JPH04131082A - Production of non-aqueous highly active enzyme - Google Patents

Abstract

PURPOSE:To reproduce the subject enzyme useful in the fields of pharmaceuticals, foods, etc., utilizing waste liquid of a production process by adding a surfactant and a lipid to a buffer solution containing enzyme, a surfactant and a lipid as solutes and cooling the mixture. CONSTITUTION:The objective enzyme can be produced by dissolving a surfactant (e.g. lauric acid) in a buffer solution (e.g. waste liquid discharged from a production process) containing >=0.7mg/ml of an enzyme, a surfactant and a lipid as salutes, dispersing a lipid (e.g. monogalactosyl diglyceride or phosphatidylcholine) in the solution and cooling preferably to 0-10 deg.C. The weight ratio of the enzyme to the total surfactant (surfactant/enzyme) is adjusted to 0.001-1,000 and the weight ratio of enzyme to total lipid (lipid/enzyme) to 0.2-100.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention is useful in the pharmaceutical industry, food industry, agriculture and fishery fields, organic intermediate raw material production fields, etc. The present invention relates to a method for producing a non-aqueous highly active enzyme used in the production of organic intermediate raw materials.

[Conventional technology]

In order to effectively utilize the excellent properties of enzymes in organic synthesis, it is essential to use organic solvents from the standpoint of dissolving substrates and products.

Conventionally, enzyme reactions in organic solvents involve using enzymes in organic solvents. Heterogeneous reaction (A, M
, K11banov et al., CHEMTEC
) 1.16.354 (1986) ) and enzymes with IR
There is a homogeneous reaction in which the substance is completely dissolved in a medium and reacts.

In a heterogeneous reaction, since the enzyme is suspended in an organic solvent, diffusion of the substrate into the enzyme is low, resulting in low reactivity. Therefore, a homogeneous reaction is desirable, but for this purpose it is necessary to solubilize the enzyme.

As a method for solubilizing the enzyme, the enzyme is modified with polyethylene glycol and solubilized in an organic medium (Y, In
ada et al, Biochem, Biophy
s,Res,Commun,,”η, 845 (19
134)), but the preparation method is complicated. In addition, complexes between enzymes and lipids are formed. Although there is a method of solubilizing the enzyme in a solvent (Japanese Patent Laid-Open No. 64-80282), there are problems such as the amount of enzyme contained in the complex is low and the yield of the complex is also low.

The present inventor previously discovered a non-aqueous highly active enzyme that is a complex of an enzyme, a surfactant, and a lipid and is soluble in organic solvents and filed a patent application (Japanese Patent Application No. 2-081040). For example, the enzyme made of the complex is prepared by dissolving the enzyme in a buffer solution with a pu of 5.0 to 9.0, adding a surfactant dissolved in the buffer solution dropwise, dispersing the lipids, and then heating the solution at a low temperature. It can be produced by allowing the composite to precipitate by standing under. However, this method has the disadvantage that the proportion of the enzyme used that forms a complex is not necessarily large, and a considerable portion remains in the waste liquid.

[Problem to be Solved by the Invention] The problem to be solved by the present invention is to remove enzymes, etc. from the waste liquid discharged from the manufacturing process of the non-aqueous highly active enzyme, which is a complex of the enzyme, surfactant, and lipid. Make effective use of
The object of the present invention is to provide a method for producing a non-aqueous highly active enzyme.

[Means for Solving the Problems] The object of the present invention is to further dissolve a surfactant in a buffer solution containing an enzyme, a surfactant, and a lipid, disperse the lipid, and cool the solution. This is achieved by a method for producing a non-aqueous highly active enzyme, which is a complex of an enzyme, a surfactant, and a lipid.

In the present invention, the buffer solution in which enzymes, surfactants, and lipids are dissolved refers to the enzyme, surfactant, and lipid dissolved in a buffer solution with a pH of 5.0 to 9.0.
A solution or dispersion of a surfactant and a lipid may also be used, but for the purpose of the present invention,
It is preferable to use the waste liquid from which No. 40 non-aqueous highly active enzyme was produced. That is, the enzyme is dissolved in a buffer solution with a pH of 5.0 to 9.0, a surfactant dissolved in the buffer solution is added dropwise to the solution, and the complex is precipitated by leaving it to cool at a low temperature. and use the solution after removing this precipitated complex. Furthermore, a waste liquid of a non-aqueous highly active enzyme produced according to the present invention may also be used.

The concentration of enzyme in the buffer is 0.7 g/111 or more.

At less than 0.7 u/d, the composite does not precipitate.

Examples of enzymes that can be used in the present invention include hydrolases, transferases, oxidoreductases, addition enzymes, and isomerases.

Examples of hydrolytic enzymes include esterase, peptidase, glycosidase, phosphatase, and amidase.

As an esterase that hydrolyzes esters, lipase or a living tissue containing lipase can be used. They may be produced by microorganisms,
It may also be obtained from animal organs, plant seeds, etc.

Peptidases that hydrolyze peptides include animal-derived pepsin, chymotrypsin, carboxypeptidase,
Examples include thermolysin; plant-derived papain, bromelin, and aminopeptidase; and bacterial-derived gelatinase and dipeptidase. Examples of glycosidases that act on Gelconte bonds of sugars include oligosaccharases such as α- and β-glycosidases, α- and β-galaxidases, polysancarases such as α- and β-amylases, and cellulases. Phosphatases involved in hydrolysis of phosphate bonds include phosphomonoesterase, phosphogesterase, pyrophosphatase, and the like. Amidases that hydrolyze amide groups include arginase, urease, glutaminase, and the like.

Enzymes other than hydrolytic enzymes include transpeptidase, transglucosidase, transpeptidase,
Examples include transferases such as transamidase and transglutaminase; oxidoreductases such as alcohol dehydrogenase and oxygenase; addition enzymes such as aconitase, enolase and aspartase; and isomerases such as isomerase.

The surfactant used in the present invention is a nonionic surfactant such as polyoxyethylene p-
t-Octyl phenyl ether; Disform CA-1
15; Polyethylene glycol monoalkyl ethers such as Nonion E-215 and Nonion P-210; Polyethylene glycol-p-alkylphenyl ethers such as Nonion NS-210 and Nonion H8-210; Nonion Shi-4, Nonion S-6, etc. Polyethylene glycol fatty acids; sorbitan fatty acids such as Span 80 and Span 85;
Examples include sorbitan ester ether fatty acids such as Tween 20 and Nonion S-221. Here, Disform CA-115, Nonion E-215, Nonion P
-210, Nonion NS-210, Nonion H320,
Nonion L-4, Nonion S-6, Span 80, Span 85, Tween 20, and Nonion LT-221 are trade names manufactured by NOF ■. Anionic acids include lauric acid, myristic acid, palmitic acid, stearic acid,
Fatty acids and their salts such as oleic acid; octyl malonate,
These include alkyl malonates such as dodecyl malonate; alkanesulfonates such as octyl sulfate, delen sulfate, and dodecyl sulfate; and α-sulfo fatty acids such as α-sulfolauric acid and α-sulfopalmitic acid. Examples of cationic salts include N-alkylpyridinium salts such as N-dodecylpyridinium salts, alkyl ammonium salts such as dodecyl ammonium chloride, and heptyltrimethylammonium chloride.

Lipids that can be used in the present invention include, for example, naturally occurring lipids such as neutral lipids, anionic lipids, and amphoteric lipids.

Neutral lipids include glyceroglycolipids such as monogalactosyl diglyceride and galactosyl glucosyl diglyceride; glycosphingolipids such as monoglycodylceramide, ceramide hexoside, and ganglioside; steroid glycosides such as sterol glycosides, cardenolide glycosides, and saponins. Examples include fatty acid sugars such as diacyltrehalose, triadylglucose, sorbitan distearate, and sucrose distearate. Anionic lipids include phospholipids such as phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid;
Examples of amphoteric lipids include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine.

Synthetic lipids include two hydrocarbon chains with 6 to 30 carbon atoms as hydrophobic parts, polyhydroxy groups such as sugars, phosphate groups,
A synthetic bilayer membrane-forming compound having a functional group such as a sulfone t11 group or an ammonium salt as a hydrophilic moiety can be used. Specifically, dialkyl phosphates, dialkyl polyethylene glycols, dialkyl sulfosuccinic acids, dialkyl glycolipids, dialkyl ammonium salts and the like can be mentioned.

The ratio of total surfactant to total enzyme is 0.001-10
00 (surfactant weight/enzyme weight), and there is no distinction between those dissolved in the buffer beforehand and those added later. A conventional method may be used to dissolve the surfactant in the buffer solution.

For fat, use 1 small amount of methanol, ethanol, propatool,
Dissolve in acetone, methyl ethyl ketone, other hydrophilic organic solvents or buffers, or use as is.

The ratio of total lipids to total enzymes is 0.2 to 100 (lipid weight/enzyme weight), and there is no distinction between those dissolved in the buffer solution in advance and those added later.

Lipid dispersion methods include stirring blades, magnetic stirrers,
A stirring device such as a homomixer may be used, or ultrasonic waves may be applied for dispersion.

After the lipids are sufficiently dispersed, the buffer solution is allowed to stand at 0 to 30°C, preferably 0 to 10°C, to precipitate and precipitate the enzyme-surfactant-lipid complex. At this time, enzymes, surfactants, and lipids are dissolved in the supernatant. This supernatant can be used again as a buffer in the present invention.

After separating this precipitate by centrifugation or filtration, it is washed with a buffer solution and then with distilled water, and then it is freeze-dried or fluidized bed-dried as it is, or it is dispersed in a small amount of distilled water and then spray-dried. A powdered non-aqueous highly active enzyme is obtained.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples.

Fruit, male side 1 Candid Cyli
After dissolving 501 g of lipase derived from S. n-dracea in 25 m of acetate buffer (0.01 M, pH 6.0), it was centrifuged to remove insoluble matter (solution a). 50μ of a nonionic surfactant (Nonion LT-221: trade name, manufactured by NOF Corporation) was added to an acetate buffer (0.01M, pH 6,
0) Melted to 25m (liquid b). Add solution B to solution A at 4°C.
Add dropwise while cooling to 4°C and stir for 1 hour.To solution A, add dropwise solution D, which is a solution of 50 μm of nonionic lipid (sorbitan distearate) dissolved in 0.5% ethanol, while cooling to 4°C, and stir. After dropping, it was left at 5°C for 24 hours. After standing, the solution in which a white precipitate was formed was centrifuged, the supernatant was removed, and the remaining solid was washed twice with vinegar M buffer and once with distilled water. This solid is then freeze-dried,
24.8 μg of powder was obtained.

Furthermore, after removing the white precipitate, liquid B was added dropwise to the supernatant liquid while cooling it to 4°C, and after stirring for 1 hour, 25 μm of nonionic lipid (sorbitan distearate) was dissolved in 0.5 d of ethanol. Solution a was added dropwise while cooling to 4°C, stirred, and left at 5°C for 24 hours after the dropwise addition.

Following the method described above, 17.91 g of white powder was obtained.

This collection operation was performed a total of four times.

Table 1 shows the results.

The enzyme of the obtained complex of enzyme, surfactant, and lipid was identified by UV spectrum and elemental analysis. This complex was insoluble in water and soluble in organic solvents such as benzene and chloroform.

Next, 1.61 g of this enzyme, surfactant, and lipid complex
was dissolved in 1.52 M of tributyrin, and 1.52 M of octatool was further dissolved, and water was added so that the water content was 1% by volume. Thereafter, the mixture was stirred at 22°C for 30 minutes. The produced octyl acetate was measured by gas chromatographic analysis.

From this value, the synthesis rate was calculated using the following formula.

Table 1 shows the yield of the complex, the enzyme content in the complex and the synthesis rate.

Example 2 Instead of the surfactant used in Example, 50μ of the surfactant shown in Table 2 was added to an acetate buffer (0.01M, pH 6,
0) It was dissolved in 25m and used as liquid b.

Thereafter, a white powder was obtained by the same operation as in Example 1.

The results are shown in Table 2.

Table 1 Table 2 [Effects of the Invention] According to the method of the present invention, a non-aqueous highly active enzyme can be further produced in high yield from the waste liquid from which the non-aqueous highly active enzyme was produced.

Claims (2)

[Claims] (1) Enzymes and surfactants characterized by dissolving a surfactant in a buffer solution containing 0.7 mg/ml or more of an enzyme, a surfactant, and a lipid, dispersing the lipid, and cooling the solution. A method for producing a nonaqueous highly active enzyme that is a complex of an agent and a lipid. (2) The ratio of the weight of total surfactant to the weight of enzyme is
0.001 to 1000 (surfactant weight/enzyme weight), and the ratio of total lipid weight to enzyme weight is 0.2
2. The method for producing a non-aqueous highly active enzyme according to claim 1, wherein the ratio is 100 to 100 (lipid weight/enzyme weight).