JPH01291794A - Modified thiol protease - Google Patents

Abstract

PURPOSE:To obtain an enzyme having enzymatic activity stable even in an organic solvent and to expand the field of industrial utilization of the enzyme, by partially substituting a free amino acid in a thiol protease (TYP) molecule with a specific modifying group. CONSTITUTION:A modifying group (C) of formula (R is polymer residue soluble in water and organic solvent) is produced by reacting (A) o-methoxypolyethylene glycol having a molecular weight of about 5,000 with (B) 2,4,6-trichlolo-o-triazine. The modifying group and a TYP such as papain are added to a buffering solution, reacted with each other at 30-37 deg.C for 1-10hr and the obtained reaction liquid is purified to obtain a modified thiol protease having the above modifying group of formula partially substituting the amino group of the TYP molecule such as papain.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a modified thiol protease that is soluble in water and organic solvents, retains enzymatic activity in organic solvents, and has the activity of generating peptide bonds. .

(Prior Art) As a peptide synthesis method, the following chemical methods have been generally used.

1) Acid chloride P method 2) Azide method 3) Mixed acid anhydride method 4) Active ester method 5) Method using a condensing agent On the other hand, the enzyme method has recently attracted attention because it has the following advantages. .

1) Not accompanied by racemate, 2) Does not require side chain protection; 3) Reaction conditions are mild and no special reaction tank is required. 4) There are few side reactions and purification is easy.

However, enzymes are proteins, and when they come into contact with organic solvents, they denature, become insoluble, and lose enzyme activity, so the use of enzyme methods has been limited to reactions in aqueous systems. Therefore, it has been difficult to synthesize peptides by enzymatic methods from substrates that are soluble only in organic solvents. In addition, in the peptide synthesis reaction using an enzymatic method in an aqueous system, hydrolysis reactions compete with each other, so there are constraints such as the need to minimize the water content in the reaction system.

(Problem to be solved) Therefore, if we can obtain an enzyme that is soluble in organic solvents and fully expresses its enzymatic activity by chemically modifying the enzyme, we can conduct the enzymatic reaction in organic solvents and eliminate water-insoluble substances. It will be used as a substrate for enzymes, significantly expanding the scope of industrial use of enzymes.

The present inventor has repeatedly conducted research from this perspective and found that thiol enzymes (EC3, 4, 22) are enzymes that catalyze the hydrolysis reaction of peptides, amides, esters, and thioesters that have a thiol group in the enzyme active site. However, by chemical modification, scales became soluble in organic solvents, and by taking advantage of their reversibility, they discovered scales that could be used to form peptide bonds.

(Structure of the Invention) The present invention provides a method for partially substituting an amino group in a thiol protease molecule with a modifying group represented by the formula (wherein R represents a polymeric residue soluble in water and an organic solvent). It is a modified thiol protease.

Examples of the above-mentioned polymer residues include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, and carboxymethylcellulose having a molecular weight of 5,000 or more, and polyethylene glycol is particularly preferred.The polymer residues include methyl, which is a hydrophobic group, Protected with an alkyl group such as ethyl or propyl or an acyl group such as acetyl or benzoyl. For example, 0-substituted polyethylene glycol, particularly 0-methoxypolyethylene glycol, is preferably used.

As a thiol protease, cathepsin (EC, 3
, 4, 22, 1), 79 nodyne (EC, 3, 4, 22
, 2), ficin (EC, 3, 4, 22, 3), zolomeline (EC, 3, 4° 22.4), xeno9pine (E
C, 3, 4, 22, 6), and the thiol group in the enzyme active site is free or protected with an activity inhibitor such as mercuric chloride, p-chloromercury-benzoic acid, or -valent metal. Thiol proteases, as well as reduced or non-reduced thiol proteases, can be used.

The method for producing the modified thiol protease of the present invention is disclosed in Japanese Unexamined Patent Publication No. 5
It is carried out according to the method for producing modified asparaginase described in Publication No. 5-135590. Taking papain as an example, as shown in the following reaction formula, 15,000 molecules of 〇-substituted polyethylene glycol (Il and 2,4,6-dolychlorosu
-) Rhianone (n) to obtain triazine (III) with 0-(f-substituted polyethylene glycol bound to the 2- and 4-positions), and an excess amount of compound (I[l) was added to denature the protein. By reacting with ・9pain under conditions that do not cause the 9-9yne molecules, for example, at +1-37°C in a loose ointment solution for 1-10 hours, the amino group in the 9-9yne molecule is converted to the formula (
The modified zepain (IV) of the present invention is obtained which is partially substituted with the modifying group of nl).

(III) (IIT') (In the formula, X represents a terminal group) Modification/4' The substitution rate of the modifying group of the weapon in the pine name is that it can be substituted with 20-1001 of the amino group in the papain molecule. and preferably about 50-70%. Unreacted amino groups can be measured using a fluorescence method using fluorescamine.

(Effects of the Invention) The modified thiol protease of the present invention can be applied to aromatic hydrocarbons such as benzene, toluene, and xylene; chlorinated hydrocarbons such as dichloromethane and carbon tetrachloride; ketones such as acetone; ethanol; It is soluble in alcohols such as D-butanol, and exhibits enzymatic activity even in these organic solvents as shown in the Examples below. When the modified thiol zolotease of the present invention is used in peptide synthesis, the substrate And there is an advantage that hydrolysis of the product does not proceed.

Therefore, the ability to perform an enzymatic reaction in an organic solvent using the modified thiol protease of the present invention significantly expands the scope of industrial use of the enzyme.

Example 1 0.2 M acetate buffer containing 50 μg/9 pine obtained from fruit emulsion of Nokupaiya (voice 4.5 > 10 t, 0
．． Add 1M aqueous sodium hydroxide solution and adjust P)i to 10L, 2.4-His(0-methoxypolyethylene glycol)-6-chloro-5-)riazine (molecular weight of polyoxyethylene glycol moiety is 5000) 0 .99
was added and reacted at 28°C for 1 hour. This is the usual method #
) f#, and 2.
4-bis(0-methoxypolyethylene glycol)-6
-Chlor-5-) A modified pa-9yne substituted with riazine was obtained.

This modified Puff 9in retained the enzyme activity of the unmodified Puff 4in in an aqueous solution.

Unmodified papain is insoluble in benzene and does not exhibit enzymatic activity in benzene, whereas the above-mentioned modified papain is soluble in benzene and does not exhibit enzymatic activity in benzene. , it is clear as follows.

Figure 1 shows 49.2mM N-benzoyl-L-alanine methyl ester (Bz-Ala-OMe) (!:
Straight chain aliphatic amines (CFi, -(a
H2), -Na,, n = 3, (Bl, 5(C1
, 7 (DJ, 11 (El)) with 100 μt each of a 45 mM benzene solution), to which was added 100 μt of a benzene solution of modified papain containing 20 mM dithiothreitol.
10 μt of the 0 reaction solution was collected at each time by adding TFC and shaking at a constant temperature of 35°C, and the absorbance at 280 nm was measured using high performance liquid chromatography (silica column). The horizontal axis shows the reaction time. , the left vertical axis is Bz
-The remaining amount (mM) of -jua-OMe, the right vertical axis shows the peptide production rate, modified papain is N-
It has been revealed that the reaction rate and yield of the reaction between benzoyl-L-alanine methyl ester and linear aliphatic amine can be efficiently catalyzed. To increase. Furthermore, it was found from A that in the absence of amine, the ester substrate did not change over time, and hydrolysis of the ester substrate did not occur.

Example 2 As ester substrates, N-benzoyl-L-alanine methyl ester (Bz-Ala-oMe), N-pensoyl-chrysine ethyl ester (BZ-Gly-OEt)
), N-pensoyl-L-tyrosine ethyl ester (B
z-Tyr-OEt) and L- as the amine component.
Tyrosine ethyl ester (L-Tyr-OEt), L-
Phenylalanine methyl ester (L-Pfie-OM
e), L-leucine ethyl ester (L-Leu-OE
t), L-cysteine ethyl ester (L-Cy, 5-
OEt), D.

L-tryptophan ethyl ester (D, L-Trp
-OEt), glycine ethyl ester (Gly-oE
t) was used to investigate the peptide synthesis reaction of modified four-pine in benzene. The reaction was carried out at 35°C using benzene containing 20mM dithiothreitol. As shown in Table 1, the results show that peptides greater than so-peptides or tripeptides were synthesized from the combination of the diester substrate and the amino acid of the amine component.

Ester substrate (15mM) L-Leu-OEt ++ Supplement (11.8mM) G17-OEt +
Ten ++ is
This shows a product in which one molecule of an amine component is added to an ester substrate.

A square indicates a product in which one or more molecules of an amine component are added to an ester substrate.

[Brief explanation of the drawing]

Figure 1 shows a peptide bond formation reaction by modified pack-in. A is a control group without amine, B is butylamine, C
indicates the addition of hexylamine, D indicates the addition of octylamine, and E indicates the addition of dodecylamine. Patent applicant Hisaharu Mihama Chisso Corporation

Claims (1)

[Claims] 1. The free amino group in the thiol protease molecule has the following formula (1) ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R is a polymeric residue soluble in water and organic solvents. A modified thiol protease partially substituted with a modifying group represented by 2. The modified thiol protease according to claim 1, wherein R in formula (1) is o-methoxypolyethylene glycol. 3. A method for producing a peptide bond in an organic solvent in the presence of the modified thiol protease according to claim 1 or 2.