JPH04365479A - Chemically modified esterase, its production and use thereof - Google Patents

Abstract

PURPOSE:To provide a chemically modified esterase having high resistance to organic solvent and capable of keeping the activity even in a high- concentration organic solvent. CONSTITUTION:The objective chemically modified esterase is produced by introducing RCO- groups (R is benzyloxy group, t-butoxy group, 9- fluorenylmethoxy group, p-methoxybenzyloxy group or 1-24C alkyl group) to 20-60% of the total amino groups of an esterase. The chemically modified esterase can be obtained by reacting an esterase with a p-hydroxyphenyl dimethylsulfonio-phenyl ester having a group to be introduced. The modified esterase can be used for the ester-hydrolysis, esterification or ester-interchange reaction.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to chemically modified esterases, methods for producing the same, and methods for using the same. Methods for using chemically modified esterases include ester hydrolysis methods, ester synthesis methods, and transesterification methods. The chemically modified esterase obtained according to the present invention is endowed with organic solvent resistance and organic solvent affinity, and can be used in many fields such as bioreactor materials.

0002

BACKGROUND OF THE INVENTION Esterase is a general term for enzymes that catalyze the hydrolysis of esters into their constituent acids and alcohols. Esterase contains lipase and a portion of protease, and is an extremely important enzyme in industry. Enzyme reactions are also very interesting, as they not only catalyze hydrolysis reactions, but also synthesis, substitution, and addition reactions.

Typical substrate esters include ordinary carboxylic acid esters and triglyceride fatty acid esters (so-called fats and oils). In particular, hydrolyzing enzymes for fats and oils are collectively called lipases among esterases, and are attracting particular attention for their use in hydrolyzing fats and oils, isolating fatty acids, and modifying lipids.

[0004] In recent years, research on the production of useful substances using enzymatic reactions has been active. Compared to general chemical reactions, enzymatic reactions are superior in that they proceed under mild conditions (temperature, pH), have high stereospecificity, and have few side reactions, but on the other hand, they are costly. There are also problems, such as the fact that it is highly effective, and its uniqueness limits its use. Among these enzymatic reactions, one that is relatively versatile and is often used in practice is the hydrolysis reaction of esters by esterase.

[0005] For example, research involves stereoselectively hydrolyzing esters to obtain optically active compounds. However, in such cases, the problem that often arises is that the target compound (substrate) to undergo the enzymatic reaction is sparingly soluble in water. Therefore, in order to dissolve the substrate, an organic solvent must be added to the reaction system.
This results in deactivation of the enzyme or reduction in activity. As a means of solving this problem, attempts have been made to chemically modify enzymes to give them affinity or resistance to organic solvents, thereby allowing enzyme reactions to occur in organic solvents without reducing or deactivating enzyme activity. It is being

[0006] As a method for chemically modifying enzymes, methods generally used include: (1) A method of acylating enzyme proteins using acyl halides or active esters (Japanese Patent Application Laid-Open No. 1983-1999);
-96084 publication), ■ A method of crosslinking a modification reagent and an enzyme using glutaraldehyde etc., ■ A method of introducing polyethylene glycols via cyanuric chloride (
Yasujima et al., Oil Chemistry, 37, 1097-1103, 1988
Several methods have been proposed, including

Among these methods, method (2) has many advantages such as easy preparation of reagents, but it often involves the use of acyl halides and N-
Active esters represented by hydroxysuccinimide ester are poorly soluble in water and are unsuitable for modifying enzymes. Furthermore, method (2) is not preferred because unnecessary reactions such as crosslinking between enzymes occur. Method (2) is a chemical modification method that has become frequently used in recent years, but it is time-consuming to prepare the modifier, and it is not possible to freely select the modifier other than by changing the degree of polymerization of polyethylene glycol. There are also other problems.

[0008] On the other hand, the present inventors have found that an endopeptidase can be efficiently modified by using an active ester represented by chemical formula 1, para-hydroxyphenyldimethylsulfoniophenyl (DSP) ester. We have already applied for a patent after discovering 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 an enzyme protein is chemically modified by the above DSP, is stable even in a water-organic solvent system. Furthermore, the above-mentioned Japanese Patent Application Laid-Open No. 62-96084 discloses a lipoxygenase in which the amino group on the surface of an enzyme protein is chemically modified with an alkyl group. 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

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel esterase chemically modified with the above-mentioned reagent, a method for producing the same, and a method for using the esterase. Methods for using this esterase include ester hydrolysis methods, ester synthesis methods, and transesterification methods. The modified esterase obtained according to the present invention has the feature that it has strong organic solvent resistance and does not lose its activity even in a highly concentrated organic solvent.

[0011]

[Means for solving the problem] It is known that some esterases such as lipase have high resistance to organic solvents and exhibit activity even in relatively high concentration organic solvents. Therefore, the reaction cannot be carried out efficiently. In order to prevent a decrease in activity and to make the esterase soluble in organic solvents, in the present invention, an RCO- group is introduced into the amino group of the esterase. (However, R is a benzyloxy group, tert-butoxy group, 9-fluorenylmethoxy group, p-methoxybenzyloxy group, carbon number C1
-C24 represents any alkyl group. )

00
12] In order to introduce an RCO- group, a DSP ester having the desired RCO- group is used in the present invention. This compound was prepared using the method disclosed in JP-A No. 63-8365 to obtain the desired DS having an RCO- group.
P esters can be prepared. Such esters include, for example, 4-(lauroyloxy)phenyldimethylsulfonium methylsulfate, 4-(palmitoyloxy)phenyldimethylsulfonium methylsulfate, 4-(benzyloxycarbonyloxy)
Phenyldimethylsulfonium methylsulfate, 4
-(tert-butyloxycarbonyloxy)phenyldimethylsulfonium methylsulfate, 4-(9-fluorenylmethoxycarbonyloxy)phenyldimethylsulfonium methylsulfate, etc., but other DSP esters may also be used in the present invention. It can be used for implementation.

[0013] To produce the modified enzyme, the enzyme is adjusted to pH 7.
DSP ester having the group to be introduced is dissolved in the Na2B4O7-HCl buffer solution of 8 and added in an amount of 0.5 to 15 equivalents based on the total amount of amino groups of the enzyme, and is reacted with the amino groups in the protein. A desired RCO- group can be introduced. The reaction temperature may be any condition as long as the enzyme is not inactivated, but preferably 0 to 40°C. After the reaction is completed, the desired modified enzyme is treated by a conventional method such as gel filtration or membrane treatment, and the enzyme fraction is recovered. The enzyme fraction is cryopreserved or freeze-dried to obtain a modified enzyme.

[0014] The DSP ester described above has high water solubility, and even under conditions other than the above, an appropriate buffer solution can be selected, and p
At H5.0 to 11.0, an RCO- group can be easily introduced into an amino group in the enzyme. Compared to the introduction of hydrophobic groups from polymers such as polyethylene glycol, which have been widely used in the past, this method allows the introduction of low-molecular hydrophobic groups, and because the reaction is conducted in an aqueous system, enzyme deactivation is less likely. It has the following advantages. The appropriate amount of groups to be introduced into the modified enzyme is 20 to 60% of the total amount of amino groups in the esterase, and a range within this range is preferable in terms of organic solvent affinity and activity maintenance.

[0015] The modified enzyme thus obtained retains the enzymatic chemical properties of the original esterase, such as substrate specificity and reactivity, and is further endowed with organic solvent resistance and affinity. Ru. The introduced group is liberated from the amino group by treatment such as acid decomposition, and the enzyme can be identified as the modified enzyme of the present invention by analyzing this by gas chromatography.

In particular, when the introduced group is a benzyloxy group, tert-butoxy group, 9-fluorenylmethoxy group, or p-methoxybenzyloxy group, it is possible to cleave the introduced group under mild conditions. can be easily regenerated into the original natural enzyme. Particularly in the case of valuable esterases, we can expect their applications to expand as the scope of use of the enzyme expands.

[0017] Unlike conventional esterases, the enzyme thus obtained has resistance to organic solvents and affinity for organic solvents, so that enzymatic reactions can be carried out in organic solvents such as hexane, benzene, ether, acetone, and cyclohexane. can. Generally, for any of the modified enzymes, 0.1 mg/ml to 10 mg is added to a neutral buffer such as a phosphate buffer.
Then, dissolve the target ester to a concentration of about 10 to 20%, collect 1 to 10 times the amount of the enzyme solution mentioned above, and mix it with the enzyme solution. and allow the enzymatic reaction to occur. The enzyme reaction can also be carried out using the present esterase as an immobilized enzyme, such as by immobilizing it on a resin or encapsulating it in a gel.

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

[0019] As a reaction using modified esterase,
Examples include ester hydrolysis reaction, ester synthesis for the purpose of esterification by reverse reaction, and transesterification reaction. Using the modified esterase of the present invention, these reactions can be catalyzed to obtain the target substance.

[0020] The present invention will be further explained in detail with reference to Examples below.

[0021]

[Example 1] This example shows an example of a method for producing a modified esterase. (1) Chemical modification of lipase using 4-(lauroyloxy)phenyldimethylsulfonium methylsulfate (La-DSP) 10 mg of lipase PN (Seikagaku Corporation) was diluted with 0.1 M N
a2 B4 O7 -HCl buffer (pH 7.8) 5m
Dissolve in l. While slowly stirring with a magnetic stirrer under ice cooling, the desired amount of La-DSP was added in three portions over 60 minutes. Thereafter, the mixture was allowed to react at 4° C. for 16 hours with slow stirring. After the reaction is completed, the reaction solution is heated to 4°C.
Centrifuge at 10,000G for 10 minutes, remove the precipitate, and centrifuge the supernatant at 200°C at 4°C using a dialysis membrane with a molecular weight cutoff of 2,000.
Dialysis was performed for 72 hours against twice the amount of deionized water. The deionized water used for dialysis was replaced with fresh water every 12 hours. After dialysis, the solution containing the target product was freeze-dried to obtain the target modified enzyme powder.

Regarding the obtained freeze-dried powder, TNBS
Law (Novoenzyme Information: November issue A
F-75/1-GB (1970), the amount of modified amino groups was quantified, and the modification rate was determined from this. Table 1 shows the amount of the modifier added, the yield of the modified enzyme, and the modification rate.

[0023]

[Table 1] ────────────
────────────
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
- Chemical modification of lipase using (palmitoyloxy)phenyldimethylsulfonium methylsulfate (Pa-DSP) Porcine pancreatic lipase (manufactured by Elastin Products) 10m
g was modified in the same manner as in Example 1 (1). Table 2 shows the relationship between the amount of modifier added, the yield, and the modification rate obtained as a result.

[0025]

[Table 2] ────────────
────────────
Amount of modifier added 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) 10 mg of lipase PN (Seikagaku Corporation) was modified in the same manner as in Example 1 (1). Table 3 shows the relationship between the amount of modifier added, the yield, and the modification rate obtained as a result.

[0027]

[Table 3] ────────────
────────────
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) 4-
(Tertiary-butyloxycarbonyloxy)phenyldimethylsulfonium methylsulfate (Boc-DSP)
Chemical modification of lipase using porcine pancreatic lipase (Elastin Products Co., Ltd.) 10 mg Example 1 (1)
Modifications were made in the same way. As a result, Table 4 shows the relationship between the amount of modifier added, the yield, and the modification rate.

[0029]

[Table 4] ──────────────
─────────
Amount of modifier added Yield*1 Modification rate (equivalent per amino group) (mg) (%)
──────────────────
──────
0.5 11.20
32.2
1.5 11.3
8 40.0
2.5
11.10 51.9
10.0
11.39 56.8
──────────────────
──────
*1 10mg of enzyme used for modification

0030
] (5) Chemical modification of lipase using 4-(9-fluorenylmethoxycarbonyloxy)phenyldimethylsulfonium methylsulfate (Fmoc-DSP) Same as Example (1) for 10 mg of lipase PN (Seikagaku Corporation) Modifications were made to. As a result, Table 5 shows the relationship between the amount of modifier added, the yield, and the modification rate.

[0031]

[Table 5] ──────────────
─────────
Amount of modifier added Yield*1 Modification rate (equivalent per amino group) (mg) (%)
──────────────────
──────
0.5 11.88
28.8
1.5 12.0
1 39.8
2.5
12.10 46.9
10.0
12.63 52.9
──────────────────
──────
*1 10mg of enzyme used for modification

[0032](
6) Dispersibility of modified lipase in organic solvents (1) above
~(5) Regarding each chemically modified lipase obtained in
Their dispersibility in organic solvents was investigated. In this method, 1 mg of modified enzyme was well suspended in 1 ml of solvent, and the amount of enzyme that did not precipitate was used as a measure of dispersibility. The results are shown in Tables 6 and 7. Note that Table 6 and Table 7 are the same.

[0033]

[Table 6]

[Table 7] In the modifier section of the table, La is 4-(lauroyloxy)phenyldimethylsulfonium methylsulfate, Pa is 4-(palmitoyloxy)phenyldimethylsulfonium methylsulfate, and 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 enzymes 1 mg of lipase modified according to Example 1 was added to phosphate buffer (pH 7).
．． 0) Dissolve in 3ml and add 1g of tristearin as a substrate to the solution as a 20W/V% organic solvent solution 3
Incubated for 20 minutes at 7°C. The amount of free fatty acids was measured by titration. The results of ester hydrolysis activity are shown in Table 8. The activity was expressed as relative activity with the unmodified enzyme as 100.

[0035]

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

[
It has been confirmed that the modified lipase of the present invention exhibits higher activity in organic solvents than unmodified lipase and maintains this activity, and is found to have excellent resistance to organic solvents.

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

[0038]

[Table 9] ────────────
────────────
enzyme
Decomposition rate (%) ────
────────────────────
unqualified
45.3
LML-30
63.9
LML-40
64.2
LML-50
69.8
PML-30
71.0
PML-40 7
0.1
PML-50 79.
7 ──────────
──────────────The abbreviations in the table indicate the following. LML-30: Lipase 30% lauroylation modification enzyme L
ML-40: Lipase 40% lauroylation modification enzyme LM
L-50: Lipase 50% lauroylation modification enzyme PML
-30: Lipase 30% palmitoylation modification enzyme PML
-40: Lipase 40% palmitoylation modification enzyme PML
-50: Lipase 50% palmitoylation modification enzyme

00
39] The modified lipase of the present invention showed a degradation rate 20% higher than that of the unmodified lipase.

[0040]

[Example 3] This example shows an example of ester synthesis. (1) Synthesis of EPA ethyl ester Add ethanol to 1 g of eicosapentaenoic acid (EPA) to make a 20% W/V ethanol solution, and add 1 mg of the modified enzyme prepared in Example 1 to the solution. 37℃, 12
The reaction was stopped after 24 hours of incubation at 0 rpm (l=5 cm). After development using HPTLC and color development with 20% sulfuric acid, the relative ratio was detected by densitometry and the reaction rate was calculated. The results are shown in Table 10.

[0041]

[Table 10] The abbreviations in the table indicate the following. ZML-10: Lipase 10% benzyloxycarbonylation modification enzyme ZML-30: Lipase 30% benzyloxycarbonylation modification enzyme ZML-50: Lipase 50% benzyloxycarbonylation modification enzyme LML-10: Lipase 10% lauroylation modification enzyme L
ML-30: Lipase 30% lauroylation modification enzyme LM
L-50: Lipase 50% lauroylation modification enzyme BML
-5: Lipase 5% Tertiary-butoxycarbonylation modification enzyme BML-10: Lipase 10% Tertiary-butoxycarbonylation modification enzyme BML-30: Lipase 30% Tertiary-butoxycarbonylation modification enzyme BML-50: Lipase 50% Tertiary-butoxycarbonylation modification enzyme 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, it was found that the present invention using this modified enzyme makes it possible to synthesize esters at a high reaction rate even in organic solvents.

(2) Synthesis of cholesterol ester 100 m of cholesterol in 10 ml of water-saturated n-hexane
g and 100 mg of oleic acid were dissolved, and 1 mg of the modified enzyme prepared according to Example 1 was added. 37℃, 120rp
m (l=5 cm), and the reaction was stopped after 24 hours. After development using HPTLC and color development with 20% sulfuric acid, the relative ratio was detected by densitometry and the reaction rate was calculated. The results are shown in Table 11.

[0044]

[Table 11] ────────────
────────
Enzyme reaction (%)
────────────
───────
Unqualified 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
──────────────────── The abbreviations in the table indicate the following. LML-10: Lipase 10% lauroylation modification enzyme L
ML-30: Lipase 30% lauroylation modification enzyme LM
L-50: Lipase 50% lauroylation modification enzyme PML
-10: Lipase 10% palmitoylation modification enzyme PML
-30: Lipase 30% palmitoylation modification enzyme PML
-50: Lipase 50% palmitoylation modification enzyme

00
45] As a result, according to the present invention using this modified enzyme,
It was found that it is possible to synthesize esters with a high reaction rate even in organic solvents.

(3) Synthesis of lactose ester 0.0
Disperse 1 g of finely ground lactose powder in 10 ml of 5M phosphate buffer (pH 7.0), and add 1 g of oleic acid.
to which 10 ml of ethyl acetate was added, and 1 mg of the modified enzyme prepared in Example 1 was added. 37℃, 120r
pm (1=5 cm) and the reaction was stopped after 24 hours. After development using HPTLC and color development with 20% sulfuric acid, the relative ratio was detected by densitometry and the reaction rate was calculated. The results are shown in Table 12.

[0047]

[Table 12] The abbreviations in the table indicate the following. ZML-10: Lipase 10% benzyloxycarbonylation modification enzyme ZML-30: Lipase 30% benzyloxycarbonylation modification enzyme ZML-50: Lipase 50% benzyloxycarbonylation modification enzyme LML-10: Lipase 10% lauroylation modification enzyme L
ML-30: Lipase 30% lauroylation modification enzyme LM
L-50: Lipase 50% lauroylation modification enzyme BML
-5: Lipase 5% Tertiary-butoxycarbonylation modification enzyme BML-10: Lipase 10% Tertiary-butoxycarbonylation modification enzyme BML-30: Lipase 30% Tertiary-butoxycarbonylation modification enzyme BML-50: Lipase 50% Tertiary-butoxycarbonylation modification enzyme 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, it was found that by the present invention using this modified enzyme, it is possible to synthesize esters at a high reaction rate even in organic solvents.

[0049]

[Example 4] This example shows an example of transesterification. (1) Transesterification using triolein and palmitic acid Triolein 10 in 10 ml of water saturated n-hexane
0 mg of palmitic acid and 100 mg of palmitic acid were dissolved, and 10 mg of the modified enzyme prepared in Example 1 was added to perform a transesterification reaction. The mixture was incubated at 37° C. and 120 rpm (1=5 cm), and the reaction was stopped after 24 hours. After separation into a fatty acid fraction and a glyceride fraction by TLC, the transesterification rate was measured by gas chromatography. The results are shown in Table 13.

[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% tert-butoxycarbonyl chemical modification enzyme BML-30: Lipase 30% tert-butoxycarbonyl chemical modification enzyme BML-50: Lipase 50% tert-butoxycarbonyl chemical modification enzyme PML-10: Lipase 10% palmitoyl chemical modification enzyme PML-30: Lipase 30% palmitoyl chemical modification enzyme PML-50: Lipase 50% palmitoyl chemical modification enzyme

As a result, it was found that by the present invention using this modified enzyme, it is possible to synthesize esters at a high reaction rate even in organic solvents.

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

[0053]

[Table 14] ────────────
────────────────
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 ────
────────────────────── Abbreviations in the table indicate the following. PML-10: Lipase 10% palmitoylation modification enzyme PML-30: Lipase 30% palmitoylation modification enzyme PML-50: Lipase 50% palmitoylation modification enzyme

As a result, it was found that by the present invention using this modified enzyme, it is possible to synthesize esters at a high reaction rate even in organic solvents.

[0055]

Effect of the invention: By carrying out the present invention, amino groups can be converted into RCO
The - group made it possible to obtain chemically modified esterases. Furthermore, using this chemically modified esterase, it becomes possible to perform enzymatic hydrolysis of esters, ester synthesis, or ester exchange in organic solvents. This modified esterase has excellent organic solvent resistance and organic solvent affinity, and can be used in bioreactors and the like.

Claims (5)

[Claims] Claim 1: 20-60% of the total amount of amino groups in the enzyme
A chemically modified esterase characterized by introducing a group represented by RCO- into (where R is a benzyloxy group, a tert-butoxy group, a 9-fluorenylmethoxy group,
p-methoxybenzyloxy group, carbon number C1 to C24
Indicates any alkyl group. ) [Claim 2] RCO-
A method for producing a chemically modified esterase characterized by introducing a group (wherein, R is a benzyloxy group,
It represents any one of a tert-butoxy group, a 9-fluorenylmethoxy group, a p-methoxybenzyloxy group, and an alkyl group having a carbon number of C1 to C24. ) 3. A method for hydrolyzing esters, which comprises hydrolyzing esters using the chemically modified esterase according to claim 1. 4. An esterification method, characterized in that esterification is carried out using the chemically modified esterase according to claim 1. 5. A method for transesterification, which comprises transesterifying an ester using the chemically modified esterase according to claim 1.