JP2657694B2 - Chemical modification enzyme and peptide synthesis method - Google Patents

Description

The present invention relates to a chemically modified enzyme for improving the function of a natural enzyme, and a method for producing a peptide using the same.

<Prior Art> Modification of a chemically reactive functional group present in a protein such as an enzyme is an effective means for chemically knowing the relationship between the biological activity and the structure of a protein. Recently, there has been an attempt to improve the function of the original protein according to the purpose by this chemical modification method, and to create a better drug or an enzyme as a catalyst. In particular, in the synthesis of organic compounds using an enzyme as a catalyst, a chemically modified enzyme having better organic solvent resistance and heat resistance than natural products is desired. In JP-A-63-8365, the applicant has disclosed a substance patent for dimethylsulfoniophenols, and in JP-A-62-263132, an active ester has been disclosed. Although these applications describe the acylation of amino acids, there is no example for enzymes. Further, a method of using an N-acyloxysuccinimide derived from N-hydroxysuccinimide for enzyme modification and introducing an acyl group into the enzyme is disclosed in JP-A-62-96084. However, this disclosure relates to lipoxygenase, and no disclosure of endopeptidase is found.

<Problems to be Solved by the Invention> Although overlapping with the above description, the chemical modification methods conventionally performed in the general formula include: 1. A method of acylating an amino group in a protein using an N-hydroxysuccinimide active ester .

2. A method of forming a crosslink between two amino groups in a molecule using glutaraldehyde.

3. A method of converting a thiol group in a protein into a disulfide bond using N-succinylimidyl-3- (2-pyridyldithio) propionate.

4. A method of introducing polyethylene glycols via cyanuric chloride.

Etc. are known. However, in these chemical modification reactions, the modifying reagent is insoluble in a water solvent which is a use condition, and an organic solvent compatible with water, such as dimethylformamide, is added to the reaction system. For this reason, some proteins may have a problem of denaturation and inactivation. In addition, regarding peptide synthesis using enzymes,
The peptide synthesis reaction is preferably carried out in an organic solvent-water system. However, under these conditions, there is a problem that the natural enzyme is largely inactivated by the organic solvent and the desired product is not produced.

<Means for Solving the Problems> The present inventors have conducted intensive studies to overcome the drawbacks of the conventional chemical modification of enzymes and the peptide synthesis by enzymatic methods. (Wherein R represents benzyloxy, tert-butoxy, 9-fluorenylmethoxy, p-methoxybenzyloxy, a C ₁ -C ₁₇ alkyl group)
For the chemical modification of enzymes using Was introduced into the enzyme, and the invention of a chemically modified enzyme was achieved. That is, the sulfonium salt represented by the above general formula has extremely high water solubility and has a pH of 5.0 to 11.0 in an appropriate buffer.
In the above, it is possible to easily introduce an R-CO- group (R is as described above) into an amino group in the enzyme. That is, as compared with the method of introducing an amphipathic polymer such as a polyethylene glycol group as in the conventional enzymatic chemical modification method, it has a feature that a relatively low-molecular and highly hydrophobic group can be introduced. In this case, the modification rate is 10-65% of the total amino group content of the enzyme.
It is. If the amount is more than this, although it is soluble in the organic solvent, the catalytic activity is deactivated because the R-CO- group inhibits the enzyme active center. If the amount is too small, the organic solvent resistance is inferior, which is not preferable. For the modification reaction, a water-only system is sufficient, and does not necessarily require an organic solvent. The reaction proceeds at a reaction temperature of room temperature or lower. As the enzyme used here, proteolytic enzymes (proteases) such as chymotrypsin, trypsin, thermolysin, and pepsin are preferable. In the modified enzyme of the present invention, the introduced group is benzyloxy,
Tertiary butoxy, 9-fluorenylmethoxy, and p-methoxybenzyloxy can be cleaved under mild conditions, and also have a characteristic that they can be easily regenerated to the original natural enzyme. Furthermore, it is presumed that the chemically modified enzyme of the present invention protects the active center inside the enzyme from an organic solvent by introducing an R-CO- group into the hydrophilic group on the enzyme surface. Therefore, the enzyme of the present invention is a chemically modified enzyme that exhibits high resistance to organic solvents and hardly changes the three-dimensional structure of a natural product as compared with a chemically modified enzyme into which a conventional polymer has been introduced. As a result, it functions as an efficient catalyst and can be used particularly for peptide synthesis.

<Function> In the enzymatic peptide synthesis using a proteolytic enzyme such as chymotrypsin, trypsin, thermolysin, pepsin, etc., an organic solvent compatible with water is added due to the low water solubility of the N-terminal protected amino acid as a substrate. To increase the reaction rate. However, since the unmodified enzyme has no resistance to organic solvents, significant inactivation is often observed, and the desired peptide may not be obtained. In this regard, since the chemically modified enzyme of the present invention has high resistance to organic solvents against dimethylformamide and the like, it is possible to generate peptide bonds in high yield.

<Examples> Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Synthesis Example 1 A method for chemically modifying chymotrypsin using 4-benzyloxycarbonyloxyphenyldimethylsulfonium methyl sulfate (Z-DSP).

Chymotrypsin A4 (BMY) 14 mg of Na ₂ B ₄ O ₇ -HCl buffer (pH 7.8) was dissolved in 5 ml, was added 42.5 to 340 equivalents of 4-benzyloxycarbonyl-oxyphenyl dimethylsulfonium methylsulfate against chymotrypsin 1
It was left still for 6 hours. Centrifuge the reaction solution (10000G, 10 minutes)
The supernatant was dialyzed for 72 hours. After lyophilization, a white powder was obtained.
1 mg of this chemically modified chymotrypsin is dissolved in 0.1 ml of Tris buffer, and 5 μl of the solution is subjected to high performance liquid chromatography (column: AApack, eluent 0.1 M citrate buffer pH 7.7, OPA
Detection system, pressure: 50Kg / cm ² , 60 ° C, flow rate 0.6μl / min)
The unmodified chymotrypsin had an elution time of 2 minutes, while the modified product migrated to 3.4 minutes. Further, the modification ratio of the amino group was measured by a trinitrobenzenesulfonic acid method. Table 1 shows the results calculated from the amount of free amino groups.

Synthesis Example 2 4- (9-fluorenylmethoxycarbonyloxy) phenyldimethylsulfonium methyl sulfate (Fm
Chemical modification method of trypsin using oc-DSP).

Trypsin (Sigma) 14 mg was added to Na ₂ B ₄ O ₇ -HCl buffer (pH
7.8) Dissolve in 5 ml and add Fmoc-DSP to trypsin
2.5 to 340 equivalents were added and allowed to stand for 16 hours. The reaction solution was centrifuged (10,000 G, 10 minutes), the supernatant was dialyzed for 72 hours, and then lyophilized to obtain a white powder. The modification rate of this chemically modified trypsin was measured by the trinitrobenzenesulfonic acid method in the same manner as in Synthesis Example 1. Table 2 shows the results calculated from the amount of free amino groups.

Example 1 Test of esterase activity of chemically modified enzyme 2 mg of chymotrypsin modified according to Synthesis Example 1 was dissolved in 1 ml of Tris buffer (pH 8.0), and the solution was treated with 2,3-di-o- ( Phenylalanyl) -α-methyl-D-glucoside 70 mg was dissolved in Tris buffer 10 ml,
Further, dimethylformamide was mixed and incubated at 40 ° C. for 30 minutes. 0.01 μl was sampled, 0.49 ml of cold water was added to stop the reaction, and 5 μl of the reaction was measured for high-performance liquid chromatography (conditions conforming to Synthesis Example 1) to determine the amount of phenylalanine released. Table 3 shows the results of the ester hydrolysis rate.

It was confirmed that the modified chymotrypsin of the present invention maintained a high activity even in 30% dimethylformamide as compared with unmodified chymotrypsin, and was found to be excellent in organic solvent resistance.

Example 2 35% benzyloxycarbonylation chymotrypsin (Z-35 chymotrypsin) benzyloxycarbonyl tyrosyl glycyl amide using (Z-Tyr-Gly-NH 2)
Synthesis of 1 mM benzyloxycarbonyl tyrosine, 0.1 M glycylamide, and 2 mg of 35% benzyloxycarbonylated chymotrypsin (Z-35 chymotrypsin) prepared in Synthesis Example 1 were dissolved in 1 ml of Tris buffer (pH 6.7), and an appropriate amount of dimethylene was dissolved. Formamide was added and the mixture was gently stirred at 20 ° C. for 48 hours. The reaction solution was sampled and subjected to high performance liquid chromatography (C18-ODS column, 278 nm, eluent: acetonitrile: water = 1: 1) to measure the production rate of Z-Try-Gly-NH ₂ . Table 4 shows the results.

Example 3 Esterase activity test of chemically modified enzyme 2 mg of trypsin modified according to Synthesis Example 2 was dissolved in 1 ml of Tris buffer (pH 8.0), and benzoylarginine methyl ester (Bz-Arg-OMe) was used as a substrate in the solution. )Ten
0 mg was dissolved in 10 ml of Tris buffer, dimethylsulfoxide (DMSO) was mixed, and the mixture was incubated at 40 ° C. for 30 minutes. In the same manner as in Example 2, the amount of benzoylarginine released from this reaction solution was measured by high performance liquid chromatography using a UV280 nm detection system. Table 5 shows the results of the ester hydrolysis rate.

Compared with unmodified trypsin, the modified trypsin of the present invention was confirmed to maintain a high activity even in dimethylsulfoxide at 40 to 60%, and was found to be excellent in organic solvent resistance.

Example 4 Benzyloxycarbonylarginylleucinamide (Z-Arg-Leu) using 30% 9-fluorenylmethoxycarbonylated trypsin (Fmoc-30 trypsin)
Synthesis of —NH ₂ ) Benzyloxycarbonyl arginine (5 mM) and leucinamide (0.5 M) were prepared using the 30% 9-fluorenylmethoxycarbonylated trypsin (Fmoc-30) prepared in Synthesis Example 2.
(Trypsin) was dissolved in 1 ml of Tris buffer (pH 6.5), an appropriate amount of dimethyl sulfoxide was added, and the mixture was gently stirred at 20 ° C. for 48 hours. The production rate of Z-Arg-Leu-NH ₂ was determined in Example 2.
Was measured in the same way as

 Table 6 shows the results.

<Effect of the Invention> The modified trypsin and modified chymotrypsin of the present invention both have better organic solvent resistance than unmodified trypsin and chymotrypsin, and have a high concentration of an organic solvent even in a peptide synthesis reaction utilizing a reverse reaction of a protease. The yield was higher than when the unmodified enzyme was used.

Therefore, the chemically modified enzyme of the present invention exhibits high organic resistance and is useful for peptide synthesis in an organic solvent.

Claims (7)

(57) [Claims] (1) A chemically modified enzyme comprising 10 to 65% of the total amino group content of the enzyme. (Where R is a benzyloxy group, a tert-butoxy group, 9-
Shown fluorenyl methoxy group, p- methoxybenzyl group, or an alkyl group of C ₁ -C ₁₇₎ 2. The enzyme according to claim 1, wherein the enzyme to be modified is endopeptidase. 3. The general formula Wherein R is a benzyloxy group, a tert-butoxy group, a 9-fluorenylmethoxy group,
p- methoxybenzyloxycarbonyl group, or an alkyl group of C ₁ -C _17. ). 4. The method according to claim 3, wherein the chemical modification is carried out at pH
A method for chemically modifying an enzyme, which is performed in an aqueous solution of 4.0 to 12.0. 5. A method for synthesizing a peptide using the chemically modified enzyme according to claim 1 or 2 as a catalyst. 6. The method according to claim 5, wherein the solvent for the peptide synthesis reaction is a mixed solvent of water and an organic solvent compatible with water.
The method described in the section. 7. The method according to claim 5, wherein the pH of the peptide synthesis reaction is from 5.0 to 11.0.