JPS6214781A - Immobilization of lipase - Google Patents

Abstract

PURPOSE:To improve the yield and stability of the activity of lipase, by microencapsulating an aqueous dispersion of lipase with amphiphatic coating film and including and immobilizing the lipase microcapsule using a hydrophobic photo-crosslinkable resin. CONSTITUTION:A lipase microcapsule is prepared by including an aqueous dispersion of lipase with an amphiphatic film in an organic solvent containing one or more amphiphatic substances. The obtained lipase microcapsule is mixed with a hydrophobic photo-crosslinkable resin prepolymer (abbreviated as PCRP) having an average molecular weight of 2,000-20,000, and the mixture is irradiated with actinic light in the presence of a photo-polymerization initiator to effect the inclusion and fixing of the lipase microcapsule in the hydrophobic photo- crosslinkable resin. The PCRP is preferably an unsaturated polyester having an average molecular weight of 2,000-20,000 and obtained by introducing photo- polymerizable ethylenic unsaturated group at both terminals of a nonionic surfactant consisting of a polyoxyethylene polyoxypropylene block polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing lipase. Specifically, lipase microcapsules are made into hydrophobic photocrosslinkable resin by microcapsulating a lipase aqueous suspension with an amphipathic film, mixing it with PCRP, and irradiating it with actinic rays in the presence of a photopolymerization initiator. The present invention relates to a method for immobilizing lipase, which is comprehensively immobilized within.

(Industrial Application Field) Lipase immobilization is achieved by performing oil and fat processing, which has conventionally been carried out under high temperature and pressure, such as decomposition of fats and oils, ester synthesis of glycerides, transesterification, etc., at room temperature and normal pressure, and This is an indispensable and important technology for industrializing this technology, and is also expected to save energy and resources.

(Prior art) As a conventional lipase immobilization method, Hans Prandenberger (Ran s, BRANDENBERGE)
R) is a covalent bonding method using polystyrene-based ion exchange resin (Rev, Ferment).

Ind.Aliment, 11,237. (1956
)) Since then, we have developed a method for enclosing lipase in microcapsules made of polymers such as polystyrene, silicone, and ethylcellulose [Kitajima, Miyano, Kondo, Industrial Chemistry Journal, P, 493, 2°72, (1969)] ), or a method of enclosing lipase in a polymeric gel such as polyacrylamide or polyvinyl alcohol [Unexamined Japanese Patent Publication No. 1987-1]
[29789] and many other research examples have been reported.

In addition, as a comprehensive immobilization method using photocrosslinkable resin,
A method in which lipase is adsorbed onto Celite, etc., and the surrounding area is surrounded by a hydrophobic photocrosslinkable resin having a main chain of polypropylene [Fukui, Nichi, Chemistry and Biology, P620, 19, 1
0. (1981)) have been reported.

(Problem to be solved by the invention) Lipase is attached to the surface of a water-insoluble carrier through covalent bonding, ionic bonding,
Alternatively, in the case of immobilization by molecular adsorption, the activity yield during immobilization is high because the lipase is on the surface, and it is significant from the reaction kinetics. The stability of lipase is low because it is directly affected by When lipase is comprehensively immobilized using a polymer, the influence of the external environment is alleviated because a polymer membrane or polymer gel is interposed between the lipase and the substrate, and highly stable immobilized lipase can be obtained. , a decrease in the kinetic reaction rate depending on the permeability of the substrate to the polymer often occurs. The industrial productivity of immobilized enzymes is expressed as the product of the activity yield and the rate of activity decrease (deterioration) over time, that is, the stability.
It can be said that it is an excellent immobilized enzyme. From this point of view, there are only a limited number of conventional immobilization methods that simultaneously satisfy both activity yield and stability, and in particular, no such method has been found for lipase. Furthermore, in the conventional lipase immobilization method, the activity expression rate is extremely low because it is not possible to maintain hydration conditions that can completely activate lipase. This is because in order to improve substrate permeability into the carrier, it is necessary to make the carrier highly hydrophobic, but if there are many hydrophobic groups, the water required for hydration is This is because it becomes difficult to get close to the immobilized lipase, and the lipase cannot be activated sufficiently.

In the microcapsule entrapment method, water necessary for lipase can be sufficiently maintained, but there are problems with the permeability of the substrate and the physical strength of the microcapsule film, making industrial production difficult.

Most of the conventional lipase immobilization methods were developed for the purpose of hydrolyzing oils and fats, and are effective for the hydrolysis of triglycerides, but they cannot be used for ester synthesis reactions using fatty acids and glycerin as substrates. In all cases, the reaction rate is low and the reaction is not effective for the purpose of ester synthesis. This is because, in the case of triglyceride hydrolysis, it is possible to homogenize the substrate by dissolving the triglyceride in a water-saturated low polar solvent such as benzene, toluene, or heptane, but when using fatty acids and glycerin as the substrate, This is because both must be passed into the carrier as an emulsion by an emulsification operation, and the substrate will not be sufficiently supplied.

(Means for solving the problem) The present inventors immobilized lipase to maintain its high activity for a long period of time, and to industrially perform oil and fat hydrolysis reactions, ester synthesis reactions, transesterification reactions, etc. As a result of intensive research aimed at achieving this goal, we found that by microcapsulating a lipase suspension in water with an amphiphilic film and entrapping and immobilizing the lipase microcapsules using a hydrophobic photocrosslinkable resin, lipase activity can be maintained for a long period of time. We have invented a method that can be maintained and industrially process oils and fats. That is, in the present invention, a lipase aqueous suspension is made into microcapsules with an amphipathic film in an organic solvent containing an amphipathic substance, and the average molecular weight is 200.
0 to 20,000 PCRP, irradiation with actinic rays in the presence of a photopolymerization initiator, and entrapping and immobilizing lipase microcapsules within a hydrophobic photocrosslinkable resin. .

The lipase used in the present invention may be derived from an animal organ or a microorganism, but from the viewpoint of industrial production,
It is desirable to use a microbial lipase that is heat resistant and can be used under a wide range of reaction conditions. As a microbial lipase, Rhizopus delemer (Rhiz.

pus delemer), Pseudomonas fluorescens (Pseudomonas f'1uor)
escens), Candida cylindracea (Cand
ida cylindracea).

Chromobacterium viscosum (Chrom.

bacterium viscosum) and the like.

The lipase aqueous suspension used in the present invention is a solution in which lipase is suspended in a buffer solution, and this solution contains various metal ions, albumin, globulin, and casein as stabilizers to maintain lipase activity. Proteins, glycerin, etc.
Polyhydric alcohol compounds such as propylene glycol, ester compounds thereof, water-dispersible phospholipids, etc. can be added.

The amphipathic substance used in the present invention is a substance that constitutes an amphiphilic film, and is broadly classified into a film-forming substance and a film-strengthening component. Lecithin derived from animals and plants.

Phosphosphoglyalides such as ethanolamine cephalin and serine cephalin, phosphosphingosides such as inositol phosphorus ai and sphingomyelin, and their compounds, alkyl polyoxyethylene glycol ethers having 4 to 30 carbon atoms , alkylsulfinyl alcohols, alkylglufsides, and alkyltrimethylammonium salts, or a mixture of two or more of them, which has a high micelle-forming ability, is a film-forming substance. However, amphiphilic films can be formed.

Film-strengthening components include cholesterol, animal steroids such as vitamin D and their esters, plant steroids such as phytosterols and their esters, bile such as cholic acid, deoxycholic acid, chenodeoxycholic acid, glucocholic acid, and taurocholic acid. Examples include acids and their salts, diacyl phosphoric acid compounds, proteins, etc., and cholesterol in particular is well known as a constituent of biological membranes and is effective in maintaining the physicochemical properties of amphiphilic membranes. These reinforcing components can be used in combination with film-forming substances to strengthen the amphiphilic film and impart chemical properties to it.

The amount of amphiphilic substance used is 0.1 to 0.1 as an organic solvent solution.
The content is preferably 3.0% by weight.

The organic solvent used in the present invention is hexane.

R prime number 4 to 2 such as heptane, octane, cyclohexane, etc.
0 aliphatic hydrocarbon compound, chloroform.

Examples include halogenated hydrocarbon compounds having 1 to 4 carbon atoms such as trichloroethylene and carbon tetrachloride, fatty acids having 2 to 20 carbon atoms such as caproic acid, caprylic acid, and oleic acid, and their esters, which inhibit photopolymerization reactions. It is desirable that the material does not contain two or more conjugated double bonds. Moreover, these organic solvents are not limited to being used alone, but may be used in combination of two or more types.

The PCRP used in the present invention is mainly composed of polyoxyethylene/polyoxypropylene block polymer, and the oxyethylene/oxypropylene ratio is 1/10 to 1.
/3 is desirable; if it is larger than 1/3, the hydrophilic dust of PCRP will increase, and the permeability of the substrate oil will decrease significantly; if it is smaller than 1/10, the lipase microcapsule The effect of the oxyethylene group that protects the surface decreases, making it impossible to stably hold the lipase microcapsules.

The hydrophobic photocrosslinkable resin of the present invention has an average molecular weight of 2000 to
20,000 PCRP is obtained by forming a three-dimensional crosslinked structure through a photopolymerization reaction, and if the average molecular weight of PCRP is smaller than 2,000, it will be difficult to secure enough space to enclose the lipase microcapsules.
If it is larger than 20,000, the lipase microcapsules tend to detach from the hydrophobic photocrosslinkable resin, which is not preferable from the viewpoint of stabilization.

Examples of the photopolymerization initiator used in the present invention include α-carbonyl alcohols such as penzoin and acetoin, acyloin ethers such as benzoin methyl ether, benzoin ethyl ether, benzoin propyl ether, and pivaloin ether, naphthol, hydroxyanthracene, etc. of polycyclic aromatic compounds are desirable, and the amount is determined by PCRP
It is preferable to use 0.01 to 10 parts by weight based on 0.01 to 10 parts by weight.

The actinic light used in the present invention has a wavelength of 220 to 700 nm, preferably a wavelength of 250 to 700 nm, which causes a photopolymerization reaction.
The light has a wavelength of 600 nm, and its light source includes a low-pressure mercury lamp, a high-pressure mercury lamp, a fluorescent lamp, a xenon lamp, and a chemical lamp.

(Function) In the present invention, the lipase aqueous suspension is microencapsulated with an amphipathic film.

The amphiphilic substance of the present invention forms a monomolecular film or a liposome-like double membrane structure with the hydrophobic group on the outside and the parent tree group on the inside, and the encapsulated lipase is contained in the amphiphilic film. Although it is presumed that the lipase is scattered on the inner surface of the film, a double membrane structure is preferable from the viewpoint of stability of the lipase.

In the present invention, the fat and oil that serves as the substrate passes through the molecular lattice of the hydrophobic photocrosslinkable resin and reaches the surface of the lipase microcapsule, where it is acted upon by the lipase within the amphipathic film, but the substrate permeability in the amphipathic film is Unlike conventional polymer membrane microcapsules, the lipase reaction does not depend on the pore size of the membrane, but rather on the solubility of the membrane components, resulting in a significant change in the polarity of the substrate and reaction product. In this system, it is possible to impart selective permselectivity to the target substance at the molecular level.

The lipase in the present invention is packaged in a sufficiently hydrated and activated state, and the water required to maintain its activity is replenished from within the microcapsules as needed.
Lipase activity can be maintained for a long period of time.

As described above, microencapsulation of lipase with an amphipathic film fundamentally solves the problem of decreased activity yield that was inevitable in conventional immobilization methods. Since the physical strength is very low, in order to make it suitable for industrial production, it was necessary to add a film-strengthening substance and protect the periphery of the film with a polymer.

The film-strengthening substance used in the present invention not only strengthens the film by increasing the hydrophobic attractive force between amphiphilic substances and the repulsive force between polar groups, but also has the effect of adjusting the solubility of the substrate fat and oil.

The PCRP used in the present invention not only allows the substrate permeability to be adjusted arbitrarily by changing the oxyethylene/oxypropylene ratio, but also has the effect of protecting the amphiphilic film surface with polyoxyethylene groups, and has heat resistance. It can be said to be the most suitable material for imparting physical strength to lipase microcapsules in terms of strength and solvent resistance.

That is, the present invention is a composite immobilization method that combines the microcapsule immobilization method using an amphipathic film and the comprehensive immobilization method using a hydrophobic photocrosslinkable resin, and the synergistic effect of the advantages of both improves the lipase activity yield and activity stability. The quality has become extremely high.

The present invention will be specifically explained in the following examples, but the present invention is not limited to these examples.

Example 1 Polyoxyethylene/polyoxypropylene block polymer (Pronon 2012 Japan Oil Visit ■M) 1.0 mol, 2,4-tolylene diisocyanate 2.0 mol, 2-hydroxyethyl methacrylate 2.3 mol 50 parts of a hydrophobic photocrosslinkable resin prepolymer consisting of n-
Add to 50 parts of hexane and dissolve.

Chromobacterium viscosum
Lipase derived from Terium viscosum (
Toyo Jozo Co., Ltd.) in 0.1M phosphate buffer (pH 7.6)
The enzyme aqueous solution suspended in the solution was poured into a chloroform solution containing 3% by weight of an egg yolk lecithin-cholesterol-dicetyl phosphate mixture (7:2:10), transferred to a rotary evaporator, and heated at 37°C with nitrogen gas aeration. Mix by rotating at the bottom. The obtained emulsion was incubated at 37°C for 3 hours, then subjected to ultrasonication at 5°C under nitrogen gas, and the lipase microcapsules were recovered by centrifugation. 10 parts of lipase microcapsules and 5 parts of n-hexane
This was added to the previously prepared PCRP solution, mixed and stirred with 2 parts of benzoin, formed into a sphere with a diameter of 2-3 m, and then exposed to ultraviolet light with a wavelength of 365 nm for 10 minutes.
Immobilized lipase was prepared by irradiation for a minute.

Example 2 10 parts of the immobilized lipase of Example 1 was added to 500 parts of refined rice oil and 500 parts of n-hexane, stirred at 37°C under nitrogen gas, and samples were taken over time. After n-hexane was distilled off using an evaporator, the acid value was measured to determine the decomposition reaction rate.

The compositional analysis of the sample was carried out according to the method of J. Blum et al. (Lipid, 5, 601. (1970)), in which 2.0 mg of the sample was dissolved in 1.0 ml of pyridine, and 0.0 ml of hexamethyldisilazane was dissolved. 2 ml and 0.1 ml of trimethylchlorosilane were added to convert into TMS, followed by gas chromatography, and the results shown in FIG. 1 were obtained.

The final reaction rate after 72 hours was 95.0%.

Example 3 600 parts of oleic acid (Extraolein 902 manufactured by NOF ■), 40 parts of purified glycerin (manufactured by Hanhon Yakuhin Kogyo ■)
A substrate solution was prepared by adding 400 parts of n-hexane to 0 parts, 10 parts of the immobilized lipase of Example 1 was added to this, and the mixture was stirred at 37°C under nitrogen aeration, and the samples were separated over time. After taking the sample and distilling off n-hexane using an evaporator,
The acid value was measured to determine the amount of residual fatty acids and the ester synthesis reaction rate. The reaction product composition was determined by gas chromatography in the same manner as in Example 2, and the results shown in FIG. 2 were obtained.

The final reaction rate after 64 hours was 78.0%.

Example 4 Oleic acid (Extraolein 90.Oleic acid 9
150 g of n-hexane for 1432 g of 2% 2 (manufactured by NOF ■), 920 g of purified glycerin (manufactured by Hanhon Yakuhin Kogyo ■)
0 ml was added to prepare a substrate solution, 10 g of the immobilized lipase of Example 1 was added thereto, and the mixture was stirred at 37° C. for 72 hours under nitrogen aeration to perform an ester synthesis reaction. After taking samples over time and distilling off n-hexane using an evaporator, the acid value was measured to determine the reaction rate, and the reaction product composition was determined by gas chromatography using the same procedure as in Example 2. The results shown in Table 1 were obtained.

Below is a blank table 1゜Reaction rate 95.0% Example 5 Stearic acid (NAA-180, stearic acid 90%2
A substrate solution was prepared by adding 500 ml of n-hexane to 430 g of purified glycerin (manufactured by Nippon Oil & Fats Corporation) and 420 g of purified glycerin (manufactured by Hanhon Yakuhin Kogyo ■). To this was added 3 g of the immobilized lipase of Example 1, and the The ester synthesis reaction was carried out by stirring for 272 hours under nitrogen aeration. The reaction rate and reaction product composition were determined by gas chromatography using the same procedure as in Example 2.

The reaction product composition was as shown in Table 2.

Table 2 Reaction rate 90.5% After the reaction was completed, the residual activity of the recovered immobilized lipase was measured, and it was found that 98.0% of the activity remained.

Margins below (Effects of the Invention) The immobilized lipase obtained by the present invention exhibits a high reaction rate almost equivalent to that of a hydrolysis reaction in an ester synthesis reaction, and even when treating high melting point lipids such as stearic acid.
There is no inferiority in the reaction rate, and it dispels the conventional concept that immobilization equals low activity.

The method of using the immobilized lipase according to the present invention is not limited to the batch method described in the examples, but it is possible to carry out the reaction in a continuous method by packing it into a column. The method of use can be selected according to the properties of the substrate.

In the ester synthesis reaction using the present invention, the amount of heat required for the reaction is 1/15-1 compared to the conventional high temperature and high pressure reaction.
15, there is no need to perform complicated operations to maintain reaction conditions, and the production rate of monoglyceride useful as a surfactant is also improved, which is advantageous in view of the high activity stability of immobilized lipase. It can be said that it is suitable for industrial production.

That is, the present invention provides an energy-saving and resource-saving oil and fat processing technology, and enables oil and fat processing to be carried out at high yields and at low cost.

[Brief explanation of the drawing]

FIG. 1 shows changes in the composition of reaction products during hydrolysis of refined rice oil expressed in mol%. FIG. 2 shows the change in the composition of the reaction product in mol% during the ester synthesis reaction in the oleic acid/glycerin system. TG...triglyceride DG...diglyceride MG...monoglyceride FA...free fatty acid

Claims (3)

[Claims] (1) In an organic solvent containing one or more amphipathic substances, lipase microcapsules are prepared by enclosing a lipase aqueous suspension with an amphiphilic film, and the resulting lipase microcapsules and average Molecular weight 2000-20
000 hydrophobic photocrosslinkable resin prepolymer (hereinafter referred to as PCR
A method for immobilizing lipase, which comprises mixing lipase microcapsules (abbreviated as P) and irradiating the mixture with actinic rays in the presence of a photopolymerization initiator to comprehensively immobilize lipase microcapsules within a hydrophobic photocrosslinkable resin. . (2) A patent in which PCRP is an unsaturated polyester with an average molecular weight of 2000 to 20000, which is obtained by introducing photopolymerizable ethylenically unsaturated groups at both ends of a polyoxyethylene/polyoxypropylene block polymer nonionic surfactant. A method for immobilizing lipase according to claim (1). (3) The method for immobilizing lipase according to claims (1) and (2), wherein the polyoxyethylene/polyoxypropylene block polymer has an oxyethylene/oxypropylene ratio of 1/10 to 1/3. .