JPS6121098A - Preparation of enzymic reaction product - Google Patents

Abstract

PURPOSE:To advance a reaction in a homogeneous phase and carry out separation after the reaction with ease, by carrying out an enzymic reaction in the presence of a substance capable of being a solvent in the supercritical state. CONSTITUTION:For example, lipase, e.g. Rhizopus delemar, is used as an enzyme, and a fat or oil, e.g. palm oil or beef tallow, is used as a substance subjected to the enzymic reaction. The enzymic reaction is carried out using carbon dioxide as a substance capable of being a solvent in the supercritical state. The lipase and the fat or oil are put into reaction vessels 1 and 1', and gaseous carbon dioxide (critical temperature; 31.1 deg.C, critical pressure; 73atm) is boosted with a liquid chromatographic pump 4, liquefied, fed to the reaction vessels 1 and 1' while controlling the pressure with valves 5-8, and reacted at about 32 deg.C under about 100atm pressure with stirring. After the reaction, the reaction vessels 1 and 1' are cooled with acetone-dry ice, and the reaction product is recovered in the dry ice. The dry ice is sublimed to extract and separate the aimed lipid from the residue.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an enzymatic reaction product, and more particularly to a method for producing an enzymatic reaction product characterized by carrying out an enzyme reaction in a supercritical state.

In the present invention, a supercritical solvent (“supercritical gas”)
(sometimes simply abbreviated as ) is used, but this is based on the critical temperature (T
c) and the fluid in a state exceeding the critical pressure (Pc).

In recent years, various extraction and separation techniques that apply the physical properties of supercritical gases have been developed. Supercritical gas extraction takes advantage of the fact that supercritical fluid exhibits excellent solubility for many substances, and the solubility changes with changes in temperature and pressure. It is applied to the extraction of nicotine, caffeine, etc.

The present inventors have conducted various studies on enzymatic reactions, but
Enzyme reactions proceed even in supercritical conditions (for example, 311°C or higher and 73 atmospheres or higher for carbon dioxide), which are harsh conditions that living organisms have never experienced before. The present invention was completed after discovering that there are advantages such as the fact that reactions that were previously carried out in a homogeneous system proceed in a homogeneous phase.

That is, the present invention is characterized in that a mixture containing an enzyme, a substance to be subjected to an enzymatic reaction, that is, a substrate, and a substance that can serve as a solvent in a supercritical state is brought to a supercritical state and maintained at a temperature at which the enzymatic reaction proceeds. This is a method for producing an enzymatic reaction product.

The enzyme used in the present invention may be any proteinaceous polymer organic catalyst produced by living cells. In particular, the present invention uses a structurally stable enzyme that maintains its structure and properties even in a supercritical solvent. is useful. Specific examples include hydrolase, transferase, lyase, synthetase, and isomerase.

A substance that undergoes an enzymatic reaction corresponding to the above-mentioned enzyme, generally known as a substrate, is used. Hereinafter, a case will be described in which gauze is used as an enzyme, and oil or fat, or one or more types of oil and fat and fatty acids are used as the substance to be subjected to the enzymatic reaction, but the present invention is not limited thereto.

First, regarding the lipase, Rhizopus deremer, Rhizoges alhizas, Aspergillus niger, Candida cylindrasose, Jootricum candium, etc. can be used. As fats and oils, iR-
Petroleum, vegetable oils such as soybean oil, rapeseed oil, olive oil, coconut oil, corn oil, cottonseed oil, and safflower oil; animal fats and fats such as beef tallow, lard, and fat-free; and synthetic oils and fats such as trilaurin, tristearin, and triolein. Further, the fatty acid is a straight chain fatty acid having 8 to 20 carbon atoms, such as palmitic acid, stearic acid, oleic acid, linoleic acid, and liluic acid, and one or more of these can be used.

When using a commercially available enzyme agent, the amount of enzyme used is 0.025 to 5 units by weight based on the raw material oil or fatty acid (5 x 102 to 5 x 105 units per 100 g of oil or fat).
(amount of enzyme with lipolytic properties) is used.

Furthermore, IJ with A? The reaction can be promoted by carrying out the transesterification reaction of fats and oils with the enzyme in the presence of an inert organic solvent and/or a carrier.

Petroleum benzine, n-hexane,
Petroleum ether and other commonly used carriers such as celite, activated carbon, alumina, silicic acid, and cellulose can be used.

In addition to the enzyme and the substance to be reacted with the enzyme, a substance that can serve as a solvent in a supercritical state is mixed.

Specifically, ethane, ethylene, propane, propylene,
Examples include nitrogen oxide, but carbon dioxide is the easiest to handle because it is a harmless inert gas and has a critical point near room temperature, which is suitable for maintaining the temperature at which the reed enzyme reaction proceeds.

Such a mixture is brought into a supercritical state and maintained at a temperature at which the enzymatic reaction proceeds. Specifically, 10℃ to 50℃
A range of is preferred. When carbon dioxide is used, it is particularly preferable to have a critical temperature (T) of 31.1°C and a critical pressure (Pc) of 73 atm, but since carbon dioxide will dissolve in a reaction system using water, the pH will also be lower. It is necessary to adjust the value to be suitable for the enzyme reaction.

Reactants can be recovered by increasing and/or decreasing the pressure and/or temperature of the reaction system. This recovery process can be applied to general supercritical extraction operations, such as a method using reduced pressure, a method that separates by increasing temperature, a method that separates by placing an adsorbent that adsorbs only the target component in a separation tank, etc. This can be done by

Since the enzyme reaction of the present invention is carried out in a supercritical state, it is easier to handle the enzyme in subsequent separation steps if it is immobilized. As carriers for immobilizing enzymes, carriers such as polysaccharides such as cellulose, porous glass, and ion exchange resins can be used.
Alternatively, it can be immobilized by covalent bonding. Most carriers are in the form of particles, but they can also be in the form of films, plates, tubes, or fibers. In addition, 2 enzymes
Methods that function in a supercritical state of solvent include methods of cross-linking by reacting with reagents having functional groups, methods of encasing the enzyme in a cell lattice, or coating it with a selectively permeable polymer film. It may be fixed. As an enzyme reaction device, a patch method may be used, but it is also possible to carry out a continuous reaction by using an immobilized enzyme column or by circulating the enzyme using an ultralacrimal membrane.

Next, the method of the present invention will be explained using the drawings. Raw materials are fed into a reaction vessel 1 whose temperature can be controlled as shown in FIG. The raw materials can be stirred using a magnetic stirrer 2. The carbon dioxide gas supplied from the carbon dioxide cylinder 3 is pressurized by the liquid chromatography pump 4 while
The pressure is controlled by the secondary pressure control valve 5, stop valve 6, trace control valve 7, safety valve 8, stop valves 6', 6", 6", etc., and then supplied to the reaction vessel 1. After the enzymatic reaction is carried out in a supercritical state for a certain period of time, the reaction vessel is cooled to convert carbon dioxide together with the reactants into 1' dry ice, and after opening the valve, the reactants are recovered by sublimating the dry ice at room temperature. Lipids can be separated from the reactant by extraction with hexane or the like.

Carbon dioxide can also be supplied via a suitable repressurization device to a second reaction vessel connected in parallel and fed with raw materials for enzyme reaction.

Incidentally, when using an immobilized enzyme, the reactor/separator system can also be configured as shown in FIG.

In this case, the substances produced by the continuous reaction can be separated in separator 1 and separator 2 by increasing and/or decreasing pressure and/or temperature by taking advantage of the characteristics of supercritical fluid. That is, an immobilized enzyme column 10 is provided in the reaction vessel 1, a substrate inlet 11 is provided in the carbon dioxide supply line, and a line 12 and a valve (three-way valve) 13 for washing after continuous reaction for a certain period of time are provided. The enzyme reaction product passes through the line filter 14, the six-way valves 15, 15', the liquid connectors 16, 16', and the separators 17, 1.
It is separated from carbon dioxide within 7'. Pressure is adjusted using six-way valves i5, 15', - next pressure regulating valve 5', and pressure gauge 9//
, 90/flow meter 18, three-way valve 13', etc., as appropriate.

In the conventional method, the reaction was carried out at room temperature and pressure, so the reaction system rarely became a homogeneous liquid with high fluidity, resulting in poor reactivity, but the enzyme reaction in the supercritical state of the present invention By performing this, it becomes possible to make the reaction system homogeneous. In addition, supercritical gases have lower viscosity and higher diffusion coefficients than liquid solvents, so they can easily penetrate solids and the time it takes for reactions to reach equilibrium is short. Furthermore, it is easy to separate the supercritical gas and the reactants, and the fractionation process is simple.

This method has the advantage that non-volatile components obtained through enzymatic reactions and reactants sensitive to heat can be processed at temperatures close to room temperature. Furthermore, it has been discovered that even in a supercritical state, which is harsh for living organisms, the activity of the enzyme does not decline and the enzymatic reaction proceeds.

Example 1 Experimental Method A stirrable liquid chromatography column packing device with an internal volume of 40 ml was used as the reaction vessels 1 and 1', two of which were connected in parallel. 1st
The figure shows a schematic diagram of the experimental setup. In this reaction vessel, 0.2 g of triolein (manufactured by Gyudon Kagaku), Rh1zopus d
e (Emar origin lipase (Seikagaku Corporation, code number 100890) 0.1 g or 0.2 g, 0
．． 3MTES buffer (manufactured by Wako Tsumugi Pharmaceutical, pH 6,8) 0.
2 ml of Celite (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) Ig was added. Liquid carbon dioxide was gradually pumped into the reaction vessel using a liquid chromatography pump (TRIROTAR, JASCO Corporation) 4, and the pressure was increased.
When the pressure reached 100 atm, stirring was started, and the reaction was carried out at 32° C. for a certain period of time shown in Table 1. During the reaction, the pump was operated continuously, and the pressure inside the reaction vessel was kept constant using the secondary pressure control valve 5 and the safety valve 8.

After the reaction is complete, cool the container with acetone and dry ice.
The reaction product was collected in dry ice, and the formation of dry ice was confirmed using a pressure gauge 9. After sublimating dry ice at room temperature, the residue was extracted with hexane to separate lipids.

Analysis method: The reaction product separated in hexane was added to a 7% hydrous Florisil Gyudon Kagaku, 60-80 mesh, 309) column, and developed with a mixed solvent of diethyl ether and hexane at a volume ratio of 20:80. Both 40-8 ml portions were collected and concentrated under reduced pressure.

A small amount of the concentrate was poured onto a TLC plate (Silica Kake 8×60°A, 5747, Merck) and developed with a mixed solvent of hexane, ethyl ether, and acetic acid in a volume ratio of 70:30:1 to detect monoglycerides and diglycerides. I confirmed that it was not.

After drying the concentrate, fatty acid methyl ester was prepared by the BF3/methanol method (NOF Standard Analysis Method 2, 4, 20.2-77), and quantitative analysis was performed by gas chromatography. Gas chromatography analysis is 2
0%DEGS Diasolid L, 80-100
A column (inner diameter 3 mm, length 2 m) packed with Mesoyu (Gas Kuro Kogyo) was used, and the inlet, detector (FID)
) and column temperatures of 200°C and 210°C, respectively.
and maintained at 190°C. The model of gas chromatography used was Hitachi 663.30.

The ratio of methyl stearate to methyl oleate was calculated. The results are shown in Table 1.

Table 1 Example 2 Using the same method as in Example 1 at a reaction temperature of 35°C, the amount of enzyme was 0.
02' and 9 were added and reacted. The results are shown in Table 2.

Table 2 Example 3 Using the same method as in Example 1, the enzyme amount was 00 at a reaction temperature of 40°C.
1 g or 0.029 was added and reacted. The results are shown in Table 3.

Procedural correction tooth (method) 1. Display of the case 1982 Patent Application No. 143138 2, Name of the invention Method for producing enzyme reaction product 3, Person making the amendment Relationship to the case Patent applicant Address 5-8 Kyobashi-chome, Chuo-ku, Tokyo (Date of dispatch)
(October 30, 1982) 7. Contents of the amendment (1) The following statement is inserted after Table 3 on page 12 of the specification.

[Brief explanation of drawings]

FIG. 1 shows a schematic diagram of the experimental apparatus used in Example 1. . FIG. 2 shows a schematic diagram of the experimental setup when using immobilized enzymes. In the figure, 1 and 1' are reaction vessels, 2 and 2- are magnetic stirrers, 3 is a carbon dioxide cylinder, 4 is a liquid chromatography pump, 5 and 5' are secondary pressure control valves, 6.6-16'' and 6. --' is a stop valve, 7 is a micro-temperature control valve, 8 is a safety valve,
9.9', 9'' and 9-'' are pressure gauges, 10 is an immobilized enzyme column, 11 is an M quality inlet, 12 is a washing line, 13 and 13- are three-way valves, 14 is a line filter, 15 and 15 ' is a six-way valve, 16 and 16- are quick connectors, 17 and 17' are separators, and 18 is a flow meter. ”

Claims (3)

[Claims] (1) An enzyme reaction product characterized by bringing a mixture containing an enzyme, a substance subjected to the enzymatic reaction, and a substance that can serve as a solvent in a supercritical state to a supercritical state and maintaining it at a temperature at which the enzymatic reaction proceeds. manufacturing method. (2) The production method according to claim (1), wherein the enzyme is lipase, and the substance subjected to the enzymatic reaction is one or more types of fats and oils or fatty acids. (3) The manufacturing method according to claim (1), wherein the enzyme is an immobilized enzyme.