0136]

[Table 17]

From the above table, starch, gum arabic and gum karaya were the most effective tested in this study and produced active biocatalysts.

Although the invention has been described in relation to particular embodiments, many alternatives, modifications and changes will be apparent to those skilled in the art. All such alternatives, modifications and variations within the spirit of the invention and the broad scope of the claims are intended to be embraced.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1a is an illustration showing lipase-catalyzed transesterification acidosis with 1,3-position specificity. P indicates glycerol-bound palmitic acid, and C indicates glycerol-bound capric acid. PA and PC indicate free palmitic acid and capric acid, respectively.

FIG. 1b is an illustration showing lipase-catalyzed ester substitution with 1,3-position specificity. P indicates glycerol-bound palmitic acid, and C indicates glycerol-bound capric acid.

FIG. 2 is an illustration showing the chemistry involved in surfactant coating of a covalently immobilized enzyme following covalent immobilization of lipase on “Eupergit C250L”.

FIG. 3 is an explanatory view showing a transesterification profile physically immobilized. The reaction conditions were tripalmitin 50 mg, capric acid 35 mg, n-hexane 10 ml.
Surfactant-coated lipase ("Saiken 100" triangle, or "Lili"
pase A10-FG "square 20 mg). The reaction system was magnetically stirred and kept at a constant temperature of 40 ° C.

FIG. 4 is an explanatory diagram showing an Arrhenius plot in the case of a transesterification reaction between palmitin and capric acid using “LilipaseA10-FG” physically immobilized on DE.

FIG. 5 is an illustration of a bar graph showing the functional stability of “LilipaseA10-FG” immobilized on celite and granulated with 2% starch.

FIG. 6: Powder “Lilipas” modified with sorbitan monostearate and immobilized on Celite
It is an explanatory view of a bar graph showing the functional stability of “eA10-FG”.

FIG. 7 is an explanatory diagram of a bar graph showing the functional stability of “LilipaseA10-FG” modified with sorbitan monostearate, immobilized on celite, and granulated with 2% starch.

FIG. 8 is an explanatory diagram of a bar graph showing the functional stability of “LilipaseA10-FG” modified with sorbitan monostearate, immobilized on celite, and granulated with ethyl cellulose.

FIG. 9 is an explanatory diagram of a bar graph showing the functional stability of “LilipaseA10-FG” modified with sorbitan monostearate, immobilized on celite, and granulated with 2% gum arabic.

FIG. 10: Modification with sorbitan monostearate, immobilization on “Amberlite IRA-900”,
FIG. 3 is an explanatory diagram of a bar graph showing the functional stability of “LilipaseA10-FG” granulated with ethyl cellulose.

FIG. 11 is an explanatory diagram of a bar graph showing the functional stability of “LilipaseA10-FG” modified with sorbitan monostearate, immobilized on celite, and granulated with 2% agarose.

FIG. 12 is an explanatory diagram of a bar graph showing the functional stability of “LilipaseA10-FG” modified with sorbitan monostearate, immobilized on celite, and granulated with 4% starch.

FIG. 13 is an explanatory diagram of a bar graph showing the functional stability of “Lilipase A10-FG” modified with sorbitan tristearate, immobilized on celite, and granulated with 2% starch.

FIG. 14 is an explanatory diagram of a bar graph showing the functional stability of “LilipaseA10-FG” modified with sorbitan monostearate, immobilized on aluminum monostearate, and granulated with 2% starch.

FIG. 15: Modification with sorbitan monostearate, immobilization on “Amberlite XAD-16”,
FIG. 4 is an explanatory view of a bar graph showing the functional stability of “LilipaseA10-FG” granulated with% starch.

FIG. 16: Modification with sorbitan monostearate, immobilization on “Amberlite XAD-7”, 2
FIG. 4 is an explanatory view of a bar graph showing the functional stability of “LilipaseA10-FG” granulated with% starch.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12N 9/96 C12N 9/96 11/08 11/08 A 11/12 11/12 C12P 7/64 C12P 7 / 64 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ) , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA , C , CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) NA02 NA03 NA27 NB22 NB38 NB45 NB62 NC03 NC04 ND02 4B050 CC07 DD03 DD04 GG10 HH02 HH03 KK06 KK07 KK20 LL01 LL02 LL05 4B064 AD87 CA33 CA34 CA38 CB24 CC04 CD04 DA01 DA10 4H059 BA12 BA13 BA02 BA30 BC33 BA33 BC33

Claims (51)

[Claims] 1. A lipase preparation comprising an insoluble matrix and a surfactant-coated lipase complex immobilized on the insoluble matrix. 2. The lipase preparation according to claim 1, wherein the surfactant-coated lipase complex is covalently, ionically or physically bound to an insoluble matrix. 3. The lipase preparation according to claim 1, wherein the insoluble matrix is selected from the group consisting of an inorganic insoluble matrix and an organic insoluble matrix. 4. An inorganic insoluble matrix comprising alumina, diatomaceous earth, celite, calcium carbonate,
The lipase preparation according to claim 3, which is selected from the group consisting of calcium sulfate, ion exchange resin, silica gel and charcoal. 5. An ion exchange resin comprising Amberlite and Dowex.
The lipase preparation according to claim 4, which is selected from the group consisting of: 6. The lipase preparation according to claim 3, wherein the organic insoluble matrix is selected from the group consisting of Eupergit, ethylsulfoxycellulose and aluminum stearate. 7. The lipase preparation according to claim 1, wherein the lipase content is 2 to 20% by weight of the surfactant-coated lipase complex. 8. The lipase preparation according to claim 1, wherein the lipase content is between 0.01 and 1.0% by weight of the preparation. 9. The lipase preparation of claim 1, wherein the surfactant-coated lipase complex comprises a fatty acid attached to a hydrophilic moiety. 10. The fatty acid is monolaurate, monomyristate, monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate,
The lipase preparation according to claim 9, which is selected from the group consisting of trilaurate, trimiristate, tripalmitate and tristearate. 11. The lipase preparation according to claim 9, wherein the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxyl group and a hydroxylated organic residue. 12. The lipase preparation according to 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 linked via an ester bond. 14. The lipase preparation according to claim 1, wherein the lipase is derived from a microorganism. The lipase may be Burkholderia sp., Candida antractica B, Candida rugosa.
, Pseudomonas sp., Candida antractica A, Porcine pancreas lipase, H
umicola sp., Mucor miehei, Rhizopus javan., Pseudomnas fluor., Candid
a cylindrcae, Aspergillus niger, Phizopus oryzae, Mucor javanicus
The lipase preparation according to claim 1, wherein the lipase preparation is derived from a species selected from the group consisting of Rhizopus sp., Rhizopus japonicus and Candida antractica. 16. The lipase preparation according to 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 on the insoluble matrix, wherein the lipase preparation is provided in an organic solvent. . 18. The organic solvent is selected from the group consisting of n-hexane, toluene, isooctane, n-octane, benzene, cyclohexane and diisopropyl ether.
A lipase preparation according to claim 17. 19. The lipase preparation according to claim 17, for use as a catalyst for esterification, transesterification and transesterification of fats and oils and alcoholysis of triglycerol and fatty alcohols. 20. The lipase preparation according to claim 19, for use as a catalyst exhibiting 1,3-position specificity with respect to triacylglycerol. 21. The lipase preparation according to claim 1, wherein said preparation is granular. 22. The lipase preparation according to claim 1, wherein the insoluble matrix is modified with a fatty acid derivative. 23. The enzyme preparation of claim 1, for use in a reaction environment without requiring the addition of water. 24. A method for improving the stability of an immobilized lipase complex coated with a surfactant, wherein the complex is granulated prior to contacting a substrate to be reacted with the complex. 25. A method for preparing a surfactant-coated lipase complex immobilized on an insoluble matrix, comprising: (a) maintaining a concentration of lipase at a constant concentration over a time period sufficient to obtain lipase coating; Contacting with a surfactant field in an aqueous medium at a constant temperature; and (b) a constant concentration of said lipase over a time period and under conditions sufficient to achieve immobilization of the lipase on the insoluble matrix. Contacting with an insoluble matrix in an aqueous medium in a desired order. 26. The method of claim 25, wherein said lipase is first contacted with an insoluble matrix and then with a surfactant field. 27. The method of claim 25, wherein the lipase is first contacted with a detergent field and then with an insoluble matrix. 28. The method of claim 25, further comprising the step of: (c) separating the matrix-immobilized surfactant-coated lipase complex formed in the aqueous solution from the aqueous solution. 29. The method of claim 28, further comprising the step of: (d) drying the surfactant-coated lipase complex immobilized on the matrix. 30. The method according to claim 29, wherein the drying is performed by lyophilization. 31. The method according to claim 29, wherein the surfactant-coated lipase complex immobilized on the matrix is dried to a water content of less than 100 ppm by weight. 32. The method according to claim 25, wherein the aqueous solution is a buffered aqueous solution. 33. A method of contacting a lipase and a surfactant field in an aqueous medium, comprising: (i) dissolving the surfactant field in an organic solvent to obtain a surfactant-dissolved solution; 26. The method of claim 25, wherein (ii) mixing the lipase and the dissolved surfactant solution in the aqueous medium. 34. The method of claim 25, further comprising sonicating the aqueous solution. 35. The insoluble matrix is alumina, diatomaceous earth, celite, calcium carbonate, calcium sulfate, ion exchange resin, silica gel, charcoal, Eupergit,
3. The composition of claim 2, wherein the substance is selected from the group consisting of ethylsulfoxycellulose, aluminum stearate, and celite or other inorganic matrix treated with a fatty acid derivative.
5. The method according to 5. 36. The method of claim 25, wherein the surfactant comprises a fatty acid attached to a hydrophilic moiety. 37. The fatty acid is monolaurate, monomyristate, monopalmitate, monostearate, dilaurate, dimyristate, dipalmitate, distearate,
37. The method of claim 36, wherein the method is selected from the group consisting of trilaurate, trimiristate, tripalmitate, and tristearate. 38. The method of claim 36, wherein the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxyl group, and a hydroxylated 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 linked 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 lipase is Burkholderia sp., Candida antarctica B, Candida rugosa.
, Pseudomonas sp., Candida antractica A, Porcine pancreatic lipase
, Humicola sp., Mucor miehei, Rhizopus javan., Pseudomnas fluor., Can
dida cylindrcae, Aspergillus niger, Phizopus oryzae, Mucor javanicu
42. The method of claim 41, wherein the method is derived from a species selected from the group consisting of s, Rhizopus sp., Rhizopus japonicus, and Candida antarctica. 44. A method for preparing a structured triacylglycerol by esterification reaction, acidolysis, ester substitution reaction, transesterification reaction or alcoholysis between two substrates, comprising an interface immobilized on an insoluble matrix. A method of contacting an activator-coated lipase complex with the substrate. 45. The method of claim 44, wherein the detergent-coated lipase complex immobilized on the matrix is contacted with the substrate in the presence of an organic solvent. 46. The method of claim 44, wherein the at least one substrate is selected from the group consisting of oils, fatty acids, triacylglycerols and fatty alcohols. 47. The oil of claim 46, wherein the oils are selected from the group consisting of olive oil, soybean oil, peanut oil, fish oil, palm oil, cottonseed oil, sunflower oil, Nigellas sativa oil, canola oil and corn oil. Method. 48. The method 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 fatty acid of claim 46, wherein the fatty acid is selected from the group consisting of oleic acid, palmitic acid, linoleic acid, linolenic acid, stearic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid and ester derivatives thereof. the method of. 50. The method according to claim 44, which is performed in a tank reactor or a fixed bed reactor. 51. A triacylglycerol prepared according to the method of claim 44 for use as a cocoa butter replacer, a specially formulated edible human milk fat-like triglyceride, or a structured triglyceride for medical use.