JPH09124570A - Glucoside derivative for enzyme modification, organic solvent-soluble enzyme modified with the same and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a glucoxide derivative which is inexpensive and readily available, has a specific chemical structure, modifies an enzyme to impart the the enzyme solubility in organic solvent as well as sustain its high enzymatic activity in the organic solvent. SOLUTION: This glucoxide derivative has a chemical structure of' the formula (R is a 6-22C hydrocarbon group), for example, N,N-dilauryl-D-gluconic acid amide. This derivative is obtained, for example, by dissolving (A) a secondary amine disubstituted with a 6-20 hydrocarbon group and (B) glucono-1,5-lactone in a solvent boiling at 60-250 deg.C such as methanol and allowing them to react with each other at the refluxing temperature. When an enzyme is modified with this compound, the weight ratio of this compound to the enzyme is 0.2-100.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a glucoxide derivative for enzyme modification, an organic solvent-soluble enzyme modified with the above derivative, and a method for producing an organic solvent-soluble enzyme using the above derivative.

[0002]

2. Description of the Related Art In recent years, as the excellent properties of enzymes have been revealed, there is a growing demand for effective utilization of them in advanced technological fields such as organic synthesis of physiologically active substances and development of new functional coatings.

However, although the enzyme catalyzes various reactions in an aqueous solution, it is generally unstable to an organic solvent and easily aggregates to be inactivated. Therefore, the utilization of enzymes in the field of using organic solvents has been insufficient.

Conventionally, several methods have been proposed for carrying out the reaction in an organic solvent while maintaining the activity of the enzyme. For example, the reverse micelle method (Ruishi et al., J. Am. Chem, Soc.
No. 7285, 1984) and polyethylene glycol method (Y. Inada et al., Biochem. Biophys. Res. Commun.
No. 122, p. 845, 1984). However, these methods have a problem that the stability of the enzyme activity is very poor and the operation is complicated. In addition, a method of forming a complex of an enzyme and a lipid and solubilizing it in an organic solvent (JP-A-64)
However, there are problems that it is difficult to prepare a synthetic lipid, and the activity of the obtained lipid complex is low.

[0005]

The present invention solves the above-mentioned conventional problems, and an object of the present invention is to provide an organic solvent-soluble enzyme which exhibits high activity in an organic solvent.

[0006]

The present invention is based on the formula

[0007]

Embedded image

[In the formula, R is independently C _{6 to} C ₂₀
Is a hydrocarbon group. ] The enzyme-modified glucoxide derivative represented by the above] is provided, whereby the above object is achieved.

The glucoside derivative for enzyme modification of the present invention can be easily synthesized by reacting a C ₆ -C ₂₀ dialkyl-substituted secondary amine with glucono-1,5-lactone. The reaction is generally 60 to 60 such as methanol.
It is carried out by dissolving the above amine and lactone in a solvent having a boiling point of 250 ° C. and refluxing.

This enzyme-modified glucoxide derivative is
It is made soluble in an organic solvent by modifying various enzymes that are hydrophilic in nature. The modification method is not particularly limited,
Generally, it is carried out by a method including a step of dissolving the above-mentioned enzyme-modified glucoside derivative in a hydrophilic medium; and a step of dropping a solution of the enzyme-modified glucoside derivative into a buffer solution containing the enzyme.

The term "hydrophilic medium" as used herein includes hydrophilic organic solvents such as methanol, ethanol, propanol, acetone and methyl ethyl ketone, as well as aqueous solutions such as buffered solutions.

For example, 1 mg of the enzyme is dissolved in 0.02 to 20.0 ml, preferably 0.1 to 2 ml of a buffer solution having a pH of 4.0 to 9.0, and is dissolved at 0 to 30 ° C., preferably 4 ° C. under cooling. so,
The glucoside derivative solution is added dropwise to the stirred enzyme solution. The glucoxide derivative solution is generally 0.002-0.1 ml, in particular, 0.1 mg / mg of the glucoxide derivative.
It is preferable that the glucoxide derivative is dissolved in a hydrophilic medium at a ratio of 005 to 0.02 ml.

The stirring can be carried out using a conventional stirring device such as a stirring blade, a magnetic stirrer and a homomixer. When added dropwise with sufficient stirring and cooling, the enzyme-glucoside derivative complex precipitates and precipitates. The precipitate is separated by centrifugation or filtration, washed with a buffer solution and distilled water, and then freeze-dried or fluidized-bed dried as it is, or dispersed in a small amount of distilled water and spray-dried to give a glucoside derivative. An organic solvent-soluble enzyme powder modified with can be obtained.

In the method of the present invention, the glucoside derivative is used in a weight ratio of 0.2 to 10 based on the weight of the enzyme.
It is preferably used in an amount of 0, particularly 0.5 to 10. When the amount used is 0.2 or less, the recovery rate of the glucoxide derivative-modifying enzyme is low, and when it is 100 or more, the enzyme activity is low.

Enzymes that can be made soluble in an organic solvent by the method of the present invention include hydrolases, oxidoreductases, transferases, leaving enzymes, isomerases and synthases.

Specific examples of the hydrolase include esterases and lipases that hydrolyze esters (either those produced by microorganisms or those obtained from animal organs, serum or plant tissues, seeds, etc.) and Living tissues containing them; pepsin, chymotrypsin, carboxypeptidase, thermolysin, casepsin and aminopeptidase derived from animal organs, papain, chymopapain, promeline and aminopeptidase derived from plant tissue, and carboxypeptidase, proteinase and dipeptidase derived from microorganisms, etc. Proteases and peptidases that hydrolyze peptide bonds such as; α and β-glucosidases, α and β-glucanases, α and β-galactosidases, α and β-amylases, cellulases and pullulas Glucosidases that hydrolyze sugar glucosidic bonds such as enzymes; phosphatases that hydrolyze phosphate bonds such as phosphomonoesterases, phosphodiesterases and pyrophosphatases; hydrolyzing amide groups such as arginase, urease and glutaminase. Amidase that decomposes;
Other examples include nucleases and collagenases.

Specific examples of the oxidoreductase include alcohol dehydrogenase, lactate dehydrogenase, glucose oxidase, cholesterol oxidase and amine oxidase.

In addition, transferases such as transphospholidase, transglucosidase, transpeptidase, transamidase and transglutaminase; leaving enzymes such as decarboxylase and lyase; isomerases such as racemase and isomerase. And synthases such as ligase may also be used in the present invention.

[0019]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Preparation Examples 1 to 6 describe the preparation of lipids 1 to 6 which are glucoxide derivatives for modifying enzymes.

Production Example 1 Preparation of lipid 1 35 g of 1,5-gluconolactone and 5 of dilaurylamine were placed in a reaction vessel equipped with a condenser, a stirrer and a nitrogen introducing tube.
7 g and 125 ml of methanol were added, and the mixture was heated under reflux for 2 hours. The obtained pale yellow solid was extracted with n-hexane using a Soxhlet extractor to obtain 45 g of a white solid. In the IR spectrum of this white solid, 1750 cm
The peak of the 1,5-gluconolactone ester of ^-1 disappeared. This substance is called lipid 1.

Production Example 2 Preparation of Lipid 2 62 g of distearylamine instead of dilaurylamine
Was used, and Lipid 2 was prepared in the same manner as in Production Example 1 except that heptane was used instead of n-hexane.

Production Example 3 Preparation of Lipid 3 Lipid 3 was prepared in the same manner as in Production Example 1 except that 56 g of coconut amine was used instead of dilaurylamine.

Production Example 4 Preparation of Lipid 4 Lipid 4 was produced in the same manner as in Production Example 1 except that 61 g of oleylstearylamine was used instead of dilaurylamine.
Was prepared.

Preparation Example 5 Preparation of Lipid 5 Lipid 5 was prepared in the same manner as in Preparation Example 1 except that 52 g of octylpalmitylamine was used instead of dilaurylamine.
Was prepared.

Production Example 6 Preparation of Lipid 6 Lipid 6 was prepared in the same manner as in Production Example 1 except that 22 g of diethylamine was used instead of dilaurylamine.

Example 1 100 mg of a lipase derived from a filamentous fungus (Mucor miehei) was dissolved in 50 ml of a phosphate buffer (0.1 M, pH 7.0) and centrifuged to remove insoluble matter (solution a).

100 mg of lipid 1 was dissolved in 1 ml of acetone (solution b). Solution b was added dropwise to solution a which was stirred at ice temperature, and stirring was continued at ice temperature for 4 hours, and then left overnight at 4 ° C. The solution containing the precipitate is centrifuged (400C, 400 ° C).
0 g, 10 min), the supernatant was removed, and the remaining precipitate was washed with phosphate buffer and distilled water. Then, this solid was freeze-dried to obtain 102.4 mg of powder. The obtained modified enzyme was identified by UV spectrum.

Example 2 Lipase 1 derived from a filamentous fungus (Rhizopus niveus) instead of the lipase derived from a filamentous fungus (Mucor miehei) used in Example 1
00 mg was dissolved in 50 ml of a phosphate buffer solution (0.1 M, pH 7.0) and spun down by centrifugation to remove insoluble matter to obtain a solution a. Next, 100 mg of lipid 2 was dissolved in 1 ml of acetone (b
liquid). Then, the same procedure as in Example 1 was carried out to obtain 91.6 mg of the modifying enzyme in powder form.

Example 3 In the same manner as in Example 1 except that 100 mg of Lipid 3 was used instead of Lipid 1, 88.9 mg of powdered modification enzyme was obtained.

Example 4 97.6 mg of a modifying enzyme in powder form was obtained in the same manner as in Example 1 except that 100 mg of lipid 4 was used instead of lipid 1.

Example 5 92.4 mg of a modifying enzyme in powder form was obtained in the same manner as in Example 1 except that 100 mg of lipid 5 was used instead of lipid 1.

Comparative Example 1 Instead of the glucoside derivative used in Example 1, 100 mg of didodecyl glutamate gluconamide was used and dissolved in 1 ml of acetone to prepare a solution b. Example 1 below
By the same operation as above, 105.2 mg of powder was obtained.

[0034] is b solution was dissolved in acetone 1.5ml using monogalactosylated diglyceride 100mg instead of Gurukokisaido derivatives used in Comparative Example 2 Example 2. Then, the same operation as in Example 2 was carried out to obtain 52.6 mg of powder.

Comparative Example 3 The same operation as in Example 1 was carried out except that 100 mg of lipid 6 was used in place of lipid 1, but no powdery modification enzyme was obtained.

Example 6 Measurement of Lipid Modifying Enzyme Activity 1 mg of the modifying enzymes obtained in Examples 1 to 5 and Comparative Examples 1 to 3 were dissolved in 5 ml of toluene to measure the lipase activity. Toluene solution containing modified enzyme 10
After adding 0 μl to an olive oil-polyvinyl alcohol emulsion and reacting at 37 ° C. for 30 minutes, the amount of free fatty acid produced was titrated with a 0.05 N potassium hydroxide solution (alkali titration of free fatty acid using olive oil as a substrate. , JIS
K0601, 1988). The lipase activity (IU) is the amount of oxygen that produces 1 μmol of free fatty acid in 1 minute at 37 ° C. The activity of the modifying enzyme at this time is shown in Table 1.

[0037]

[Table 1]

From these results, it can be seen that the glucoside derivative-modifying enzyme of the present invention has higher enzyme activity than the conventional ones.

[0039]

The enzyme-modified glucoside derivative of the present invention has a simpler synthetic method than conventional modified lipids and can be obtained in large quantities at low cost. Further, the modified enzyme using the glucoxide derivative can also retain a higher enzyme activity in the organic solvent than the conventional enzyme.

Claims (7)

[Claims] (1) Formula (1) [In the formula, each R is independently a C _{6 to} C ₂₀ hydrocarbon group. ] The glucoside derivative for enzyme modification shown by these. 2. The enzyme-modified glucoside derivative according to claim 1, wherein R is independently selected from the group consisting of C _{6 to} C ₂₀ alkyl groups and alkenyl groups. 3. An organic solvent-soluble enzyme modified with the glucoxide derivative for enzyme modification according to claim 1. 4. An organic solvent comprising a step of dissolving the enzyme-modified glucoside derivative according to claim 1 in a hydrophilic medium; and a step of dropping a solution of the enzyme-modified glucoside derivative into a buffer solution containing the enzyme. A method for producing a soluble enzyme. 5. The method of claim 4, wherein each R is independently selected from the group consisting of C _{6 to} C ₂₀ alkyl and alkenyl groups. 6. The method according to claim 4, wherein the enzyme is selected from the group consisting of hydrolases, oxidoreductases, transferases, leaving enzymes, isomerases and synthases. 7. A weight ratio of 0.2 to 0.2 based on the weight of the enzyme.
The method according to claim 4, wherein the glucoxide derivative for enzyme modification is used in an amount of 100.