JP2001333789A - Method for hydrolysis with lipase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To examine the effect of the addition of an organic solvent on the hydrolysis with a lipase in an aqueous solution in order to enlarge the use of the lipase as a catalyst. SOLUTION: This method is a method for the hydrolysis with the lipase, in which the hydrolysis is performed in an aqueous solution by making the lipase derived from a microorganism act as a substrate, wherein one organic solvent or more selected from the group consisting of dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, and dimethoxyethane is/are added to the reaction system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lipase hydrolysis method, and more particularly, to a method in which a specific organic solvent is added to a reaction system when a hydrolysis reaction is carried out by allowing a microorganism-derived lipase to act on a substrate in an aqueous system. Thereby changing the substrate specificity of the enzyme and efficiently performing the hydrolysis reaction.

[0002]

2. Description of the Related Art A lipolytic enzyme lipase is an enzyme involved in fat decomposition and storage in a living body, and is widely distributed in the living world. Usually, in vivo, lipase catalyzes a reaction for decomposing and synthesizing fat. Lipases include those with low regiospecificity that cleave all three fatty acids bound to glycerin and those with high regiospecificity that cleave only the fatty acids at positions 1 and 3 of glycerin. It is also known that by adjusting the conditions of the enzymatic reaction, not only the decomposition of fat but also the action of catalyzing the reaction of synthesizing fat from glycerin and fatty acids is achieved. In addition, lipase has a transesterification effect, and this reaction is used in various industrial fields such as the food industry.

[0003] The enzymatic feature of such lipase is that it has a low substrate specificity and acts on many other compounds in addition to the original substrate, fat. For this reason, lipase is also used as a catalyst for synthesizing various useful substances, and many reports on it have been made. One of the characteristics of lipase is that it has high resistance to organic solvents. Therefore, conducting an enzyme reaction in a system containing an organic solvent is also being studied.
This method not only inactivates lipase in organic solvents, but also
Since many substrates on which lipase acts are generally insoluble or poorly soluble in water, the advantage is that lipase can easily act on the substrate by adding an organic solvent to the reaction system to solubilize the substrate. is there. In addition, in the transesterification reaction or the condensation reaction, the enzymatic reaction proceeds more easily in an organic solvent free of water, and the hydrolysis reaction does not occur, so that the yield of products of these reactions increases.

[0004] Actually, when a lipase reaction system is prepared in a 100% organic solvent containing no water, and dimethyl sulfoxide (hereinafter sometimes abbreviated as DMSO) or the like is added thereto, the reaction is accelerated. (Biotechnology and Bioengineering, Vol. 49, p. 87-
92 (1996)). However, in this method, as described above, the reaction system is a system in which a specific organic solvent is added to an organic solvent, and furthermore, only the transesterification reaction is targeted, and there is no mention of the hydrolysis reaction.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for increasing the enzymatic activity and performing an efficient enzymatic reaction by changing the substrate specificity in a lipase hydrolysis reaction system. is there. The present inventors have been studying to solve the above problems, in the course of performing a hydrolysis reaction by allowing a lipase to act on a substrate in an aqueous system,
The reaction characteristics (substrate specificity) of lipase to various substrates were analyzed by adding various organic solvents.

As a result, the reaction characteristics of lipase hydrolysis are as follows: dimethylformamide (hereinafter sometimes abbreviated as DMF), DMSO, 1,4-dioxane and dimethoxyethane (hereinafter sometimes abbreviated as DME). ), The reaction rate was increased for substrates with high hydrophobicity, while the reaction rate was decreased for substrates with low hydrophobicity. That is,
In some cases, the lipase hydrolysis activity was improved or reduced, and this was not due to a change in the solubility of the substrate but to a change in the steric structure of the enzyme. Furthermore, even when lipases derived from various organisms are used, changes in substrate specificity due to DMF or DMSO as described above (changes in hydrolysis reaction characteristics).
It was clarified that the present invention can be applied not only to fats but also to fatty acid ester compounds and fluorescent substrates. The present invention has been completed based on such findings.

[0007]

According to the first aspect of the present invention, when a hydrolysis reaction is carried out by allowing a microorganism-derived lipase to act on a substrate in an aqueous system, dimethylformamide, dimethylsulfoxide, 1,4- A hydrolysis method using a lipase, comprising adding at least one organic solvent selected from dioxane and dimethoxyethane. The present invention according to claim 2 is the method according to claim 1, wherein the substrate is a fluorescent substrate, a fat or a fatty acid ester compound.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, when a hydrolysis reaction is carried out by allowing a microorganism-derived lipase to act on a substrate in an aqueous system, the reaction system is selected from DMF, DMSO, 1,4-dioxane and DME. At least one organic solvent is added. Note that the present inventors have found that when a water-soluble organic solvent such as tetrahydrofuran (hereinafter sometimes abbreviated as THF), acetonitrile, acetone or the like is used instead of the above-mentioned organic solvent, the substrate specificity of lipase is reduced. No change in gender was observed. Also, DM
When F and DMSO are compared, DMSO has a greater effect on substrate specificity.

The hydrolysis reaction with lipase is carried out in an aqueous system, and the substrate to be used may be any one which can be a substrate for lipase, and examples thereof include fluorescent substrates, fats, fatty acid ester compounds and the like. Examples include 4-methylumbelliferones, monoglycerides, triglycerides, and fatty acid ester compounds of sugar. Specific examples thereof include 4-methylumbelliferyl oleate (hereinafter, may be abbreviated as 4MUO), 4-methylumbelliferyl palmitate (hereinafter, may be abbreviated as 4MUP), and 4-methylumbelliferyl palmitate. Veriferyl nanoate (hereinafter sometimes abbreviated as 4MUN), 4-methylumbelliferyl heptanoate (hereinafter sometimes abbreviated as 4MUH), 1 monocaprin, 1 monocaprylin, 1 monolaurin, monoolein , Triglycerides, sucrose laurate and the like.

[0010] The amount of the organic solvent to be added to the aqueous system is not particularly limited.
30%, preferably 15 to 25%, and in the case of DMSO, 20 to 45%, preferably 30 to 40%;
In the case of 4-dioxane, 10 to 30%, preferably 15 to 30%
In the case of DME, it is 7.5 to 25%, preferably 10 to 20%.

As the lipase, an enzyme of microbial origin is used, for example, Mucor mieh (Mucor mieh)
ei), Candida rugosa, Pseudomonas fluoresc
ence), Aspergillus oryza
e), those derived from Rhizopus niveus, Rhizopus delemar and the like are preferred.

According to the method of the present invention, DMF, DMS
The reaction characteristics of the lipase with respect to the substrate when O, 1,4-dioxane or DME is added include, for example, a lipase derived from microorganism Mucor mihei as an enzyme, and an acyl-
When 4 methyl umbelliferones are used, the hydrolysis activity of the enzyme is increased for strongly hydrophobic substrates such as 4 MUO and 4 MUP. However, 4MUN and 4MUH
For a substrate having a low hydrophobicity such as, for example, the hydrolysis activity is significantly inhibited. That is, when an organic solvent such as DMF is added to the reaction system, the substrate specificity of the lipase changes depending on the nature of the substrate. Such a change in the substrate specificity of lipase, that is, a change in the reaction characteristics of hydrolysis, is caused by Mucor
Similarly, when enzymes derived from the various microorganisms described above were used in addition to lipase derived from mihei, the degree of the change tended to depend on the type of enzyme. Also,
At the same time as the substrate specificity, the enzymatic analysis showed that, in the system containing the above organic solvent, the maximum rate (Vmax) of lipase to the substrate also changed. However, even when porcine pancreatic lipase was used instead of the microorganism-derived enzyme, the promotion of the hydrolysis activity by the addition of the organic solvent was not recognized.

It is evident that factors that influence the lipase hydrolysis activity vary not only with the nature of the substrate, the origin of the enzyme and the concentration of the organic solvent added, but also with the type of organic solvent. became. For example, when 4 MUO is used as a substrate, the hydrolysis activity of lipase is 20% higher than that in a 100% buffer solution.
F, approximately doubled in 30% DMSO to 15% 1,4-
It is promoted about 1.8-fold in dioxane and about 1.4-fold in 20% DME.

The change in the substrate specificity of lipase in a system containing such an organic solvent is caused not only when a fluorescent substrate such as 4MUO is used as the substrate, but also when various monoglycerides (one monocaprin, one monocaprylin, one monolaurin) are used. , Monoolein, etc.), triglycerides and sucrose laurate as substrates.

[0015]

Next, the present invention will be described in detail with reference to examples. Example 1 Various organic solvents (DMF, DMSO, 1,4-) were used as a lipase by using lipase derived from Mucor Mihei (Fluka) and using 4-methylumbelliferyl oleate (4MUO) having fluorescence as a substrate. Dioxane, DM
The change in the reaction characteristics of lipase in the reaction system to which E) was added was examined.

The composition of the enzyme reaction solution is lipase (10 ^-5 to
Buffer containing 10 ^-1 mg / mL (0.04 M Britto)
n-Robinson buffer (pH 7.0) 50 μL and a substrate solution ( ⁴ MUO, substrate concentration 10 ⁻⁴ to 10 ⁻² mM) 50 μL, and a buffer obtained by adding various organic solvents to a predetermined concentration. 900 μL was added. For example, an enzyme solution (lipase derived from Mucor miehei, 0.
1 μg / mL), 50 μL, substrate solution ( ⁴ MUO, 10 ⁻⁴⁾
mM), 700 μL of 0.04 M Britton-Robinson buffer (pH 7.0) and organic solvent (DMF) 2
Consists of 00 μL. As a control, a reaction solution to which no organic solvent was added was prepared.
μL, substrate solution ( ⁴ MUO, 10 ⁻⁴ mM) 50 μL,
0.04M Britton-Robinson buffer (pH 7.0) 9
00 μL.

The above enzyme reaction solution was incubated at 37 ° C. for 20 minutes in a water bath, and a lipase was used to hydrolyze the fluorescent substrate. Subsequently, 1 mL of 0.1 N HCl was added to stop the enzyme reaction, and then 2 mL of 0.1 M sodium citrate was added to restore the pH of the reaction solution.
Was added. By the above enzymatic reaction, the substrate 4MUO
Is broken down into oleic acid and 4-methylumbelliferone,
The fluorescence intensity of the released 4-methylumbelliferone was measured using a fluorescence spectrophotometer (JASCO FP-770, manufactured by JASCO Corporation). The excitation wavelength was set at 320 nm, and the observation wavelength was set at 450 nm. On the other hand, the fluorescence intensity of a known concentration of 4-methylumbelliferone was previously measured in the same manner, and a calibration curve was prepared. Using this calibration curve, the amount of 4-methylumbelliferone released by the lipase hydrolysis reaction was estimated, and the enzyme activity was calculated from this. The enzyme activity was determined as a relative lipase hydrolysis activity when the control was taken as 100. The results are shown in FIGS.
It is shown in (c).

Comparative Example 1 The procedure of Example 1 was repeated except that THF, 1,3-dioxane, dimethoxymethane, dimethoxypropane, anisole, methanol, ethanol, propanol or butanol were used as the organic solvent. The results are shown in FIGS.

As apparent from FIGS. 1 (a) to 1 (d),
The lipase derived from Mucor mihei had an increased hydrolysis activity when DMF, DMSO, 1,4-dioxane, and DME were added as organic solvents. However, THF, 1,
When 3-dioxane, dimethoxymethane, dimethoxypropane, anisole, methanol, ethanol, propanol and butanol were used, no improvement in the hydrolysis activity was observed, and the results were inferior to the control. On the other hand, when examining the effect of the type of lipase, similar results were obtained for lipases derived from other microorganisms other than the lipase derived from Mucor mihei, but porcine pancreatic lipase was effective in any organic solvent. I couldn't.

From the above, in order to increase the hydrolysis activity of lipase derived from microorganisms, among water-soluble organic solvents, DMF, DMSO, 1,4-dioxane, D
It became clear that it was preferable to use ME. Furthermore, it was also revealed that the amount of the lipase hydrolyzes the lipase in the direction of promotion or inhibition. In addition, even when the amount of the enzyme or the substrate concentration was changed, the same tendency as described above was shown.

When an organic solvent having an effect of promoting the activity of lipase derived from Mucor mihei is added to the reaction system, the concentration of the organic solvent and the size of the maximum activity when the enzyme activity is maximized are as follows: It depended on the alcohol of the organic solvent to be added. For example, when the lipase exhibits the maximum activity, the concentration of the organic solvent added is 20% for DMF, DMSO
35%, 1,4-dioxane 15%, DME 20%
Met. In addition, when compared with the hydrolysis activity in 100% buffer, the activities in the presence of these organic solvents are twice (20% DMF) and twice (35% DMS), respectively.
O) 1.8 times (15% 1,4-dioxane), 1.4
(20% DME).

Example 2 Four types of acyl 4-methylumbelliferones having fluorescence and different acyl side chains (4MUO, 4MUP, 4MUN, 4MU) were used as substrates for the lipase hydrolysis reaction.
Using H), the effect of the addition of an organic solvent on the lipase hydrolysis activity was examined when the concentration of the substrate was changed in the range of 0 to 20 μM. Except that DMF was used as the organic solvent to be added to the reaction system, all the procedures were performed under the same conditions as in Example 1. The concentration of DMF was set to 20%, and a test without DMF was performed as a control. FIGS. 2A to 2D show changes in fluorescence intensity when a lipase and a substrate are reacted in a reaction solution containing or not containing 20% DMF. In the figure, (a) shows the case where 4MUO was used as the substrate, (b)
Shows the case where 4MUP is used, (c) shows the case where 4MUN is used, and (d) shows the case where 4MUH is used.

As is clear from FIG. 2, when 4 MUO or 4MUP which is a highly hydrophobic substrate is used, 20% D
In the reaction system containing MF, the fluorescence intensity is increased and the hydrolysis activity is promoted by about twice, but 4MUN which is a substrate having low hydrophobicity
When 4 MUH or 4 MUH was used, the fluorescence intensity of the reaction system containing 20% DMF was significantly lower than that of the control, indicating that the hydrolysis activity of the enzyme was inhibited. In particular, when 4 MUH was used as the substrate, its fluorescence intensity was close to 0, and it was revealed that hydrolysis hardly occurred. From the above, it has been clarified that, even when the same organic solvent is used, the hydrolysis activity of lipase is promoted or inhibited depending on the properties of the substrate used. Similar results were obtained when DMSO, DME, and 1,4-dioxane were used instead of DMF as the organic solvent.

Next, DMF, DMSO,
Table 1 shows changes in the maximum rate of hydrolysis of the enzyme with respect to various substrates when a predetermined amount of each of DME and 1,4-dioxane was added to the reaction solution.

[0025]

[Table 1] Table 1

As is clear from the table, in the case of the lipase derived from Mucor miehei, the maximum rate of hydrolysis of the enzyme when four kinds of organic solvents are added increases in the case of a strongly hydrophobic substrate. 4 to 2.1 times. On the other hand, the maximum rate of hydrolysis decreases when the substrate is weakly hydrophobic.

Example 3 In this example, the effect of a fatty acid ester compound as a substrate on the hydrolysis activity of lipase (derived from Mucor Mihei, manufactured by Fluka) when DMSO was added to the reaction solution as an organic solvent was examined. did. The composition of the enzyme reaction solution was lipase (10 ^-5 to 10 ^-1 mg / m 2).
L) containing 5 μL of a buffer solution (0.04 M Britton-Robinson buffer, pH 7.0) and 5 μL of a substrate solution (sucrose laurate ester, substrate concentration of 10 ⁻³ to 10 ⁻¹ mM). 90 μL of a buffer containing 90 μL of a known concentration of DMSO was added to the solution (control) or 90 μL of the solution. The hydrolysis reaction by lipase is 3
Performed at 7 ° C. for 30 minutes.

After completion of the enzymatic reaction, 200 μL of a DMSO solution (5 mM) of 9-bromomethylacridine was added, and a fluorescent labeling reaction of the carboxyl group of lauric acid released by the enzymatic reaction with 9-bromomethylacridine was started. The fluorescent labeling reaction was performed at room temperature for 20 minutes, and the fluorescently labeled lauric acid was detected at a fluorescent wavelength (425 nm) by high performance liquid chromatography. The excitation wavelength is 365
nm. FIGS. 3A and 3B show high-performance liquid chromatograms of a fluorescently labeled product of lauric acid with 9-bromomethylacridine (elution time: 6.3 min peak) present in the reaction solution after completion of the enzyme reaction. FIG. 3A shows the results of the hydrolysis reaction in 100% buffer, and FIG. 3B shows the results of 30% DMSO.
The results of the hydrolysis reaction in the solution are shown respectively. By dissolving a known concentration of lauric acid in a solution having the same composition as the reaction solution and performing fluorescence detection under the same conditions as a calibration curve, quantification of lauric acid generated by the enzyme reaction is performed. It was converted to activity.

Example 4 The effect of an organic solvent on the hydrolysis activity of lipase when monoolein (monoglyceride), a kind of lipid, was used as a substrate was examined. Except that monoolein (concentration: 10 ^-4 to 10 ^-1 mM) was used as a substrate, all the procedures were performed under the same conditions as in Example 3. After completion of the enzyme reaction, a solution of 9 bromomethylacridine in DMSO (5 mM) was added to 20
The carboxyl group of oleic acid released by the enzyme reaction was fluorescently labeled by adding 0 μL, and the fluorescently labeled product (peak at an elution time of 11.6 minutes) was quantified by high performance liquid chromatography to convert to enzyme activity. The results are shown in FIGS. 4A and 4B. FIG. 4A shows the result of the hydrolysis reaction in a 100% buffer, and FIG. 4B shows the result of the hydrolysis reaction in a 30% DMSO solution. On the other hand, when monocaprin having a different fatty acid side chain length was used as a substrate, 30% DMSO was used.
In the presence, the lipase hydrolysis activity was suppressed. This suggests that even when the substrate is a lipid or sugar fatty acid ester, the organic solvent present in the reaction solution affects the substrate specificity of lipase.

Example 5 Using lipases derived from various organisms, the influence on the hydrolysis activity when DMSO was added as an organic solvent to the reaction solution was examined. That is, the effect on the hydrolysis activity of various enzymes when 4 MUO was used as the substrate and the concentration of the organic solvent was changed was examined. The results are shown in FIGS.
(G).

FIGS. 5 (a) to 5 (g) show the hydrolysis activity of lipases derived from various organisms in terms of relative hydrolysis activity with respect to a control containing no organic solvent as 100.
The symbols (a) to (g) in the figure indicate the origin of the enzyme,
(A) is derived from Mucor Mihei (trade name: "Lipase", manufactured by Fluka), (b) is derived from Candida Lugosa (trade name: "Lipase AY" manufactured by Amano Pharmaceutical Co., Ltd.),
(C) is from Pseudomonas fluorescens (trade name: "Lipase" manufactured by Fluka), (d) is Risops.
Delemar-derived (trade name: "Lipase", manufactured by Seikagaku Corporation), (e) is derived from Aspergillus oryzae (trade name: "Lipase", manufactured by Fluka), (f) is derived from Rhizopus nibeus (product) Name: "Lipase", manufactured by Nagase Seikagaku Corporation), (g) is derived from pig pancreas (trade name:
"Lipase type II", manufactured by Sigma).

As is clear from the figure, the addition of DMSO to the reaction mixture increased the hydrolysis activity of the six types of lipases derived from microorganisms. The activity was maximized at around 50%. It was also revealed that the magnitude of the effect on the hydrolysis activity varies depending on the origin of the lipase. On the other hand, it was shown that the activity of pig pancreatic lipase was not increased by the addition of DMSO, and the activity was inhibited depending on the concentration of the addition. From the above, it was found that the hydrolysis activity was increased by adding an organic solvent when an enzyme derived from a microorganism was used.

Example 6 In this example, the lipase hydrolysis activity on fluorescent substrates having different structures was examined. That is, 20% D
Four kinds of fluorescent substrates (1) 4MUO, (2) 4M were used in a reaction solution containing MF and a 100% control buffer solution.
The hydrolytic activities of lipases derived from various organisms on UP, (3) 4MUN and (4) 4MUH were compared. The results are shown in FIGS. In addition, the symbols (a) to
(G) shows the origin of the enzyme, each of which is given in Example 5
Is the same as the symbol in. As is clear from the figure, with respect to the six enzymes derived from microorganisms, the substrate specificity of the hydrolysis reaction could be changed by adding DMF.

[0034]

According to the method of the present invention, by adding an organic solvent to a lipase hydrolysis reaction system at a predetermined concentration, the substrate specificity of the enzyme can be adjusted according to the nature of the substrate and the type of lipase derived from various organisms. Can be changed to promote or inhibit the hydrolysis reaction.

[Brief description of the drawings]

1 (a) to 1 (d) are graphs showing the effect of various organic solvents on the lipase hydrolysis activity (relative activity).

FIGS. 2A to 2D show changes in fluorescence intensity when reacting with lipase in a reaction solution containing 20% DMF and a reaction solution not containing 20% DMF while changing the concentrations of four types of substrates. It is a graph.

FIG. 3 shows a high performance liquid chromatogram, wherein A is 100
% Is the result of the hydrolysis reaction in the buffer, B is 30% DMS
The result of the hydrolysis reaction in the O solution is shown.

FIG. 4 shows a high performance liquid chromatogram, wherein A is 100
% Is the result of the hydrolysis reaction in the buffer, B is 30% DMS
The result of the hydrolysis reaction in the O solution is shown.

FIGS. 5A to 5G are graphs showing hydrolytic activities (relative activities) when the concentration of DMSO in an enzyme reaction solution is changed using lipases derived from various microorganisms and porcine pancreas. It is.

FIGS. 6A to 6G show four types of fluorescent substrates (1) 4M using lipases derived from various microorganisms and pig pancreas.
UO, (2) 4MUP, (3) 4MUN, (4) 4MU
It is a graph which compared the hydrolysis activity with respect to H.

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[Procedure amendment]

[Submission Date] September 18, 2000 (2009.1)
8)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0025

[Correction method] Change

[Correction contents]

[0025]

[Table 1] Table 1

Claims (2)

[Claims] Claims: 1. In carrying out a hydrolysis reaction by allowing a microorganism-derived lipase to act on a substrate in an aqueous system, at least one selected from dimethylformamide, dimethylsulfoxide, 1,4-dioxane and dimethoxyethane is used in the reaction system. A method for hydrolyzing with lipase, comprising adding an organic solvent as described above. 2. The method according to claim 1, wherein the substrate is a fluorescent substrate, a fat or a fatty acid ester compound.