JP3301489B2 - How to use chemically modified esterase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel method of using a chemically modified esterase. The use of chemically modified esterase refers hydrolysis method of the ester. The chemically modified esterase obtained by the present invention has organic solvent resistance and organic solvent affinity, and can be used in various fields such as bioreactor materials.

[0002]

2. Description of the Related Art Esterase is a general term for enzymes which catalyze the hydrolysis of esters and hydrolyze them into their constituent acids and alcohols. Esterase contains lipase
It is a very important enzyme in industry. It catalyzes not only the hydrolysis reaction but also the synthesis, substitution and addition reactions, and the enzymatic reaction is also very interesting.

[0003] Typical esters of the substrate include ordinary carboxylic acid esters and triglyceride fatty acid esters (so-called fats and oils). In particular, fats and oils hydrolyzing enzymes are collectively referred to as lipases among esterases, and are particularly attracting attention such as hydrolysis of fats and oils, isolation of fatty acids, and modification of lipids.

[0004] In recent years, research on the production of useful substances utilizing enzymatic reactions has been active. Compared to general chemical reactions, enzymatic reactions are superior in that they can proceed under mild conditions (temperature, pH), are rich in stereospecificity, and have few side reactions. However, there are also problems such as the point that the usage is high and the specificity limits the use.
Among such enzymatic reactions, a reaction that is relatively versatile and often used in practice is an esterase hydrolysis reaction by esterase.

[0005] For example, studies include stereoselectively hydrolyzing esters to obtain optically active compounds. However, in such cases, the problem is often that the target compound (substrate) to be subjected to the enzymatic reaction is poorly soluble in water. Therefore, in order to dissolve the substrate, an organic solvent is unavoidably added to the reaction system, which inactivates or lowers the activity of the enzyme. As a means to solve this problem, a technique that chemically modifies the enzyme, imparts affinity or resistance to the organic solvent, and allows the enzyme reaction to be performed in the organic solvent without reducing or inactivating the enzyme activity has been attempted. Have been.

As a method generally used as a chemical modification method of such an enzyme, a method of acylating an enzyme protein using an acyl halide or an active ester (Japanese Patent Laid-Open No.
No. 96084), a method of crosslinking an enzyme with a modifying reagent using glutaraldehyde or the like, and a method of introducing polyethylene glycols via cyanuric chloride (Yasushima et al., Yuki Kagaku, 37 , 1097 to 1103, 1988).
Several methods have been proposed.

[0007] Among these methods, there are many advantages such as easy preparation of reagents, but often, acyl halide and N-
Active esters typified by hydroxysuccinimide esters are poorly soluble in water and are not suitable for enzyme modification. In addition, this method is not preferable because unnecessary reactions such as cross-linking between enzymes occur. Is a chemical modification method that has been frequently used in recent years, but it takes time to prepare the modifier, and the modifier cannot be freely selected other than changing the degree of polymerization of polyethylene glycol. There is also a problem.

On the other hand, the inventors of the present invention have proposed a method of chemically modifying an endopeptidase by using an active ester represented by Chemical formula 1 and parahydroxyphenyldimethylsulfoniophenyl (DSP) ester as a chemical modification method of an enzyme. A patent application has been filed for finding that it can be chemically modified (Japanese Patent Application No. 1-55053).

The above-mentioned DSP is a known compound disclosed as an acylating reagent in JP-A-63-8365. The present inventors have confirmed that endopeptidase in which the amino group of the enzyme protein has been chemically modified by the DSP is stable even in an aqueous-organic solvent system. Also, in the above-mentioned JP-A-62-96084,
A lipoxygenase in which an amino group on the surface of an enzyme protein is chemically modified with an alkyl group is disclosed. However, a chemically modified esterase obtained by acylating an amino group with an acylating agent as disclosed in the present invention has not been reported so far.

[0010]

An object of the present invention is to provide a novel use of an esterase chemically modified with the above-mentioned reagent. The use of this esterase refers hydrolysis method of the ester. The modified esterase obtained in the present invention has a feature that it has high resistance to organic solvents and does not lose its activity even in a high-concentration organic solvent.

[0011]

It is known that some esterases such as lipase are highly resistant to organic solvents and show activity even in relatively high concentrations of organic solvents. Accordingly, the reaction cannot be performed efficiently. In the present invention, an RCO- group is introduced into the amino group of the esterase in order to prevent the activity from lowering and to make it soluble in an organic solvent. (However, R is a benzyloxy group, a tert-butoxy group, a 9-fluorenylmethoxy group, a p-methoxybenzyloxy group, a carbon number C ₁ to
It represents any one of an alkyl group is a C _24. )

In order to introduce an RCO group, in the present invention, a DSP ester having an intended RCO group is used. This compound can be used to prepare a DSP ester having an intended RCO-group by the method disclosed in JP-A-63-8365. Such esters include, for example, 4- (lauroyloxy) phenyldimethylsulfonium methyl sulfate,
(Palmitoyloxy) phenyldimethylsulfonium methylsulfate, 4- (benzyloxycarbonyloxy) phenyldimethylsulfoniummethylsulfate, 4- (tert-butyloxycarbonyloxy) phenyldimethylsulfoniummethylsulfate, 4-
(9-Fluorenylmethoxycarbonyloxy) phenyldimethylsulfonium methylsulfate and the like, but other DSP esters can also be used in the practice of the present invention.

In order to produce the modified enzyme, the enzyme must be at pH 7.
8 in a Na ₂ B ₄ O ₇ -HCl buffer solution, and a DSP ester having a group to be introduced was added in an amount of 0.5 to 15 equivalents based on the total amino group amount of the enzyme, followed by reaction. The desired RCO-group can be introduced into the amino group. The reaction temperature may be any condition as long as the enzyme is not inactivated, but is preferably from 0 to 0.
40 ° C is suitable. After completion of the reaction, the desired modified enzyme is treated by a conventional method such as gel filtration or membrane treatment, and the enzyme fraction is collected. The enzyme fraction is cryopreserved or lyophilized to obtain a modified enzyme.

The above-mentioned DSP ester has high water solubility, so that an appropriate buffer is selected under conditions other than those described above, and p
In H5.0 to 11.0, an RCO- group can be easily introduced into an amino group in the enzyme. This method can introduce a low-molecular-weight hydrophobic group as compared with the introduction of a high-molecular hydrophobic group such as polyethylene glycol, which has been widely used, and the enzyme is less inactivated because it is an aqueous reaction. Has the advantage of The appropriate amount of the group to be introduced into the modifying enzyme is from 20 to 60% based on the total amino group content of the esterase, and this range is preferable from the viewpoint of maintaining the affinity for the organic solvent and maintaining the activity.

The modified enzyme thus obtained has the same enzyme chemical properties as the original esterase, such as substrate specificity and reactivity, and is further imparted with organic solvent resistance and affinity. You. The introduced group is released from the amino group by a treatment such as acid decomposition, and can be identified as the modified enzyme of the present invention by analyzing the amino group by gas chromatography.

In particular, when the introduced group is a benzyloxy group, a tert-butoxy group, a 9-fluorenylmethoxy group or a p-methoxybenzyloxy group, the introduced group can be cleaved under mild conditions. It can be easily regenerated to the original natural enzyme. Particularly in the case of a valuable esterase, the use of the enzyme can be expected to be expanded by expanding the range of use of the enzyme.

Since the thus obtained enzyme has resistance to organic solvents and affinity for organic solvents, unlike conventional esterases, the enzyme reaction can be carried out in an organic solvent such as hexane, benzene, ether, acetone or cyclohexane. it can. Generally, 0.1 mg / ml to 10 mg of any of the present modified enzymes is added to a neutral buffer such as a phosphate buffer.
/ Ml, and then the desired ester is dissolved to a concentration of about 10 to 20%, and 1 to 10 times the amount of the above enzyme solution is collected and mixed with the enzyme solution. Then, an enzymatic reaction is performed. Further, the present esterase can be subjected to an enzymatic reaction as an immobilized enzyme which is immobilized on a resin or encapsulated in a gel.

[0018] The modified esterase is usually lyophilized,
Can be stored as a spray dried enzyme. If necessary, the solution can be stored as it is after the modification reaction or frozen.

The reaction using the modified esterase includes:
Examples include ester synthesis and transesterification for the purpose of esterification by ester hydrolysis by reverse reaction and reverse reaction. The target substance can be obtained by catalyzing these reactions using the modified esterase of the present invention.

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

[0021]

Example 1 In this example, a production example of a modified esterase will be described. (1) Chemical modification of lipase using 4- (lauroyloxy) phenyldimethylsulfonium methylsulfate (La-DSP) 10 mg of lipase PN (Seikagaku Corporation) with 0.1 M Na
₂ B ₄ O ₇ -HCl buffer (pH 7.8) are dissolved in 5 ml. The desired amount of La-DSP was added in three portions for 60 minutes while slowly stirring under ice-cooling with a magnetic stirrer. Thereafter, the reaction was carried out at 4 ° C. for 16 hours with slow stirring. After completion of the reaction, the reaction solution
The precipitate was removed by centrifugation at 0 G for 10 minutes, and the supernatant was dialyzed against a 200-fold volume of deionized water at 4 ° C. for 72 hours using a dialysis membrane having a molecular weight cutoff of 2,000. The deionized water used for dialysis was replaced with a fresh one every 12 hours.
After dialysis, the solution containing the target substance was freeze-dried to obtain the target modified enzyme powder.

The lyophilized powder obtained was subjected to TNBS
Law (NovoEnzyme Information: November AF
At -75/1 ア ミ ノ GB (1970), the amount of the modified amino group was quantified, and the modification rate was determined from this. Table 1 shows the amount of the modifying agent added, the yield of the modifying enzyme, and the modification ratio.

[0023]

[Table 1] 量 Amount of modifier added Yield ^{* 1} Modification rate (equivalent per amino group) (mg) (%) ── ──────────────────── 0.5 11.18 31.4 1.0 12.00 35.8 1.5 12.28 36.1 2.0 12.93 50.5 2.5 12.81 56.4 5.0 12.90 57.8 10.0 12.89 56.9 15.0 12.87 57.5 ────── ──────────────── * 1 Yield from 10mg of raw material lipase

(2) 4- (palmitoyloxy) phenyldimethylsulfonium methylsulfate (Pa-D
Chemical modification of lipase using SP) Pig pancreatic lipase (Elastin product) 10m
g was modified in the same manner as in Example 1 (1). As a result, Table 2 shows the relationship between the obtained additive amount, yield, and modification rate.

[0025]

[Table 2] 量 Modifier addition amount Yield ^{* 1} Modification rate (equivalent per amino group) (mg) (%) ── ──────────────────── 0.5 12.00 30.8 1.5 11.42 38.4 2.5 12.02 54.1 10.0 12.11 55.0 ────────────────── ──── * 1 Yield from 10mg of raw material lipase

(3) Chemical modification of lipase using 4- (benzyloxycarbonyloxy) phenyldimethylsulfonium methylsulfate (Z-DSP) About 10 mg of lipase PN (Seikagaku Corporation) in the same manner as in Example 1 (1) Modifications were made. As a result, Table 3 shows the relationship between the obtained additive amount, the yield, and the modification rate.

[0027]

[Table 3] 量 Amount of modifier added Yield ^{* 1} Modification rate (equivalent per amino group) (mg) (%) ── ──────────────────── 0.5 11.10 36.6 1.5 10.98 38.9 2.5 11.38 57.1 10.0 11.29 58.8 ────────────────── ──── * 1 10mg of enzyme used for modification

(4) Chemical modification of lipase using 4- (tert-butyloxycarbonyloxy) phenyldimethylsulfonium methyl sulfate (Boc-DSP) 10 mg of porcine pancreatic lipase (Elastin Products)
Was modified in the same manner as in Example 1 (1). As a result, Table 4 shows the relationship between the obtained additive amount, the yield, and the modification rate.

[0029]

[Table 4] 量 Amount of modifier added Yield ^{* 1} Modification rate (equivalent per amino group) (mg) (%) ── ──────────────────── 0.5 11.20 32.2 1.5 11.38 40.0 2.5 11.10 51.9 10.0 11.39 56.8 ────────────────── ──── * 1 10mg of enzyme used for modification

(5) Chemical modification of lipase using 4- (9-fluorenylmethoxycarbonyloxy) phenyldimethylsulfonium methylsulfate (Fmoc-DSP) Example of 10 mg of lipase PN (Seikagaku Corporation)
Modification was performed in the same manner as (1). As a result, Table 5 shows the relationship between the obtained additive amount, the yield, and the modification rate.

[0031]

[Table 5] 量 Amount of modifier added Yield ^{* 1} Modification rate (equivalent per amino group) (mg) (%) ── ──────────────────── 0.5 11.88 28.8 1.5 12.01 39.8 2.5 12.10 46.9 10.0 12.63 52.9 ────────────────── ──── * 1 10mg of enzyme used for modification

(6) Dispersibility of Modified Lipase in Organic Solvent For each of the chemically modified lipases obtained in the above (1) to (5), their dispersibility in organic solvents was examined. In this method, 1 mg of the modified enzyme was well suspended in 1 ml of a solvent, and the amount of the enzyme that did not precipitate was used as a measure of dispersibility. The results are shown in Tables 6 and 7. Tables 6 and 7
Are the same.

[0033]

[Table 6]

[Table 7] La in the column of the modifier in the table is 4- (lauroyloxy)
Phenyldimethylsulfonium methylsulfate, Pa is 4- (palmitoyloxy) phenyldimethylsulfonium methylsulfate, Z is 4-
(Benzyloxycarbonyloxy) phenyldimethylsulfonium methylsulfate, Boc is 4-
This shows that (tert-butyloxycarbonyloxy) phenyldimethylsulfonium / methylsulfate was used as a chemical modifier.

[0034]

Example 2 This example shows an example of ester hydrolysis using the modified esterase obtained in Example 1. (1) Esterase activity test of chemically modified enzyme 1 mg of lipase modified according to Example 1 was dissolved in 3 ml of phosphate buffer (pH 7.0), and 1 g of tristearin as a substrate was added to the solution at 20 W / V% organic level. It was added as a solvent solution and incubated at 37 ° C. for 20 minutes. The amount of free fatty acids was measured by titration. Table 8 shows the results of the ester hydrolysis activity. The activity was shown as a relative activity with the unmodified enzyme taken as 100.

[0035]

[Table 8] The abbreviations in the table indicate the following. ZML-10: lipase 10% benzyloxycarbonyl modification enzyme ZML-30: lipase 30% benzyloxycarbonyl modification enzyme ZML-50: lipase 50% benzyloxycarbonyl modification enzyme BML-50: lipase 50% tert-butoxycarbonyl Modification enzyme LML-40: lipase 40% lauroylation modification enzyme PML-40: lipase 40% palmitoylation modification enzyme

The modified lipase of the present invention shows higher activity in an organic solvent than unmodified lipase, and it has been confirmed that the activity is maintained, and it has been proved that the modified lipase is excellent in organic solvent resistance.

(2) Ester hydrolysis by modified lipase 0.1 mg of the lipase modified in Example 1 was dissolved in 0.5 ml of phosphate buffer (pH 7.0), and 50 mg of stearyl stearate was used as a substrate in the solution. Was dissolved in 0.5 ml of cyclohexane. This was incubated at 37 ° C. for 20 minutes, and the released stearic acid was separated and quantified by HPLC to determine the decomposition rate.

[0038]

[Table 9] 酵 Enzyme decomposition rate (%) ───────────────── ───── Unmodified 45.3 LML-30 63.9 LML-40 64.2 LML-50 69.8 PML-30 71.0 PML-40 70.1 PML-50 79.7 ──────────────────略 The abbreviations in the table indicate the following. LML-30: Lipase 30% Lauroylating Modifier LML-40: Lipase 40% Lauroylating Modifier LML-50: Lipase 50% Lauroylating Modifier PML-30: Lipase 30% Palmitoylating Modifier PML-40: Lipase 40 % Palmitoylation-modifying enzyme PML-50: Lipase 50% palmitoylation-modifying enzyme

[0039] The modified lipase of the present invention exhibited a degradation rate of at least 20% higher than that of the unmodified lipase.

[0040]

Embodiment 3 In this embodiment, an example of ester synthesis will be described. (1) Synthesis of EPA ethyl ester Ethanol was added to 1 g of eicosapentaenoic acid (EPA) to prepare a 20% W / V ethanol solution, and 1 mg of the modified enzyme prepared in Example 1 was added. 37 ° C, 12
The reaction was incubated at 0 rpm (l = 5 cm) and stopped after 24 hours. After developing using HPTLC and developing color with 20% sulfuric acid, the relative ratio was detected by densitometry, and the reaction rate was calculated. Table 10 shows the results.

[0041]

[Table 10] The abbreviations in the table indicate the following. ZML-10: Lipase 10% benzyloxycarbonyl modification enzyme ZML-30: Lipase 30% benzyloxycarbonyl modification enzyme ZML-50: Lipase 50% benzyloxycarbonylation modification enzyme LML-10: Lipase 10% lauroylation modification enzyme LML-30: lipase 30% lauroyl modification enzyme LML-50: lipase 50% lauroyl modification enzyme BML-5: lipase 5% tert-butoxycarbonyl modification enzyme BML-10: lipase 10% tert-butoxycarbonyl modification enzyme BML-30: Lipase 30% tertiary butoxycarbonylation modification enzyme BML-50: Lipase 50% tertiary butoxycarbonylation modification enzyme PML-10: Lipase 10% palmitoylation modification enzyme PML-30: Lipase 30% palmitoylation Modification enzyme PML-50: Lipase 50% palmitoylation modification enzyme

As a result, according to the present invention using this modified enzyme, it was found that an ester can be synthesized at a high conversion even in an organic solvent.

(2) Synthesis of cholesterol ester Cholesterol 100 in 10 ml of water-saturated n-hexane
mg and 100 mg of oleic acid are dissolved, and 1 mg of the modified enzyme prepared in Example 1 is added. 37 ° C, 120r
After incubation for 24 hours at pm (l = 5 cm), the reaction was stopped. After developing using HPTLC and developing color with 20% sulfuric acid, the relative ratio was detected by densitometry, and the reaction rate was calculated. Table 11 shows the results.

[0044]

[Table 11] 酵 Enzyme reaction (%) ─────────────────── Unmodified 2.5 PML-10 35.3 PML-30 38.8 PML-50 41.5 LML-10 10.1 LML-30 9.7 LML-50 11.9 略 Abbreviations in the table are as follows Indicates that. LML-10: lipase 10% lauroylation-modifying enzyme LML-30: lipase 30% lauroylation-modifying enzyme LML-50: lipase 50% lauroylation-modifying enzyme PML-10: lipase 10% palmitoylation-modifying enzyme PML-30: lipase 30 % Palmitoylation-modifying enzyme PML-50: Lipase 50% palmitoylation-modifying enzyme

As a result, according to the present invention using this modified enzyme, it was found that an ester can be synthesized at a high reaction rate even in an organic solvent.

(3) Synthesis of lactose ester 1 g of finely ground lactose powder was dispersed in 10 ml of 0.05 M phosphate buffer (pH 7.0), and further mixed with 10 ml of ethyl acetate to which 1 g of oleic acid was added. Example 1
Add 1 mg of the modified enzyme prepared in the above. 37 ° C, 1
Incubation was performed at 20 rpm (1 = 5 cm), and the reaction was stopped after 24 hours. After developing using HPTLC and coloring with 20% sulfuric acid, the relative ratio was detected by densitometry,
The reaction rate was calculated. Table 12 shows the results.

[0047]

[Table 12] The abbreviations in the table indicate the following. ZML-10: Lipase 10% benzyloxycarbonyl modification enzyme ZML-30: Lipase 30% benzyloxycarbonyl modification enzyme ZML-50: Lipase 50% benzyloxycarbonylation modification enzyme LML-10: Lipase 10% lauroylation modification enzyme LML-30: lipase 30% lauroyl modification enzyme LML-50: lipase 50% lauroyl modification enzyme BML-5: lipase 5% tert-butoxycarbonyl modification enzyme BML-10: lipase 10% tert-butoxycarbonyl modification enzyme BML-30: Lipase 30% tertiary butoxycarbonylation modification enzyme BML-50: Lipase 50% tertiary butoxycarbonylation modification enzyme PML-10: Lipase 10% palmitoylation modification enzyme PML-30: Lipase 30% palmitoylation Modification enzyme PML-50: Lipase 50% palmitoylation modification enzyme

As a result, according to the present invention using this modified enzyme, it was found that an ester can be synthesized at a high conversion even in an organic solvent.

[0049]

Embodiment 4 In this embodiment, an example of transesterification will be described. (1) Transesterification using triolein and palmitic acid 100 ml of triolein in 10 ml of water-saturated n-hexane
g and 100 mg of palmitic acid were dissolved, and transesterification was carried out in addition to 10 mg of the modified enzyme prepared in Example 1. The mixture was incubated at 37 ° C. and 120 rpm (1 = 5 cm). After 24 hours, the reaction was stopped. After separation by TLC into a fatty acid fraction and a glyceride fraction, the transesterification rate was measured by gas chromatography. Table 1 shows the results
3 is shown.

[0050]

[Table 13] The abbreviations in the table indicate the following. ZML-10: Lipase 10% benzyloxycarbonyl chemical modification enzyme ZML-30: Lipase 30% benzyloxycarbonyl chemical modification enzyme ZML-50: Lipase 50% benzyloxycarbonyl chemical modification enzyme LML-10: Lipase 10% lauroyl chemical modification enzyme LML-30: lipase 30% lauroyl chemical modification enzyme LML-50: lipase 50% lauroyl chemical modification enzyme BML-5: lipase 5% tert-butoxycarbonyl chemical modification enzyme BML-10: lipase 10% tertiary butoxycarbonyl chemical modification enzyme BML-30: Lipase 30% tertiary butoxycarbonyl chemical modification enzyme BML-50: Lipase 50% tertiary butoxycarbonyl chemical modification enzyme PML-10: Lipase 10% palmitoyl chemical modification enzyme PML-30: Liper 30% palmitoyl chemically modified enzyme PML-50: Lipase 50% palmitoyl chemically modified enzyme

As a result, according to the present invention using the modified enzyme, it was found that an ester can be synthesized at a high conversion even in an organic solvent.

(2) Transesterification using phosphatidylcholine and EPA 5 mg of 1-palmitoyl-2-stearoylphosphatidylcholine and 20 mg of EPA were dissolved in 1 ml of n-hexane, and PML-10 and PML were prepared according to Example 1.
A transesterification reaction was performed using -30 and PML-50. The mixture was incubated at 37 ° C. and 120 rpm (1 = 5 cm), and the transesterification rate after 48 hours and the decomposition rate to lyso-form were measured. The transesterification rate was determined by TLC, and the decomposition rate was determined by gas chromatography using HPLC.
Was measured. Table 14 shows the results.

[0053]

[Table 14] 酵 Enzyme exchange rate (%) Decomposition rate (%) ──────── ────────────────── Unqualified 0.0 5.8 PML-10 3.3 20.5 PML-30 13.5 19.3 PML-50 10.8 21.5 ─────────────略 The abbreviations in the table indicate the following. PML-10: Lipase 10% palmitoylation modifying enzyme PML-30: Lipase 30% palmitoylation modification enzyme PML-50: Lipase 50% palmitoylation modifying enzyme

As a result, according to the present invention using the modified enzyme, it was found that an ester can be synthesized at a high reaction rate even in an organic solvent.

[0055]

According to the present invention, the amino group is replaced with RCO
-Groups have made it possible to obtain chemically modified esterases. In addition, enzymatic hydrolysis of an ester in an organic solvent, ester synthesis, or ethesyl exchange can be performed using the chemically modified esterase. The modified esterase has excellent organic solvent resistance and organic solvent affinity, and can be used in bioreactors and the like.

 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-23467 (JP, A) JP-A-1-68282 (JP, A) JP-A-62-151190 (JP, A) 96084 (JP, U) I AZUSE et al. , Chemistry Express, 4 (8), 539-542 (1989). Yanaihara ed. , "Peptide Chemistry 1989", Protein Research Foundation, Osaka, 391-394 (JICST acceptance date 1990. 3.19)

Claims (1)

(57) [Claims] 1. A method for hydrolyzing an ester, which comprises hydrolyzing the ester using a chemically modified esterase having a group represented by RCO- introduced into 20 to 60% of the total amount of amino acids in the enzyme. (Where R is a benzyloxy group, a tert-butoxy group, 9
- indicates either fluorenyl methoxy group, a p- methoxybenzyl group. )