JPH10248558A - Enzymatic reaction in supercritical fluid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an industrial process capable of carrying out reaction at a high speed, yield and selectivity and simplifying purification by coating the surface of an enzyme with a lipid monomolecular film and carrying out enzymatic reaction in a supercritical fluid. SOLUTION: Enzymatic reaction in supercritical fluid is carried out by a lipid-modified enzyme hardly causing deactivation which is obtained by coating the surface of an enzyme such as glycosidase with a lipid monomolecular film. This method is useful for synthesis of a physiologically active substance such as an ester or a peptide, an optically active organic intermediate raw material, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an enzyme reaction method using a lipid-modifying enzyme in which an enzyme surface is coated with a lipid monolayer when an enzyme reaction is carried out in a supercritical fluid.

[0002]

2. Description of the Related Art In the pharmaceutical industry, food industry, agriculture and fisheries field, polymer field and organic intermediate raw material manufacturing field, utilizing the excellent properties of enzymes, bioactive substances such as esters and peptides, Industrial production methods for synthesizing various compounds such as optically active organic intermediate raw materials have been studied. In these studies, the use of organic solvents has recently been actively studied from the viewpoint of the solubility of substrates and products of enzyme reactions.

The enzyme reaction in an organic solvent includes a heterogeneous reaction in which an enzyme is suspended in an organic solvent for reaction, and a homogeneous reaction in which the enzyme is completely dissolved in an organic solvent and reacted. Crude enzymes are usually used in heterogeneous reactions,
Since the enzyme is suspended in the organic solvent, the diffusion of the substrate into the enzyme is low and the reactivity is low. Examples of homogeneous reactions include:
A method in which an enzyme is modified with polyethylene glycol and solubilized in an organic solvent (Y. Inada et al., Biochem. Biophys. Res.
mmun., vol. 122, 845, (1984)), but the preparation method is complicated and the enzyme activity is low. Further, a method of forming a complex of an enzyme and a lipid and solubilizing the same in an organic solvent (JP-A-64-80282)
), But there is a problem that the amount of enzyme contained in the complex is low.

[0004] As described above, various methods using an organic solvent have been studied, but the reaction rate is low and the method is not suitable for industrial production. In addition, there are problems in quality, such as the residual organic solvent in the product, and problems in the environment, and it is difficult to say that it is a global environmental conservation type process.

On the other hand, an organic catalytic reaction in a supercritical fluid
It has been attracting attention and being actively studied as an efficient process of energy saving and high productivity, and a process for preserving the global environment. The supercritical fluid refers to a fluid phase that does not condense even when pressure is applied at a critical point or higher, in addition to the three states of gas, liquid, and solid phase. By changing the pressure, the density can be continuously changed from a state close to a gas to a state close to a liquid, and various physical properties such as viscosity, diffusion coefficient and polarity of the fluid substance can be changed. It is expected that selectivity can be controlled. In addition, since the reaction can be performed in a state close to the gas phase, an improvement in the reaction rate is also expected due to the low viscosity and high diffusivity of the supercritical fluid. In addition, when the pressure is adjusted to normal pressure after the completion of the reaction, the medium becomes a gas, and the product remains in the reactor. Therefore, if the reaction is performed in a supercritical fluid, the subsequent purification process can be simplified.

As for the enzymatic reaction in the supercritical fluid, hydrolysis, esterification and oxidation using low-toxicity carbon dioxide as a medium have been studied (O. Aaltonen et al.).
al., CHEMTECH, vol. 21, p. 240, (1991)). There is also a report on a reaction in a supercritical fluid using immobilized lipase (JP-A-61-21098, JP-A-8-256783). However, in these cases, the reaction efficiency in a supercritical fluid was poor in any case because the enzyme was deactivated or the immobilization was performed with a primary focus on separation from the product.

[0007]

The problem to be solved by the present invention is that a reaction is carried out in a supercritical fluid using a lipid-modifying enzyme in which the surface of the enzyme is coated with a lipid monolayer, whereby the reaction can be performed at a high speed. It is an object of the present invention to provide an environmentally friendly industrial process which can be carried out with a high yield and a high selectivity, and can be subjected to a simple purification method.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that a reaction is carried out in a supercritical fluid using a lipid-modifying enzyme in which the surface of an enzyme is coated with a lipid monolayer, so that a simple organic solvent can be obtained. As a result, the present inventors have found that the reaction can be performed at a higher speed, a higher yield, and a higher selectivity without deactivating the enzyme as compared with the reaction in the above, and completed the present invention.

[0009] That is, the present invention is a method for enzymatic reaction in a supercritical fluid, which comprises using a lipid-modifying enzyme obtained by coating an enzyme surface with a lipid monolayer.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The enzymes that can be used in the present invention are not limited to specific ones in the class of enzymes, and include hydrolases, transferases, oxidoreductases, addition enzymes, isomerases, and the like. Synthetic enzymes can be mentioned.

[0011] Specifically, first, as for the hydrolase, lipase can be mentioned as an esterase that hydrolyzes the ester. The lipase may be produced by a microorganism, or may be obtained from animal organs, plant seeds, or the like.

Examples of proteases and peptidases that hydrolyze peptide bonds include pepsin, chymotrypsin, carboxypeptidase, aminopeptidase, dipeptidase, papain, promerin, subtilisin, and thermolysin.

Among glycosidases acting on glycosidic bonds of sugar, α- and β-glucosidase, α- and β-
Galactosidase, α- and β-mannosidase, β-
Examples include oligosaccharases such as fructosidase and various amylases, polysaccharases such as cellulase and inulinase.

The phosphatases involved in the hydrolysis of the phosphate bond include phosphomonoesterase, phosphodiesterase, pyrophosphatase and the like.

Examples of enzymes other than hydrolases include transferases such as phosphotransferase, glycosyltransferase, peptidyltransferase, aminotransferase, and transglutaminase; oxidoreductases such as alcohol dehydrogenase, amine oxidase, and monophenol monooxygenase; and aldolase. , Aconitase, enolase, aspartase and the like; and isomerases such as racemase, mutase and cis-trans isomerase.

When any of the above enzymes is used, the enzyme does not necessarily need to be purified, but may be a crude enzyme,
Alternatively, it may be an extract of an organ or a cell of an organism containing a large amount of the enzyme.

The lipids usable in the present invention include, for example, naturally occurring lipids such as neutral lipids such as glyceroglycolipids such as monogalactosyl diglyceride and galactosyl glucosyl diglyceride, monoglycosylceramide, and the like.
Glycosphingolipids such as ceramide hexoside and ganglioside, sterol glycosides, cardenolide glycosides, steroid glycosides such as saponin, and fatty acid glycosides such as diacyltrehalose and triacylglucose can be exemplified. Examples of the anionic lipid include phospholipids such as phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid, and examples of the amphoteric lipid include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine.

On the other hand, examples of the synthetic lipid include synthetic bimolecular film-forming compounds. That is, carbon number 6 ~
Compounds having 30 hydrocarbon chains as hydrophobic parts and functional groups such as polyhydroxy groups such as sugars, phosphate groups, sulfonic acid groups and ammonium salts as hydrophilic parts, or intermolecular interactions such as biphenyl groups Is a single-chain compound containing a functional group having the following formula in the hydrocarbon chain. Specific examples thereof include dialkyl phosphate, dialkyl type polyethylene glycol, dialkyl sulfosuccinic acid, dialkyl type glycolipid, and dialkyl ammonium salt.

The lipid-modifying enzyme of the present invention refers to an enzyme whose surface is coated with a lipid monomolecular film, and which is chemically bonded to the surface of the enzyme and a hydrophilic group of lipid by a hydrogen bond. As expected. A lipid-modifying enzyme having this feature is disclosed in
It can be easily obtained by the method described in 64-80282. That is, the enzyme is dissolved in a buffer having a pH of 5.0 to 9.0. Lipids in small amounts of methanol, ethanol,
Dissolve in propanol, acetone, methyl ethyl ketone and other hydrophilic organic solvents, mix in a buffer to prepare a dissolved suspension of lipids, add dropwise to the previously prepared enzyme-containing buffer under cooling, and add the lipid-modifying enzyme. Precipitate. After separating the precipitate by centrifugation or filtration, the precipitate is washed with a buffer solution, then with distilled water, and then freeze-dried or fluidized-bed dried as it is,
Alternatively, a powdery lipid-modifying enzyme can be obtained by spray-drying after dispersing in a small amount of distilled water. Further, in order to improve the enzyme content in the lipid-modifying enzyme, a dispersant such as a surfactant can be added as a third component according to the method described in JP-A-3-280880. In this case, an enzyme-surfactant-lipid complex is used for the reaction. In addition, in order to effectively utilize enzymes and lipids without waste, after mixing the enzyme-containing buffer and the lipid-containing buffer, the lipid-modified enzymes, enzymes and lipids remaining by simply distilling water under reduced pressure were included. The residue can be used as it is in the enzyme reaction.

The supercritical fluid which can be used in the present invention has a critical temperature of 15 judging from the conventional knowledge of thermostable enzymes.
It is believed that it is limited to those below 0 ° C. Specifically, carbon dioxide is the most common, but ethane,
Examples thereof include ethylene, propane, propylene, xenon, fluoroform (Freon 23), and sulfur hexafluoride. Any media can be used in combination as needed. In addition, in consideration of the solubility of the substrate, it may be preferable to carry out the reaction in a two-phase system in which water is added to these media at a weight ratio of 0.1 to 400%.

The method of the reaction is as follows. The lipid-modifying enzyme and the substrate prepared above are placed in a suitable vessel such as a pressure-resistant stainless steel or glass, and the vessel is sealed. The enzyme reaction is performed at a predetermined temperature while maintaining a predetermined pressure.
The pressure during the reaction is 4 to 50 MPa, preferably 4 to 20 M
Pa, the temperature during the reaction is 5 to 150 ° C, preferably 10 to 10 ° C.
100 ° C. After the completion of the reaction, the medium can be removed as a gas simply by gradually reducing the pressure of the system and returning it to normal temperature and normal pressure.

[0022]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 (1) Preparation of lipid-modifying enzyme 50 mg of Bacillus circulans- derived β-galactosidase (manufactured by Daiwa Kasei) was added to an acetate buffer (10 mM, pH 6.0).
0) An enzyme solution dissolved in 25 ml was prepared and cooled to 4 ° C. A solution prepared by dissolving 50 mg of didodecyl-ND-glucono-L-glutamic acid ester (chemical formula 1) in 0.5 ml of ethanol was added dropwise to the enzyme solution, and the mixture was stirred at 4 ° C for 1 hour. Thereafter, the mixture was left at 4 ° C. for 15 hours, and a white precipitate formed was collected by centrifugation, and washed twice with the above acetate buffer and once with distilled water. The washed solid was freeze-dried to obtain 21.0 mg of a powder. The obtained lipid modifying enzyme was identified by UV spectrum and elemental analysis.
The protein content of this lipid-modifying enzyme was 10.0%.

[0023]

Embedded image

(2) Enzyme Reaction in Supercritical Fluid Next, 1 mg of this lipid-modifying enzyme and p-nitrophenyl-β-D- as a sugar donor were placed in a 10-ml pressure-resistant glass container (manufactured by Pressure-resistant Glass Industry). Galactopyranoside (1 μmo
l), 5-phenylpentanol (10
μmol) and 5 ml of water, and then 5 ml of carbon dioxide was injected from a carbon dioxide gas cylinder into the container with a liquid sending pump while cooling to -5 ° C or lower. The reaction was carried out at a temperature of 35 ° C. while maintaining the pressure at 15 MPa, the amount of p-5-phenylpentyl-β-D-galactopyranoside produced was measured by high performance liquid chromatography, and p-nitrophenyl-β- The rate of sugar transfer from D-galactopyranoside was determined. The results are shown in Table 1 (Table 1). As a comparative example, when the unmodified enzyme was used, the desired product could not be obtained, and the reaction rate was very slow in the reaction (normal pressure, 35 ° C.) in isopropyl ether using a simple organic solvent. .

[0025]

[Table 1]

Example 2 Using a lipase D derived from Rhizopus delemar (manufactured by Seikagaku Corporation), a lipid-enzyme complex was formed in the same manner as in Example 1 to obtain a lipid-modifying enzyme having a protein content of 4.2%. Obtained.

In the pressure-resistant glass container used in Example 1, 1 mg of this lipid-modifying enzyme, 0.5 mmol of monolaurin,
5 mmol of lauric acid is added, and 10
While cooling the carbon dioxide of ml to -5 ° C or lower, the solution was injected into the container by a liquid sending pump. Thereafter, the esterification reaction of monolaurin was carried out at 40 ° C. and 15 MPa. The produced lauric triglyceride was quantified by liquid chromatography. As a result, the initial rate of the esterification reaction was 0.1.
It was calculated as 94 mmol / min.mg-protein. As a comparative example, when benzene was used as an organic solvent and reacted at normal pressure and 40 ° C., the initial velocity was 0.16 mm.
ol / min · mg-protein.

Example 3 In the substrate of Example 2, 0.5 mmol of (R)-or (S) -1- was used instead of 0.5 mmol of monolaurin.
Asymmetric selective esterification of lauric acid was carried out under the same conditions as in Example 2 using phenylethanol. The generated lauric acid-1-phenylethyl ester was quantified by gas chromatography. As a result, high R-isomer selectivity (k _R / k _S ) in the reaction in supercritical carbon dioxide
= 77) was observed.

[0029]

The reaction is carried out in a supercritical fluid using a lipid-modifying enzyme having the enzyme surface coated with the lipid monolayer of the present invention, whereby the reaction can be carried out at high speed and with high yield without deactivating the enzyme. It can be performed with high selectivity.

Claims (2)

[Claims] 1. A method for enzymatic reaction in a supercritical fluid, comprising using a lipid-modifying enzyme obtained by coating an enzyme surface with a lipid monolayer. 2. The enzyme reaction method according to claim 1, wherein the enzyme is glycosidase or lipase.