KR20020010129A - Surfactant-lipase complex immobilized on insoluble matrix - Google Patents

Abstract

Lipase preparations comprising lipase compounds coated with a surfactant immobilized on an insoluble substrate and an inert substrate.

The manufacturing method and the usage of the new product have been revealed.

Description

SURFACTANT-LIPASE COMPLEX IMMOBILIZED ON INSOLUBLE MATRIX < RTI ID = 0.0 >

The modification of enzymes in oil and fat structure and composition is a tremendous industrial and clinical interest. This process is carried out using position-specific lipases in intermolecular esterification reactions or esterification exchange reactions using substrates like fats or oils (Macrea, AR, 1983, J. Am. Oil Chem. Soc. 60: 291-294) .

Using an enzymatic process, the traditional chemical intermolecular esterification reaction can be combined with the required fatty acyl group at a particular position of the triacylglycerol molecule since there is no position-specificity.

Traditionally, chemical reactions are promoted by sodium metal, sodium alkoxides, cobalt chloride, which catalyze acyl transfer between triglycerides because of the production of triglycerides with randomly distributed fatty acyl moieties (Erdem-Senatalat, A. , Erencek, E. and Erciyes, AT, 1995, J. Am. Oil Chem. Soc. 72: 891-894).

In recent years, numerous studies have made possible applications of lipases such as promising biocatalysts for other esterification reactions in organic intermediates (Wisdom, RA, Dunhill, P., and Lilly, MD, 1987, Biotechnol.Bioeng.29: 1081-1085).

Lipase with 1,3-positional properties catalyzes the hydrolysis of fats and oils to produce free fatty acids and glycerol. However, recent studies have also shown that lipases with 1,3-positional properties can catalyze two types of esterification reactions in micro-aqueous organic intermediates (Quinlan, P. & Moore, S., 1993, INFORM 4: 580-585). The first of these reactions is an intermolecular esterification reaction or an acidolysis reaction in which the free fatty acid reacts with other triglycerides to produce new triglyceride molecules. The second type of reaction is an esterification exchange reaction in which two different triglyceride molecules react to generate new triglyceride molecules (Fig. 1a-b). The sn-2 position of the reacting triglycerides does not change in the reaction of these two enzymes.

In general, the water concentration plays an important role in determining the activity of the enzyme. (Valiverty, R. H., Halling, P.J. and Macrae, A.R., 1993, Biotechnol. Lett. 15: 1133-1138), also affect the equilibrium state of the reaction carried out in the hydrophobic organic solvent. Since water is important in the activity of the enzyme in both the hydrolysis as well as the synthesis reaction, such as the trade-off between the hydrolysis and synthesis of triglycerides, the concentration of water is lowered to minimize undesirable reactions, Lt; / RTI >

For example, at high concentrations of water, which is at least 5% of the weight of the solvent, hydrolysis proceeds because lipases have their natural hydrolysis activity. However, at low concentrations of water, less than 1% of the solvent weight, lipase catalyzes the reverse reaction. That is, it is synthesized.

A typical range of moisture concentration required for promoting the intermolecular esterification reaction between different oils in an organic intermediate is 1-10 weight percent (wt%) of the hydrophobic organic solvent. The hydrolysis reaction is also promoted because this water concentration produces an undesirable partial glyceride (mono and diglycerides) in the range of 10-20 wt% of the initial triglyceride concentration, such as byproducts.

Opportunities to exploit the location characteristics of lipase are particularly enormous in the food and oily chemical industries for high-value special fat products. For example, triglycerides of cocoa butter substitute, simulated breastfed and other nutritional quality triglycerides are obtained by enzymatic use of lipase with 1,3-positional properties (Vulfson, EN, 1993, Trends Food Sci 4: 209-215).

In view of the above description, it is necessary to develop a newly structured triglyceride together with an intermediate having no adverse effect generated from an alpha-3 unsaturated fatty acid or a saturated fatty acid and an alpha-3 polyunsaturated fatty acid. For example, a molecule of MCTs with one of their acyl groups substituted for a complete long chain fatty acid provides the nutritional benefits of both MCTs and LCTs. This approach is formed by mixing the acyl forms of unsaturated fatty acids, EPA, DHA, or α-linolenic acid at the Sn-2 position of triglyceride molecules with intermediate chain fatty acyl groups at the sn-1 and sn-3 positions (Odle, J., 1997, J.Nutr.127: 1061).

Unsaturated fatty acids mixed with the above-mentioned triglyceride molecules show some health benefits regarding cardiovascular disease, immune diseases and inflammation, allergies, diabetes, kidney disease, depression, brain development and cancer. Moreover, medium chain fatty acids mixed with the same triglyceride molecules are of great importance in certain clinical uses, particularly in the provision of useful energy sources for the promotion absorption and solubility of cholesterol in blood vessels and body consumption.

Many different approaches to the use of lipases in organic intermediates have been attempted to activate them and improve performance. Those containing the availability of lipase powder are suspended in a fine aqueous organic solvent or system of ideality and the natural lipase is absorbed in the three air qualities of the fixed bed reactor and the fluidized bed reactor (Malcata, et al., 1990, J. Am. 890-910). Furthermore, lipases have been parasitized in reversed micelles, and some lipases studied bind polyethylene glycol or hydrophobic residues in organic solvents to increase their solubility and dispersibility.

It has been found that none of the above approaches apply to all enzymatic systems. However, in many cases the performance of lipase in the hydrophobic organic system has improved on activity, characterization, stability and dispersibility.

In a recent study, the development of coated surfactant lipase preparation has been reported (e.g., Basheer, S., Mogi, K. and Nakajima, M., 1995. Biotechnol.Bioeng.45: 187-195). This enzymatic mutation converts the activity and complete inactivation lipase to a highly active biocatalyst for the esterification of triglycerides and fatty acids in organic intermediates. Newly developed surfactant lipase compounds have been investigated further and have been used for intermolecular esterification reactions in organic solvent systems to produce structurally important triptolides that are critical in medical applications (Tanaka, Y., Hirano, J.and Funada, T., 1994, J. Am. Oil. Chem. Soc. 71: 331-334).

In another approach to the problem, various immobilized enzyme reactor systems have been used as lipase-catalyzed reactions in micro-aqueous solution hydrophobic organic intermediates (see, for example, Basheer, S., Mogi, K. and Nakajima, M., 1995. Process. Biotchmistry 30: 531-536). These include fixed bed reactors, fluidized bed reactors and slurry reactors. In the published study, lipase immobilized on inorganic substrates is used in both pollen-based reactor systems and fixed-bed bioreactor systems. However, the lipase used is not a coated surfactant and has the same limitations as a free lipase system. These limits are as follows:

1. difficulty in regeneration of the enzyme after completion of the process;

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

3. the problem of regeneration of spent enzymes;

4. Low synthetic activity of free lipase in organic solvents.

Neither of the steps described above satisfactorily solves the technical problems that directly correspond to the intermolecular esterification and esterification exchange reactions of fats and oils. It is therefore an object of the invention to produce an article of lipase which is capable of undergoing esterification catalysis in fats and oils having an effect greater than the existing method.

It is an object of the present invention to produce lipase products which are fixed in a matrix and mixed with a surfactant by coating.

A further object of the present invention is to produce an article of lipase that can be used repeatedly on an industrial scale with minimal loss of activity.

It is a further object of the present invention to provide a method for producing a lipase compound coated with a surfactant and an insoluble immobilized substrate as described above.

It is a further object of the present invention to provide a process for the production of structural triglycerides using the above-described insoluble immobilized substrate and a lipase compound coated with a surfactant.

Other objects and advantages of the invention will be apparent from the detailed description.

The present invention relates to lipase compounds coated with a surfactant immobilized on an insoluble substrate and to be used as a biocatalyst for catalytic reactions such as interesterification or esterification exchange reactions of oils or fats of hydrophobic organisms. The new process involves two steps. In the first step, the enzyme is coated with a surfactant. In the second step, the enzyme is immobilized on the selected substrate. These steps can be performed in a different order.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is illustrated by way of example only with reference to the accompanying drawings in which:

FIG. 1A shows an intermolecular esterification reaction catalytic reaction caused by a lipase having 1,3-position characteristics. P represents glycerol combined with palmitic acid and C represents glycerol combined with capric acid. PA and CA show glass palmitate and capric acid.

Figure 1B shows the transesterification reaction catalyzed by a lipase having 1,3-positional properties. P represents glycerol combined with palmitic acid, and C represents glycerol combined with capric acid.

Figure 2 shows that the enzyme covalently immobilized with a surfactant binds to a lipase that is covalently immobilized in the oil purged C 259L caused by the coating.

Figure 3 shows the intermolecular esterification reaction of physically immobilized lipase. The reaction conditions are 35 mg of lipase and capric acid coated with 20 mg of a surfactant fixed to diatomaceous earth in 10 ml of normal hexane, and 50 mg of tripalmitin. The reaction system is stirred and thermostatically controlled at 40 占 폚.

Figure 4 shows the Arrhenius curve for the intermolecular esterification reaction of tripalmithine and caproic acid with a lipophase A10-FG coated with a DE-physically immobilized surfactant.

Figure 5 shows a histogram showing the functional stability of the lipolytic A 10FG granulated with Celite and fixed with 2% starch.

Figure 6 shows a histogram showing the functional stability of lipase A 10FG modified with sorbitan monostearate and fixed on celite.

Figure 7 shows a histogram showing the functional stability of lyrease A 10FG modified with sorbitan monostearate and fixed to celite and granulated with 2% starch.

Figure 8 shows a histogram showing the functional stability of lyrease A 10FG modified with sorbitan monostearate and fixed to celite and granulated with ethylcellulose.

Figure 9 shows a histogram showing the functional stability of lyrease A 10FG modified with sorbitan monostearate and fixed to celite and granulated with 2% gum arabic.

Figure 10 shows a histogram showing the functional stability of lyrease A 10FG, modified with sorbitan monostearate, fixed to Amberlite IRA-900, and granulated with ethylcellulose.

Figure 11 shows a bar graph showing the functional topology of lyrease A 10FG, modified with sorbitan monostearate, fixed to celite, and granulated with 2% agarose.

Figure 12 shows a histogram showing the functional stability of lyrease A 10FG modified with sorbitan monostearate and fixed to celite and granulated with 4% starch.

Figure 13 shows a histogram showing the functional stability of lyrease A 10FG modified with sorbitan monostearate and fixed to celite and granulated with 2% starch.

Figure 14 shows a histogram showing the functional stability of lyrease A 10FG modified with sorbitan monostearate and fixed to aluminum monostearate and granulated with 2% starch. The graph also shows results comparable to those obtained by fixing the enzyme to aluminum monostearate and granulating 2% starch without modifying the enzyme.

Figure 15 shows a histogram showing the functional stability of lyrease A 10FG modified with sorbitan monostearate, fixed to Amberlite XAD-16 and granulated with 2% starch.

Figure 16 shows a bar graph showing the functional stability curve of lyrease A 10FG modified with sorbitan monostearate, fixed to Amberlite XAD-7 and granulated with 2% starch.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a process for the preparation of lipase or phosphogypsum which is catalyzed by an intermolecular esterification reaction between an oil and a fat and an esterification exchange reaction and which is coated with a fixed surfactant in an organic or inorganic insoluble substrate used in the alcoholysis of the fatty alcohol Lipase < / RTI > The present invention also prepares the enzyme preparation in granular form to demonstrate increased stability of activity. In particular, the present invention is used to produce structural triglycerol having the required nutritional and biochemical properties. The present invention relates to a method for producing a lipase or phospholipase compound coated with a surfactant immobilized on an insoluble substrate and a method for producing a lipase or phospholipase compound coated on a lipase or a phospholipase compound coated with a surfactant immobilized on an insoluble substrate Method.

The principles and operation of the present invention may be better understood with reference to the following detailed description.

The present invention, which does not limit the details and arrangement of components in this application, illustrates the following description and illustrations in the drawings. The invention may be practiced in other embodiments or in various ways.

Lipase or phospholipase coated with a surfactant according to the present invention is immobilized to an insoluble substrate by three different methods: (i) immobilization through hydrophobic (physical) absorption in an inorganic or organic insoluble substrate; (ii) (Iii) immobilization via immobilization in an insoluble substrate such as a milk pudding (organic substrate).

Lipase coated with a fixed surfactant prepared according to the sequence described has a one-step intermolecular exchange reaction between triglycerides and fatty acids and a one-step between triglyceride and fatty alcohols for the production of wax esters, as well as two other triglyceride molecules, Is used to catalyze the esterification exchange reaction between the other oils of the catalyst.

Coated lipases with a lipophilic surfactant, such as, but not limited to, fatty acid ester forms that cause activation of the lipases used in most of the organic synthesis and transformation processes, Change. The lipase enzyme is a surfactant lipase that is immobilized on an organic substrate and an inorganic substrate in order to develop a bioreactor in which an intermolecular esterification reaction or an esterification exchange reaction of an effective enzyme which can be recovered easily or continuously is used.

Biocatalytic batches have been used to find lipases coated with organic and inorganic substrate-immobilized surfactants that exhibit high intermolecular-exchange esterification activity and only weak activation loss in five consecutive intermolecular esterification reactions. Allowing repeated use thereof without significant loss of activation has been known for granulation of substrate boundaries and surfactant-treated enzymes to improve the stability of the enzyme.

Fixed surfactant lipase compounds prepared according to the present invention are used in the production of structural triglycerides with potential applications in the medical and food industries. Significant triglycerides synthesized according to the process of the present invention are produced by intermolecular esterification of long chain triglycerides such as hard segments of palm oil with short chain fatty acids such as capric acid. Lipase coated with a fixed surfactant, in which the reactants cause a catalytic reaction, produce a significant amount of product with 1,3-positional properties to the important triglycerides. Mono and di-glycerides are also produced in hydrolysis side reactions, and their percentage is typically less than 7 wt% of the initial triglyceride concentration. The operational stability of surfactant-lipase compounds immobilized on other solid substrates is very high, especially after the selective granulation step, and no significant enzyme activity is observed.

Intermolecular esterification reactions using fixed surfactant compounds are carried out in order to obtain fats of particular character in relation to their physical properties. For example, liquid olive oil transesterifies with a hard part of palm olein to obtain a mixed oil to prepare better olive oil based on margarine. This enzyme process selectively uses a chemical oil hydrogenation reaction or an intermolecular esterification reaction.

Thus, in accordance with the teachings of the present invention, there is provided a lipase preparation comprising an insoluble substrate and a lipase compound coated with a surfactant immobilized on an insoluble substrate.

As used in the specification and claims, " lipase " means that this particular enzyme is not limited, but includes similar enzymes such as phospholipase, protease, glycosidase. Other suitable enzymes are obvious to the skilled chemist, and thus are not listed here.

According to a preferred embodiment of the compound, it is immobilized on an insoluble substrate through hydrophobic (physical) interactions, ionic interactions or intrinsic immobilization.

In a preferred embodiment of the present invention, the insoluble substrate is an inorganic insoluble substrate. "Insoluble" is not soluble in polar solvents (eg water) and nonpolar (hydrophobic) solvents. Preferably, the inorganic insoluble substrate according to the present invention is aluminum, aluminum stearic acid, celite, calcium carbonate, silica gel, activated carbon, calcium sulfate, ion exchange resins which are not limited to Amberlite or Dowex. With respect to physical immobilization, the most commonly used inorganic insoluble substrate is diatomaceous earth (DE). The inorganic insoluble substrate used for ionic fixation is the strong ion exchangers Amberlite and Dowex.

Suitable organic solid substrates in accordance with the present invention include ethyl sulfoxylcellulose of the covalent immobilization and ionic interaction. Other suitable organic solid substrates are used without exceeding the scope of the invention.

In another preferred embodiment of the invention the lipase represents 2-20 wt%, mainly 5-11 wt%, of the lipase compound coated with the surfactant. Another preferred embodiment of the invention is that the lipase represents 0.01-1 wt% of the preparation. Primarily, lipase represents about 0.7 wt% of the preparation.

The surfactant used according to a preferred embodiment of the invention is a lipid comprising a fatty acid converted to a hydrophilic moiety. The fatty acid may be at least one selected from the group consisting of monolaurate, monomyristate, monoparmemate, monostearate, dillate, dimyristate, dipalmitate, distearate, trirrate, , And tristearic acid. The hydrophilic moieties include, but are not limited to, sugars, sorbitol, sucrose, glucose and lactose, phosphate groups, carboxyl groups or oxy organic moieties. Typically, fatty acids and hydrophilic moieties are converted through ester linkages.

According to another preferred embodiment of the invention, the lipase is derived from a microorganism and a multicellular organism. Species known to be used in lipase extraction include, but are not limited to, Brucolieria sp., Canna di Dianthracica B, Canna didaru lososa, Seedomonas sp., Candida anthracis A, Pork pancreatic lipase, Fumiko sp, It is also possible to use a mixture of Rifus zaponikus and Candida anthracica, which are known to be useful in the treatment of diseases such as asthma, hay, lithopathic candidiasis, saudo fluoracea, cannadidocillinic strain, aspergillus niger, .

According to a preferred embodiment of the invention, the lipase product retains the lipase catalytic activity in the organic solvent. The lipase catalytic activity includes hydrolysis, esterification reaction, intermolecular esterification reaction, esterification exchange reaction, acid decomposition and alcoholysis with 1,3-position in relation to triglycerides. The organic solvent is typically a hydrophobic solvent which is not limited to n-hexane, toluene, isooctane, n-octane, benzene, cyclohexane and di-iso-propyl ether.

The present invention provides a method for preparing a lipase compound coated with a surfactant immobilized on an insoluble substrate. The method follows the following steps. In the first step, the lipase, the insoluble substrate and the surfactant are brought into contact with the buffer solution as an aqueous solution. Second, conditions (e. G., Sonic disruption) provide for the formation of lipase compounds coated with a surfactant immobilized on a substrate. Two optional steps are useful in this context. In the first, the lipase first interacts with the surfactant, and then the lipase coated with the surfactant interacts with the substrate. Secondly, lipase first interacts with the substrate and then the lipase immobilized on the substrate interacts with the surfactant.

According to a preferred embodiment, the method comprises separating a lipase compound coated with a surfactant immobilized to a substrate from an aqueous solution.

According to another preferred embodiment of the invention, the method comprises drying a lipase compound coated with a surfactant immobilized on a substrate.

Drying is carried out mainly through freeze drying, fluidization or an oven. Lipase compounds coated with a surfactant immobilized on a substrate after drying mainly contain a water content of 100 ppm or less in weight. Preferably 50 ppm, more preferably 20 ppm or less.

In a preferred embodiment of the method according to the invention, the insoluble substrate and the surfactant in aqueous solution, which are in contact with the lipase, dissolve the surfactant in an organic solvent (e. G. Ethanol) to obtain a solution of the surfactant dissolved, By mixing the surfactant solvent (e.g., water droplets) with the lipase; Sound wave crushing as a result of suspension; And adding an insoluble substrate to the aqueous solution. Optionally, the lipase first interacts with the insoluble substrate and interacts with the substrate.

According to a preferred embodiment, the insoluble substrate of the lipase preparation described above is converted into a fatty acid derivative. This allows the immobilization of hydrophobic lipases to hydrophobic carriers such as aluminum stearic acid, celite treated with fatty acid derivatives and non-polar or weakly polar ion-exchange resins to prepare highly active enzymes.

According to the present invention, it is an object of the present invention to provide a structural triglyceride preparation process by esterification reaction, esterification exchange reaction, intermolecular esterification reaction, acid decomposition or alcoholysis of alcohol between two substrates to produce a substrate and a substrate coated with a surfactant immobilized on an insoluble substrate Lipase < / RTI > compounds. It is in the organic solvent that the substrates and lipase compounds coated with the surface-immobilized surfactant contact.

In a preferred embodiment, at least one of the substrates is an oil, a fatty acid or a triglyceride. The oil may be any of the oils listed above. Fatty acids are medium chain fatty acids or short chain fatty acids or ester derivatives. For example, suitable fatty acids are oleic acid, palmitic acid, linolenic acid, stearic acid, arachidonic acid, icosapentanoic acid, docosahexanoic acid and their ester derivatives.

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

According to the present invention there is provided a process for the preparation of an oil / fat (e. G., An oil or fat) by intermolecular esterification or esterification exchange reaction between at least oil / fat cobalt substrates by contacting lipase compounds coated with a surfactant, ≪ / RTI > triglycerides) are provided.

According to the present invention, a long chain triglyceride (LCT), which produces a wax ester by degradation of at least two substrates between at least two substrates by contact with a lipase compound coated with a surfactant immobilized on a substrate and a substrate in an organic solvent, Lt; RTI ID = 0.0 > (LCFAL). ≪ / RTI >

According to a preferred embodiment, the lipase compound coated with a substrate-immobilized surfactant exhibits 2-30 wt% of the substrate. In another preferred embodiment, the oil / fat substrate is a liquid oil and a solid fat. The oil may be any of those listed above in natural or hydrogenated form.

According to the present invention, there is provided a prepared triglyceride according to the above process. The triglycerides provide applications such as cocoa buttermilk, fat-like milk, and triglycerides for specific foods or structural triglycerides for medical applications.

According to the present invention, there is provided a preparation comprising lipase and an organic solvent. Lipases with esterification reactions (intermolecular and exchange esterification reactions), acid decomposition, alcohol decomposition and hydrolysis catalytic activity in relation to substrates, esterification reaction products and hydrolysis products. The hydrolysis product is not more than 7 wt%, preferably not more than 5 wt%, more preferably not more than 3 wt% of the product.

A double modification of the natural lipase by the coating of the surfactant (1) and the immobilisation of the insoluble substrate (2) is an object of the present invention; only when compared to either of these two treatments, the esterification exchange reaction and the intermolecular esterification reaction The result of an elevated synergistic effect on the enzyme's ability to drive the catalytic reaction. It has been found that it is possible to improve the catalytic stability of the double-modified lipase for the esterification reaction by providing enzyme preparation in granular form.

The present invention primarily refers to lipase preparations comprising a lipase compound coated with an inert substrate and a surfactant in said insoluble substrate.

Fixation of lipase compounds in insoluble substrates can be accomplished by several different methods. However, according to a preferred embodiment of the present invention, the coated surfactant lipase compound is covalently, ionically, physically bound to an insoluble substrate.

The present invention includes the use of many types of substrates, and the substrates described above are selected from the group consisting of an inorganic insoluble substrate and an organic insoluble substrate.

In a preferred embodiment of the present invention, the inorganic insoluble substrate is selected from the group consisting of aluminum, diatomaceous earth, celite, calcium carbonate, calcium sulfate, ion exchange resins, silica gel and activated carbon. The above-mentioned ion exchange resin may be any suitable material, but in the preferred embodiment is selected from the group consisting of Amberlite and Dowex.

In a preferred embodiment of the present invention, the organic insoluble substrate is selected from the group consisting of milk purge, ethylsulfoxylcellulose and aluminum stearic acid, although any suitable organic insoluble substrate may be used.

In a preferred embodiment, the component of the lipase is 2-20 wt% of the lipase compound coated with the surfactant. In a more preferred embodiment, the component of the lipase is from 0.01 to 1.0 wt% of the preparation.

The present invention provides lipase production of lipase compounds coated with a surfactant comprising a fatty acid converted to a hydrophilic moiety. In a preferred embodiment, the fatty acid is selected from the group consisting of monolaurate, monomyllistic acid, monoparmemic acid, monostearic acid, tri palmitic acid and tristearic acid. In a preferred embodiment, the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxyl group and oxy organic moieties. In a more preferred embodiment, the sugar is selected from the group consisting of sorbitol, glucos and lactose. In a preferred embodiment, the fatty acid and the hydrophilic moiety are linked through a bond in a suitable form, but the fatty acid and the hydrophilic moiety are converted through the ester bond.

In the preferred embodiment, although the lipase is obtained or derived from a suitable raw material, the lipase is derived from the microorganism. Many different species of microorganisms and multicellular organisms can be used as raw materials of lipases for the lipase production of the present invention. However, the present invention is not limited to the use of the compounds of the present invention for the treatment and / or prophylactic treatment of Brucellosis sp., Kannadida anthracica B, Kannadidarugosa, Seedomonas sp., Candida anthracis A, Pork pancreatic lipase, Fumiko sp. , Candida albicans, Aspergillus niger, Rispuss oryzae, Mucor javanicus, Rizophus sp., Rizophus japonikus and Candida anthracica.

The present invention refers to the production of lipase comprising the insoluble substrate provided in an organic solvent and the insoluble substrate immobilized on a lipase compound coated with a surfactant in lipase production. In a preferred embodiment, the organic solvent is selected from the group consisting of n-hexane, toluene, isooctane, n-octane, benzene, cyclohexane and di-isopropyl ether. The present invention refers to the use of lipase production as a catalyst for esterification of oils and fats, intermolecular esterification and esterification exchange reactions, and alcoholysis of triglycerol and fatty alcohols. In a preferred embodiment, the preparation of lipases is used as a catalyst with 1,3-positional properties with respect to triglycerol.

In another aspect, the present invention refers to lipase manufacture as described above in the form of granulating the preparation.

The present invention also provides a lipase preparation wherein the insoluble substrate is modified with a fatty acid derivative as described above.

In yet another aspect of the present invention refers to the preparation of enzymes for use in a reaction environment without the addition of water.

The invention also includes a method for enhancing the stability of a lipase compound coated with an immobilized surfactant comprising a reacted substrate and a granulated substrate that is more important than contact.

In another aspect, the invention provides a method of producing a lipase compound coated with a surfactant immobilized on an insoluble substrate. The required steps are:

(a) contacting lipase in a medium aqueous solution with a surfactant at a concentration and temperature for a period of time sufficient to coat the lipase;

(b) contacting and contacting the lipase in a medium aqueous solution having an insoluble substrate for a sufficient period of time and under conditions and concentrations to obtain a fixed lipase in the substrate;

In a preferred embodiment of the above-mentioned method, the lipase first contacts the insoluble substrate and then contacts the surfactant. In another preferred embodiment, the lipase is first contacted with a surfactant and then contacted with an insoluble substrate.

In a preferred embodiment of the present invention, the method described above consists of the separation of a lipase compound coated with a surfactant immobilized on a substrate from a formed aqueous solution. In a preferred embodiment, the method also comprises drying the lipase compound coated with the surface active agent immobilized on the substrate. The drying step may be effected by any convenient method, but in a preferred embodiment, the drying is effected by freeze drying. In another preferred embodiment, the lipase compound coated with a substrate-immobilized surfactant is dried to a moisture content of 100 ppm or less.

In another preferred embodiment, the aqueous solution used in the above described method is a buffer solution.

In another preferred embodiment of the above-described method, the lipase and the surfactant are contacted with the intermediate aqueous solution:

(i) dissolving the surfactant in an organic solvent to obtain a dissolved surfactant solution; And

(ii) mixing the lipase and the surfactant solution in the intermediate aqueous solution

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

In another preferred embodiment of the method of the present invention, the insoluble substrate is selected from the group consisting of aluminum, diatomaceous earth, celite, calcium carbonate, calcium sulfate, ion exchange resins, silica gel, activated carbon, milk purge, ethylsulfoxycellulose, Celite or other inorganic substrate.

In another preferred embodiment, the surfactant of the method comprising fatty acids is converted to a hydrophilic moiety. In a more preferred embodiment, the fatty acid is selected from the group consisting of monolaurate, monomyristate, monopammitate, monostearate, triampamate and tristearate.

In a preferred embodiment of the method of the present invention, the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxyl group and oxy organic moieties. 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 method of the present invention, the fatty acid and the hydrophilic moiety are bonded via an ester linkage.

In a preferred embodiment of the method of the present invention, the rapa agent is derived from an organism. In a more preferred embodiment, the rapa agent is derived from multicellular microorganisms. In a preferred embodiment, the lipase may be derived from a suitable host, but the lipase may be selected from the group consisting of Brucolieria sp., Canna dida anthracica B, Canna di dru rosa, Seedomonas sp., Candida anthracis A , Fusarium spp., Mucor melehei, Rhizopus sp., Seedofluor., Cannadidocylindrakas, Aspergillus niger, Rispus ozije, Mucor javanicus, Rhizopus sp., Risofus zaponi Lt; RTI ID = 0.0 > Candida < / RTI >

In another aspect, the present invention provides a process for producing a lipase comprising the steps of esterification, acidolysis, esterification exchange reaction, intermolecular esterification reaction, Refers to the process for the production of structural triglycerides by digestion.

In a preferred embodiment of this process, the coated lipase compound immobilized to the substrate is contacted with a substrate present in the organic solvent.

In another preferred embodiment of this process, at least one of the substrates is selected from the group consisting of oils, fatty acids, triglycerol and fatty alcohols. Many different types of oils can be used in this process, but in a preferred embodiment, the oils are selected from the group consisting of olive oil, soybean oil, peanut oil, fish oil, palm oil, cottonseed oil, sunflower oil, nigella lacetti butter oil, canola oil and corn oil . In another preferred embodiment, the fatty acids are selected from the group consisting of intermediate chains and short chain fatty acids and their ester derivatives. In a preferred embodiment, the fatty acid is selected from the group consisting of oleic acid, palmitic acid, linolenic acid, stearic acid, arachidonic acid, icosapentanoic acid, docosahexanoic acid and their ester derivatives.

In a preferred embodiment, when the process described above is carried out in a suitable fluid receptacle, the process is carried out in a storage or fixed bed reactor.

The invention also encompasses triglycerides prepared according to the process described above for use as a cocoa butter substitute for special foods or structured triglycerides, or as triglycerides such as breast milk for medical applications.

The following description is made by way of the following examples together with the description of the invention and the description of the invention in an unlimited trend in the reference.

Experimental course

matter

In this study, we try to produce other natural lipases. Table 1 lists not only the raw materials and suppliers of their species, but also commercially available lipase products used in this study. All the fatty acids and triglycerides used in this study are at least 99% pure, as obtained from Purolka (Switzerland) and reported by the supplier. Olive oil, sunflower oil, palm oil, canola oil, corn oil and nigella lacetti bar oil are obtained from suppliers in the provinces of Israel and Galilee. Fish oil, tri (hydroxymethyl) aminomethane and inorganic substrates are used as supports for lipase compounds coated with a surfactant containing diatomaceous earth. Aluminum and silica gel are obtained from Sigma (USA). Analytical grade N-hexane and all used solvents will be Biolab (Israel). Sucrose fatty acid esters including sorbitan monolaurate, sorbitan monomyristate, sorbitan monopalmitate, and sorbitan monostearate and sucrose fatty acid esters containing mono, di and tristearate sucrose ester mixtures of various HLB prices Fatty acid esters are obtained from Kao Pure Chemical Industries (Tokyo, Japan). Tri (hydromethyl) aminomethane (Tris) is obtained from Sigma (USA). Inorganic and organic substrates used as supports for modified lipases include diatomaceous earth (DE), aluminum and silica gel; Ion exchange resin is purchased from Sigma, USA. Oil purge C and oil purge C250L (macroporous, spherical, approximately 150-200 μm in diameter) are purchased from Rohm (Germany).

Table 1

Commercial name Raw material manufacturer Lilipase A-10FG Rispus zakonicus NR 400 Nagase, Japan Saiken 100 Rispus Zakonipus Nagase, Japan Lipase EC Aspergillus niger Ammono, Japan Lipase AY Candida Rousse Ammono, Japan Lipase LP Chromobacterium biscola breath Asahi Chemical Industry, Japan Lipase PS Shea HelpSepashia Ammono, Japan Lipase F Lithopus oriente Ammono, Japan Lipase F EC Lithopus oriente Extract Chemicals - Germany New race F Risofusnibens Ammono, Japan Lipase G Psilylium camembertity Ammono, Japan Lipase A Aspergillus niger Ammono, Japan Lipase M Mukur jabenikus

Lipase modification and immobilization through physical adsorption

The natural enzyme is firstly coated with a lipid surfactant or other enzyme activator such as gum arabic or polyethylene glycol. Typical enzymatic modification and immobilization procedures are as follows: Unprocessed enzymes (lipase, phospholipase, protease, and glycosidase, containing approximately 150 mg / L protein) are incubated with 1 L phosphate or tri- And stirred at < RTI ID = 0.0 > 10 C < / RTI > for 30 minutes. A lipid surfactant or other enzyme active agent (0.5 g) dissolved in ethanol (20 ml) or other solvent is added dropwise to the stirred solution. The resulting enzyme solution is pulverized at high frequency for 15 minutes and then vigorously stirred at 10 ° C for 3 hours. Insoluble organic (20 g, such as polypropylene, aluminum stearic acid or chitin) or inorganic substrate (20 g, such as celite, aluminum, silica gel or ceramic support) is added to the stirred enzyme solution. The solution is stirred at 10 DEG C for 5 hours or more. The precipitate is collected by centrifugation or filtration at 12000 rpm and then treated by one of two different methods:

1. Freeze dry the wet precipitate overnight at -20 ° C. The formed powder has a particle size of 50-1000 μm and is used for the batch enzyme reaction or becomes a granule to obtain a modified and fixed enzyme consisting of individual particles. The granulation process may be carried out using various adhesion reagents such as starch, methyl or ethyl cellulose, rubber, agarose or other adhesives. For example, starch granulation is induced as follows: starch solution (4 g starch / 20 ml water) turns to gel at 70 캜. The gel is cooled to 60 ° C and then the modified, fixed enzyme wet powder is visible. The mixture is leached at 40-60 ° C for 48 hours, dried and unified at a rapid rate. The immobilized enzyme is filtered to obtain particles in the range of 50-1000 mu m. These granulated enzymes are mainly used in tightly packed columns.

2. The wet sediment formed after deformation and immobilization is directly granulated with starch or other adhesion reagent as described above.

Enzyme modification and immobilization through ion absorption

The modification, immobilization and granulation processes described above can also be converted to ion exchange resins and used. The types of resin used are: strong and weak basic cation exchange resins, strong and weak acidic anion exchange resins and weakly polar and nonpolar ion exchange resins. Examples of commercially available resins (purchased from Sigma, USA) used in the experiments are: 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, Dianon SA-10A, Exola Cellulose, Sephadex and Sulfoxethyl Cellulose. Typically, the modified and immobilized enzyme is prepared according to the procedure described above.

Enzyme modification and immobilization through covalent bonding

Two different immobilization procedures were selected. According to the first, the enzyme is primarily coated with a surfactant, and then the lipase-surfactant compound is covalently linked to a lipid substrate having an active oxirane group. Natural lipase (1 g protein) is dissolved in a liter of phosphate buffer solution pH 5.8. The enzyme solution is actively stirred by a magnetic stirrer at 10 DEG C for 30 minutes. Sorbitan monostearic acid dissolved in 30 ml of ethanol is added dropwise to the stirred enzyme solution. The synthetic colloidal enzyme solution is sonicated for 10 minutes and stirred at 10 < 0 > C for 3 hours. 250 g (250 g) of either milk purge C or milk purge C and 12 ml of a 5% hydrogen peroxide solution are added to the enzyme solution and the synthetic suspension is hintshaked for 1 minute and then incubated at 23 ° C for 48 hours. The precipitate is filtered and washed with tris or phosphate buffer solution pH 5.8 and lyophilized overnight.

According to a second procedure, the lipase is covalently first bound to the lipid substrate and then the bound lipase is coated with a surfactant. The chemistry involved in this process is shown in FIG.

Natural lipase (1 g protein) is dissolved in 1 liter of Tris or phosphate buffer solution pH 5.8. The enzyme solution is actively stirred by a magnetic stirrer at 10 DEG C for 30 minutes. 250g (500g) of duffer C or dufage C and 12ml of 5% hydrogen peroxide are added to the enzyme solution and the synthetic suspension is handshaked for 1 minute and then incubated at 23 ° C for 48 hours. Sorbitan monostearate (0.5 g) dissolved in 30 ml ethanol is added dropwise to the suspension with shaking. The synthetic suspension is sonicated for 10 minutes and incubated at 10 ° C for 6 hours. The precipitate is filtered, washed with tris or phosphate buffer solution pH 5.8, and lyophilized overnight.

Because of the regulation of the activity of covalently immobilized lipases, fixed processes occur without the surfactant being added to the enzyme solution.

Natural lipase (1 g) is dissolved in 1 liter Tris or phosphate buffer solution pH 5.8. The enzyme solution is vigorously agitated with a magnetic stirrer at 10 DEG C for 30 minutes. A solution of 250 pg (500 g) or 5% hydrogen peroxide (250 g) is added to the enzyme solution and the synthetic suspension is handshaked for 10 minutes and then incubated at 23 ° C for 48 hours. The precipitate is filtered, washed with tris or phosphate buffer solution pH 5.8, and lyophilized overnight.

Protein measurement according to the Bradford method ensures that all enzymes prepared according to this method maintain 0.9-1.5 wt% protein.

Reaction model

Three different reaction models are used to test the activity of the modified and immobilized enzymes and compare it with the native enzymes and the immobilized enzymes without modification.

1. Esterification between Raw Acid and Dodecyl Alcohol in Organic Solvent Systems or Solventless Systems

The esterification reaction is initiated by adding a 10 mg lipase preparation to 10 ml normal hexane containing 200 mg raus acid and 186 mg dodecyl alcohol. The reaction is stirred at 40 占 폚. Samples are filtered with microfiltration (45 μm) and mixed with an equal volume of normal hexane solution containing normal hexa tecan, such as an internal standard.

2. Esterification exchange reaction between two triglycerides; tripolmitin and tristearin in organic solvent system or solventless system.

The esterification exchange reaction begins with the addition of a 10 mg lipase preparation to 10 ml normal hexane containing 40 mg tri-palmitin and 40 mg tris-stearin. The reaction is stirred at 40 占 폚. Samples are filtered with microfiltration (45 μm) and mixed with an equal volume of normal hexane solution containing normal hexa tecan, such as an internal standard.

3. Alcohol decomposition reaction between olive oil and cetyl alcohol in organic solvent system and solventless system.

The alcoholysis begins with the addition of a 10 mg lipase preparation to 10 ml normal hexane containing 500 mg olive oil and 500 mg cetyl alcohol. The reaction is stirred at 40 占 폚. The sample (50 μl) is periodically removed, filtered through a microfiltration (45 μm) and mixed with an equivalent amount of a normal hexane solution containing tridecanone, such as an internal standard.

Unless otherwise indicated, all experiments are performed under the conditions described above. Each esterification reaction is carried out repeatedly. In all experiments, normal hexane is dried with molecular sieves containing at least 6 mg / l water. Therefore, the water content in all reaction systems is below 30 mg / L.

Protein component

The protein components of modified lipase and modified and fixed lipase are determined by the microkehjdahl method.

Enzyme Activity in Batch Systems

The activity of the native enzyme and the immobilized enzyme in the activated degenerate enzyme, the enzyme immobilized and modified in the insoluble substrate, the insoluble substrate, is tested using a 1 ml vial containing the substrate. The vial is shaken at 40 DEG C and the sample is analyzed after a certain time interval. The response rate is determined by substrate conversion of less than 7% per mg of protein.

Stability of modified and fixed enzyme

The small stability of the keratin-modified and immobilized enzyme is tested in a jacketed column reactor (0.5 cm ID, 15 cm length) using an alcoholysis of olive oil and cetyl alcohol in normal hexane as in the reaction model. The enzyme particles are loaded into the column and the substrate solution circulates again through the filled enzyme. The circulation is stopped after an hour and the reaction solution is analyzed. Each working solution is discarded and the immobilized enzyme immobilized is washed with an organic solvent (normal hexane) before charging the new substrate solvent.

Experimental result

Example 1

Protein components of lipase products

The protein components of other natural lipases, modified lipases with sorbitan monostearate (SMS), celite-fixed lipases and celite-fixed SMS-modified lipases are measured as described above. The results are shown in Table 2 below.

The results show wide variability in protein composition among various manufactures. Protein content varies from 0.05% to 1.12% by weight, depending on the enzyme used in the preparation, coated with a surfactant and immobilized on a substrate. Similar enzymes, similar modifications seen in lipases, are treated with other lipid surfactants or other activating reagents and optionally immobilized on celite. The results of this study are shown in Table 3.

Table 2

Lilipase A-10FG

enzyme Protein content (%) Natural lipase 4.6 Lipase + SMS 1.62 Lipase + SMS / cellulite 0.11 Lipase / cellite 0.1

Lipase M

enzyme Protein content (%) Natural lipase 0.96 Lipase + SMS 1.8 Lipase + SMS / cellulite 0.12 Lipase / cellite 0.1

Lipase G - Amano 50

enzyme Protein content (%) Natural lipase 9.65 Lipase + SMS 1.11 Lipase + SMS / cellulite 0.06 Lipase / cellite 0.29

Lipase A - Amano 6

enzyme Protein content (%) Natural lipase 21.63 Lipase + SMS 4.85 Lipase + SMS / cellulite 0.225 Lipase / cellite 0.2

Lipase siken 100

enzyme Protein content (%) Natural lipase 4.84 Lipase + SMS 2.87 Lipase + SMS / cellulite 0.06 Lipase / cellite 0.04

Lipase F P-15

enzyme Protein content (%) Natural lipase 49.7 Lipase + SMS 9.31 Lipase + SMS / cellulite 0.70 Lipase / cellite 0.47

Lipase F-EC

enzyme Protein content (%) Natural lipase 49.7 Lipase + SMS 7.21 Lipase + SMS / cellulite 1.12 Lipase / cellite 0.2

Lipase EC

enzyme Protein content (%) Natural lipase 36.8 Lipase + SMS 6.51 Lipase + SMS / cellulite 0.48 Lipase / cellite 0.39

Lipase PS

enzyme Protein content (%) Natural lipase 7.5 Lipase + SMS 0.72 Lipase + SMS / cellulite 0.16 Lipase / cellite 0.08

Lipase new race F

enzyme Protein content (%) Natural lipase 25.1 Lipase + SMS 2.17 Lipase + SMS / cellulite 0.24 Lipase / cellite 0.013

Lipase LP

enzyme Protein content (%) Natural lipase 6016 Lipase + SMS 4.6 Lipase + SMS / cellulite 0.65 Lipase / cellite 0.21

Lipase AY - Amano 30

enzyme Protein content (%) Natural lipase 5.14 Lipase + SMS 2.61 Lipase + SMS / cellulite 0.05 Lipase / cellite 0.05

Table 3

enzyme Protein content (%) Lipase + sorbitan monostearate lipase + sorbitan monostearate / celite 1.6140.112 Lipase + sucrose ester HLB = 5 lipase + sucrose ester HLB = 5 / cellite 1.00.128 Lipase + sucrose ester HLB = 11 lipase + sucrose ester HLB = 11 / cellite 0.150.051 Lipase + sucrose ester HLB = 16 lipase + sucrose ester HLB = 16 / cellite 0.10.04 Lipase + sorbitan monolaurate lipase + sorbitan monolaurate / cellite -0.128 Lipase + sorbitan tristearate lipase + sorbitan tristearate / cellite 1.4360.134 Lipase + sorbitan trioleate acid lipase + sorbitan trioleate acid / cellite -0.115 Lipase + monooleate lipase + monooleate / cellite -0.140 Lipase + lecithin lipase + lecithin / cellite -0.16 Lipase + stearic acid lipase + stearic acid / cellite 1.650.108 Lipase + octadecanoic acid lipase + octadecanoic acid / cellite 1.170.102 Lipase polyoxyethylene-8-stearic acid lipase polyoxyethylene-8-stearic acid / 2.350.115 Lipase + polyethylene glycol lipase + polyethylene glycol / cellite -0.10 Lipase Arabic Rubber Lipase Arabic Rubber / Celite -0.172

Example 2

Esterification reaction, esterification exchange reaction and alcohol decomposition activity of lipase products

The comparison makes the activity of enzymes of various lipase production. For the purposes of this comparison, the results (shown in Table 4) are expressed as reaction rate (ri).

Table 4

Lilipase A-10FG

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0.16 0.01 0 Lipase + SMS 7.43 5.2 2.1 Lipase + SMS / cellulite 27.4 18.1 7.1 Lipase / cellite 4.5 0.8 0.4

Lipase M

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0.2 0 0 Lipase + SMS 6.7 4.8 2.0 Lipase + SMS / cellulite 22.3 16.4 6.7 Lipase / cellite 3.2 0.1 0.3

Lipase PS

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0.3 0 0 Lipase + SMS 5.2 4.3 1.6 Lipase + SMS / cellulite 18.3 12.5 6.5 Lipase / cellite 4.5 0.4 0.2

Lipase LP

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0.3 0 0 Lipase + SMS 4.9 3.3 1.4 Lipase + SMS / cellulite 15.5 9.5 2.8 Lipase / cellite 2.3 0.2 0.35

Lipase EC

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0 0 0 Lipase + SMS 0.7 1.9 1.9 Lipase + SMS / cellulite 1.9 10.3 5.4 Lipase / cellite 0.1 1.1 0.3

Lipase AY Marano 30

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0 0 0 Lipase + SMS 2.2 0.3 0.7 Lipase + SMS / cellulite 16.3 0.8 1.3 Lipase / cellite 0.9 0.05 0.4

Lapase G

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0 0 0 Lipase + SMS 0.1 0.15 0 Lipase + SMS / cellulite 1.4 0.4 0 Lipase / cellite 0.1 0 0

Lipase A

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0 0 0 Lipase + SMS 0.9 0.5 0.1 Lipase + SMS / cellulite 3.0 1.2 0.6 Lipase / cellite 0.3 0.1 0

Lipase F-AP15

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0.3 0 0 Lipase + SMS 6.7 4.9 1.9 Lipase + SMS / cellulite 26.4 12.7 5.4 Lipase / cellite 0.8 0.93 0.3

Lipase F-EC

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0.4 0 0 Lipase + SMS 7.1 3.4 1.5 Lipase + SMS / cellulite 23.5 10.5 5.4 Lipase / cellite 1.9 0.6 0.6

Lipase siken 100

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0.15 0 0 Lipase + SMS 6.9 4.2 2.4 Lipase + SMS / cellulite 29.3 10.3 8.2 Lipase / cellite 1.2 3.2 0.4

Lipase new race F

enzyme Reaction rate (esterification reaction) (μmol / min.mg protein) Reaction rate (esterification exchange reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) natural 0 0 0 Lipase + SMS 1.3 1.3 0.4 Lipase + SMS / cellulite 6.4 6.8 0.5 Lipase / cellite 0.2 0.1 0

These results show that the natural lipase used in this study lacks the esterification and transesterification reactions that can be measured with the low moisture concentration used. On the other hand, the deformation of the surfactant sorbitan monostearate and the immobilization on the celite are the result of the esterification reaction and the transesterification reaction. When enzymes are presented in both of these treatments, it has been studied that the rate of reaction of esterification and transesterification increases to a greater level. From the numerical results shown in Table 4, it is clear that no synergy can be expected between the two stages of transformation. For example, treatment of the surfactant and immobilization on the substrate.

In Fig. 3, when inter-molecular esterification reaction of tripalmitic acid and capric acid is used to cause the catalyst, Saiken-100 (triangle) and lyrease A10-FG (quadrangle) fixed to diatomite and immobilized with surfactant, . The measured intermolecular esterification reaction rate is 0.096-0.104 mmol / min per mg of the biocatalyst.

Example 3

Fatty Acid Properties of Lipase Compounds Coated with Fixed Surfactant

The properties of lipase compounds coated with surfactants immobilized on inorganic substrates of the present invention with respect to other fatty acid substrates are tested by monitoring the intermolecular esterification reaction of fatty acids of various chain lengths with tripalmitin. The results are summarized in Table 5. The reaction conditions are as follows:

The intermolecular esterification reaction activity of a natural lipase product is tested in normal hexane using triplymethine (50 mg) as a triglycerol substrate and capric acid (35 mg) as a medium chain fatty acid substrate. The intermolecular esterification reaction activity of 20 mg of lipase (5-10% protein component) coated with 10 mg non-fixed surfactant in normal hexane (10 ml) or lipase (0.2-2% protein component) coated with an inorganic substrate surfactant Are tested using the same substrate.

Table 5

fatty acid Fatty acid chain length Intermolecular esterification reaction rate (mmol / min.mg biocatalyst) Butyrate (C4) 0.055 Hexanoic acid (C6) 0.06 Octanoic acid (C8) 0.09 Decanoic acid (C10) 0.104 Lauric acid (C12) 0.15 Myristic acid (C14) 0.2 Palmitic acid (C16) 0.25 Stearic acid (C18) 0.26 Oleic acid (C18: 1) 0.3 Arachidonic acid (C20) 0.28 Behenic acid (C22) 0.28

From Table 5, it can be seen that the lipase compound coated with the fixed surfactant according to the present invention catalyzes the intermolecular esterification reaction of the tri-palmitin with the fatty acid having the 1.3-position characteristic. The concentration of the hydrolysis product does not exceed 5 wt% of the initial tricapatite concentration.

The intermolecular esterification reaction activity of a lipase compound coated with a surfactant immobilized on an inorganic substrate according to the present invention is affected by the fatty acid selected for use as a substrate. Thus fatty acids with long alkyl chains such as palmitic acid and stearic acid are more basic than lipase compounds coated with surfactants fixed to DE rather than fatty acids with short alkyl chains.

Example 4

Effect of Surfactant Selection on Lipase Compounds Coated with Surfactants Immobilized on Inorganic Substrates

The sorbitan fatty acid esters selected to coat the lipase in Table 6 show that they affect the intermolecular esteractivation activity of the lipase compounds coated with the surfactant immobilized on celite.

When sorbitan monostearate is used to coat, the intermolecular ester reaction activity of the compound is increased. However, shorter fatty acid chain lengths in sorbitan esters result in a decrease in the activity of the compound.

Table 6

Lilipase A-10FG

enzyme Reaction rate (ester reaction) (μmol / min.mg protein) Reaction rate (transesterification reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) Natural lipase Lipase / Cellite 0.164.5 0.010.1 00.4 Lipase + sorbitan monostearate lipase + sorbitan monostearate / celite 7.4327.4 5.7515.5 1.22.3 Lipase + sucrose ester HLB = 5 lipase + sucrose ester HLB = 5 / cellite 23.622.4 4.57.5 3.55.5 Lipase + sucrose ester HLB = 11 lipase + sucrose ester HLB = 11 / cellite 31.325.3 12.530 6.28.2 Lipase + sucrose ester HLB = 16 lipase + sucrose ester HLB = 16 / cellite 8.516.2 212.5 1.14.2 Lipase + sorbitan monolaurate lipase + sorbitan monolaurate / cellite 6.27.1 411.5 0.62.1 Lipase + sorbitan tristearate lipase + sorbitan tristearate / cellite 10.122.5 431 1.97.2 Lipase + sorbitan trioleate acid lipase + sorbitan trioleate acid / cellite 18.327.3 7.520 2.69.3 Lipase + sorbitan monooleate lipase + monooleate / celite 6.28.5 2.510 1.44.2 Lipase + lecithin lipase + lecithin / cellite 10.216.2 3.510 1.153.2 Lipase + stearic acid lipase + stearic acid / cellite 6.313.4 3.7514 1.65.3 Lipase + octadecanoic acid lipase + octadecanoic acid / cellite 6.214.6 311.5 0.813.4 Lipase polyoxyethylene-8-stearic acid lipase polyoxyethylene-8-stearic acid / 6.513.5 4.416 2.16.2 Lipase + polyethylene glycol lipase + polyethylene glycol / cellite 5.49.2 00.4 00.4 Lipase Arabic Rubber Lipase Arabic Rubber / Celite 9.311.1 25 0.51.1

Experiment 5

Physical bonding of modified lipase in inorganic substrates

The esterification, transesterification and alcoholysis of lipase A-10FG immobilized on a variety of inorganic substrates is a modification of sorbitan monostearate and the activity of immobilized lipase A-10FG on other inorganic substrates is compared and measured. The results are shown in Table 7.

Table 7

Enzyme / insoluble substrate ^* Reaction rate (ester reaction) (μmol / min.mg protein) Reaction rate (transesterification reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) Natural Lipase A10-FG Lipase + SMS 0.167.43 0.015.2 02.1 Lipase + SMS / Celite (acid-wash) Lipase / Celite (acid-wash) 27.44.5 18.10.8 7.10.4 Lipase + SMS / Celite (acid-free) Lapase / Celite (acid-free) 25.43.5 13.50.5 6.00.1 Lipase + SMS / fatty acid treated celllite ^* lipase / fatty acid treated celllite 41.319.7 27.49.5 17.34.6 Lipase + SMS / aluminum lipase / aluminum 23.21.6 11.10.3 5.40.4 Lipase + SMS / aluminum monostearate lipase / aluminum monostearate 45.319.2 29.19.8 19.26.1 Lipase + SMS / silica lipase / silica 21.33.2 10.80.4 5.70.2 Lipase + SMS / calcium carbonate lipase / calcium carbonate 18.62.4 12.30.4 6.10.2 Lipase + SMS / calcium sulfate lipase / calcium sulfate 16.60.3 11.30.2 5.60.3

^* Fatty acids (or their derivatives), the processing of Celite (Celite of hydrophobicity) are prepared as follows: Celite (100g) is hyeontaek in phosphate buffer solution (100ml) of pH5.7. Free fatty acids or derivatives thereof (stearic acid or esters per fatty acid) dissolved in ethanol (5 g / 30 ml) are added dropwise to a vigorously stirred suspension at 70 ° C. The suspension is stirred for 2 hours, filtered, washed with water, and lyophilized.

The most suitable carriers for the immobilized enzyme used in this study are aluminum monostearate and fatty acid treated celite. As shown in Table 7, unmodified lipase or fatty acid treated celite fixed to aluminum monostearate exhibits good reaction activity in all three reaction models. The results shown in this table show that enzymes with surfactants in the first step are modified and then the enzyme modified in the insoluble substrate treated with fatty acids causes a further increase in the activity of the enzyme. As shown in the above table, the activity of the lipase modified and fixed in the fatty acid derivative in the insoluble substrate treated with the fatty acid derivative (celite treated with the aluminum monostearate, the fatty acid derivative) was lowered to that of the immobilized lipase Lt; / RTI >

Example 6

Effect of selection of ion exchange resin on enzyme activity

The lysozyme A-10FG immobilized on a variety of ion exchange resins and the esterification, transesterification and alcoholysis of the modified enzyme with sorbitan monostearate before immobilization were compared. The results of this comparison are shown in Table 8.

Lipase / ion exchange resin * Reaction rate (ester reaction) (μmol / min.mg protein) Reaction rate (transesterification reaction) (μmol / min.mg protein) Reaction rate (alcohol decomposition) (μmol / min.mg protein) Dowex 22 Dowex 22 + SMS 4.932.2 4.723.1 1.96.3 Dowex 1x2-400 Dowex 1x2-400 + SMS 6.929.6 3.525.4 1.16.1 Dowex 2x8-100 Dowex 2x8-100 + SMS 5.521.3 4.716.2 2.27.3 Cellulose Phosphate Cellulose Phosphate + SMS 3.56.3 0.21.5 0.11.1 AmberLight IRA-95 AmberLight IRA-95 + SMS 7.626.2 0.812.5 0.92.3 AmberLight IRA-200 AmberLight IRA-200 + SMS 9.228.6 3.118.9 1.45.3 AmberLight IRA-900 AmberLight IRA-900 + SMS 6.137 4.228.3 2.38.6 Dianon SA-10A Dianon SA-10A + SMS 6.137 2.819.5 1.211.3 Etoella Cellulose Etoella Cellulose + SMS 5.429.5 2.312.2 1.75.7 Sephadex Sephadex + SMS 1.52.9 0.20.9 0.10.8 Sulfoxyethylcellulose Sulfoxyethylcellulose + SMS 5.632.3 3.217.0 2.512.6 Amberlite XAD-7 (weak polarity) Amberlite XAD-7 + SMS 8.337.3 4.529.5 3.817.5 Amberlite XAD-16 (non-polar) 6.7 6.5 4.3 Amberlite XAD-16 + SMS 33.4 24.4 14.3

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

These results show that the esterification and ester exchange reactions of the immobilized modified enzyme are increased. Moreover, from Table 8, ion exchange resins containing hydrophobic groups in their structure act as carriers of the enzyme modified with surfactants.

Example 7

Biosynthesis of structured triglycerides from other oils using lipase compounds coated with a surfactant immobilized on an inorganic substrate

Table 9 shows the intermolecular esterification reaction rates between the normal hexane system and the other duck and capric acid in the 60 DEG C solventless system. The intermolecular esterification reaction is catalyzed by a DE-immobilized surfactant lipase A10-FG compound.

Surfactant-lyophase compounds immobilized on diatomaceous earth catalyze the intermolecular esterification of various oils with normal hexane and capric acid in solvent-free systems. When olive oil is used, an intermolecular esterification reaction is obtained. Similar to the reaction rate obtained when the intermolecular esterification reaction is carried out in a solventless system.

Table 9

Raw material of oil R.R. (mmol / min · mg biocatalyst) In normal hexane R.R. (mmol / min · mg biocatalyst) No solvent system Olive oil 0.52 0.73 Fish oil 0.4 0.53 Sunflower oil 0.3 0.42 palm oil 0.2 0.31 Canola oil 0.32 0.45 Corn oil 0.24 0.41 Nigelasetiba oil 0.2 0.46

Enzymatic activity in organic solvents is maintained by granulation with other coupling reagents such as starch, ethyl cellulose, and rubber. The granulation process also promotes flow rate in the column filled with the enzyme. The binders used in these figures are as follows:

7 starch 2%

8 Ethylcellulose 2%

9 Arabic rubber 2%

10 Ethylcellulose 2%

11 Agarose 2%

12 starch 4%

13-16 Starch 2%

Immobilization performed in Celite is shown in FIGS. 5-9 and 12-13. Immobilization to Amberlite IRA-900 shows immobilization of aluminum monostearate in FIGS. 10 and 11 and FIG. 14, and Immobilization XAD- 16 and FIG. 16 show immobilization of Amberlite XAD-7. Except for the experiment shown in Figure 13 in all cases where all lipases are immobilized by a surfactant, the surfactant is sorbitan tristearate and the surfactant used in this treatment is sorbitan monostearate.

Example 9

Covalent linkage between modified lipase and unmodified lipase in a milk purge

Lipase is covalently linked to the lipid according to the manufacturer's description. Covalent bonding is described in two steps described above.

Table 10

Lilipase A10-FG

Enzyme form Conversion Rate (%) Natural lyrease A10-FG 2 Lipitor 4 Lee Lipase + Yu from fuzzy 19 Lipophase + SMS in the fluid after 48 hours 26

Lilipase LP

Enzyme form Conversion Rate (%) Natural lyrease A10-FG One Lipitor 2 Lee Lipase + Yu from fuzzy 14 Lipophase + SMS in the fluid after 48 hours 18

Lipitorase PS

Enzyme form Conversion Rate (%) Natural lyrease A10-FG One Lipitor 2 Lee Lipase + Yu from fuzzy 8 Lipophase + SMS in the fluid after 48 hours 12

Other lipases exhibit other intermolecular esterification reaction activity similarly treated. This result belongs to the other raw materials of lipase used. All natural lipases exhibit a very low intermolecular esterification reaction activity under the conditions described because their activity is weakly increased when they are covalently immobilized in milk purges. When the lipase is coated with a surfactant, their intermolecular esterification reaction activity is weakly increased. A very large conversion in its intermolecular esterification reaction product with 1,3-positional properties after 2 hours is when the lipase is first covalently immobilized in the oil puddle and then coated with a surfactant. The lipase A10-FG coated with a surfactant and immobilized in a milk puff yields a high intermolecular esterification reaction activity in the three lipases tested in Table 10. [

Example 10

Effect of binding agent on enzyme activity

As described earlier, other binders are used for granulation of transformed-fixed lipaena. Table 11 shows the average conversion rates of the olive oil of its wax ester in the first and second runs using different binders to granulate the lipase A10-FG immobilized on celite and modified with sorbitan monostearate. Percent of all conjugates is the 2% dry weight of the particles. In these experiments, the granulated enzyme is loaded into the column and used 10 times.

The use of lipolytic A-10FG granulated with other binders (2%) after being modified with sorbitan monostearate and fixed in celite is the average rate of change of olive oil in its wax ester in the first and second runs. Reaction conditions: 100 ml of normal hexane containing 2 g of olive oil and 2 g of cetyl alcohol is recirculated at a circulation rate of 2.5 ml / min and at a temperature of 40 ° C above the fixed enzyme bed. Column size: length 12 cm, inner diameter 0.75 cm,

Table 11

concrete Conversion Rate starch 92 Ethyl cellulose 44.6 Methyl cellulose 38.1 Agarose 16.8 gelatin Inactive Polyvinylpyrrolidone Inactive gum arabic 49.1 Jan rubber 33.2 Karaya rubber 49.5 Tragacanth rubber 20.1 Locas rubber 33.6

From the table above, starch, gum arabic and karaya rubber are the most useful combinations tested in this study to produce active biocatalysts.

Claims (51)

Lipase preparations that constitute a lipase compound immobilized on insoluble and insoluble substrates and coated with a surfactant. 2. The lipase preparation according to claim 1, wherein the lipase compound coated with the surfactant is covalently or ionically or physically immobilized on the insoluble substrate. The preparation of lipase according to claim 1, wherein the insoluble substrate is selected from the group consisting of an inorganic insoluble substrate and an organic insoluble substrate. 4. The lipase product according to claim 3, wherein the inorganic insoluble substrate is selected from the group consisting of aluminum, diatomaceous earth, celite, calcium carbonate, calcium sulfate, ion exchange resin, silica gel and activated carbon. 5. The article of manufacture of claim 4, wherein the ion exchange resin is selected from the group consisting of Amberlite and Dowex. 4. The lipase preparation according to claim 3, wherein the organic insoluble substrate is selected from the group consisting of milk purge, ethylsulfoxyxycellulose and aluminum stearic acid. The preparation of lipase according to claim 1, wherein the component of the lipase is 2-20 wt% of the lipase compound coated with the surfactant. The preparation of lipase according to claim 1, wherein the component of the lipase is 0.01-1.0 wt% of the product. The preparation of lipase according to claim 1, wherein in the lipase compound coated with a surfactant, the surfactant comprises a fatty acid bound to a hydrophilic moiety. 10. The composition of claim 9, wherein the fatty acid is selected from the group consisting of monolaurate, monomyristate, monoparmate, monostearate, dillate, dimyristate, dipalmitate, distearate, , Triampharmate and tristearate. ≪ RTI ID = 0.0 > 15. < / RTI > The product of claim 9, wherein the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxyl group, and an oxy organic moiety. 12. The lipase preparation according to claim 11, wherein the sugar is selected from the group consisting of sorbitol, sucrose, glucose, and lactose. 10. The lipase preparation according to claim 9, wherein the fatty acid and the hydrophilic moiety are bonded through an ester bond. The lipase preparation according to claim 1, wherein the lipase is derived from a microorganism. The method of claim 1, wherein the lipase derived from a particular species Brewer call terrier sp., Khanna Dida not track urticae B, Khanna Dida base test, build even Pseudomonas sp., Candida not track urticae A, pig pancreatic lipase, Fumiko La spp., Mucor melehei, Rispops spp., Seedofluor., Cannadidocylindrakas, Aspergillus niger, Rispops oidze, Mucor javanicus, Rizophus sp., Risofus zaponikus Candida albicans, Candida albicans, and Candida albicans . 15. The lipase preparation of claim 14, wherein the lipase is derived from a multicellular organism. Wherein the lipase preparation comprises an insoluble substrate immobilized on the insoluble substrate and a lipase compound coated with a surfactant provided in the organic solvent. 18. The article of manufacture of claim 17, wherein the organic solvent is selected from the group consisting of n-hexane, toluene, isooctane, n-octane, benzene, cyclohexane and di-isopropyl ether. 18. The lipase product according to claim 17, characterized by the use of an oil and fat esterification reaction, an intermolecular esterification reaction, and an ester exchange reaction and a catalytic reaction for triglycerol and fatty alcohol alcoholysis. 20. The manufacture of a lipase according to claim 19, characterized by the use to produce a catalytic reaction with 1,3-positional properties with respect to triacylglycerol. The product of claim 1, wherein the product is granulated. The lipase preparation according to claim 1, wherein the insoluble substrate is modified with a fatty acid derivative. The lipase preparation according to claim 1, characterized by its use in a reaction environment without water addition. A method of enhancing the stability of a fixed and coated lipase compound with a surfactant comprising granulation prior to contacting to react with the substrate. (a) contacting the lipase in an intermediate solution with the surfactant at a time and concentration, temperature sufficient to obtain the coated lipase; And (b) contacting the lipase in an intermediate solution with an insoluble substrate under conditions for a time sufficient to obtain immobilization of the lipase at the substrate ≪ / RTI > is coated with a surfactant immobilized on an insoluble substrate comprising the steps < RTI ID = 0.0 > of: < / RTI > 26. The method of claim 25, wherein the lipase is first contacted with an insoluble substrate and then contacted with a surfactant. 26. The method of claim 25, wherein the lipase is first contacted with a surfactant and then contacted with an insoluble substrate. 26. The method of claim 25, (c) Separation of lipase compounds coated with a surfactant fixed to the substrate from the aqueous solution formed ≪ / RTI > 29. The method of claim 28, (d) drying a lipase compound coated with a surfactant fixed to the substrate ≪ / RTI > 30. A process according to claim 29, characterized in that the drying is effected by freeze drying 30. The method of claim 29, wherein the lipase compound coated with the substrate-immobilized surfactant is dried to a moisture content of less than 100 ppm by weight. 26. The method of claim 25, wherein the aqueous solution is a buffer solution. 26. The method of claim 25, (i) dissolving the surfactant in an organic solvent to ensure a dissolved surfactant solution (ii) mixing the lipase and the dissolved surfactant solution in the intermediate aqueous solution Characterized in that the lipase and the surfactant are brought into contact with the intermediate aqueous solution. 26. The method of claim 25, further comprising sonicating the aqueous solution. 26. The method of claim 25, wherein the insoluble substrate is selected from the group consisting of aluminum, diatomaceous earth, celite, calcium carbonate, calcium sulfate, ion exchange resins, silica gel, activated carbon, milk purge, ethylsulfoxylcellulose, celite treated with aluminum stearic acid and fatty acid derivatives, ≪ / RTI > inorganic substrate. 26. The method of claim 25, wherein the surfactant comprises a fatty acid bonded to a hydrophilic moiety. 37. The composition of claim 36, wherein the fatty acid is selected from the group consisting of monolaurate, monomyristate, monoparmate, monostearate, dillate, dimyristate, dipalmitate, distearate, ≪ / RTI > tricyclodecanoic acid, tricyclodecanoic acid, tristearic acid, tristearic acid, 37. The method of claim 36, wherein the hydrophilic moiety is selected from the group consisting of a sugar, a phosphate group, a carboxyl group, an oxy organic moiety. 39. The method of claim 38, wherein the sugar is selected from the group consisting of sorbitol, sucrose, glucose and lactose. 37. The method of claim 36, wherein the fatty acid and hydrophilic moieties are linked in an ester linkage. 26. 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. 42. The method according to claim 41, wherein the lipase is selected from the group consisting of Brucolieria sp., Canna diadancetracica B, Canna di dulcis sp., Sedomonas sp., Candida anthracis A, Pork pancreatic lipase, Fumiko sp, , Rhizopus spp., Rhizopus spp., Candida spp., Candida spp., Candida spp., Candidiasis spp., Aspergillus niger, Rhizopus oryzae, Mucor javanicus, Rhizopus sp. ≪ / RTI > is derived from a species selected from the group. The process of preparing triglycerol structured by esterification, acidolysis, transesterification, and intermolecular esterification between two substrates composed of a substrate and a lipase compound coated with a surfactant immobilized on an insoluble substrate. 45. The process of claim 44, wherein the lipase compound coated with a surface-immobilized surfactant is in contact with a substrate in an organic solvent. 45. The process of claim 44, wherein at least one of the substrates is selected from the group consisting of oils, fatty acids, triglycerol, and fatty alcohols. 47. The process of claim 46, wherein the oil is selected from the group consisting of olive oil, soybean oil, peanut oil, fish oil, palm oil, cottonseed oil, sunflower oil, nigella sativa oil, canola oil and corn oil. 47. The process of claim 46, wherein the fatty acid is selected from the group consisting of an intermediate chain and short chain fatty acids and an ester derivative thereof. 47. The process 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, icosapentanoic acid, docosahexanoic acid and their ester derivatives. 45. The process of claim 44, carried out in a tank reactor or a fixed bed reactor. 44. The process of claim 44, wherein the triglyceride is selected from the group consisting of cocoa butter substitutes, triglycerides such as breast milk for special diets, or triglycerides prepared for use as structured triglycerides for medical applications.