JPH05184374A - Non-aqueous enzymatic reaction process - Google Patents

Abstract

PURPOSE:To provide an enzymatic reaction process to use a chemically modified enzyme as a catalyst in an organic solvent and to provide a process for the recovery and reuse of the used enzyme. CONSTITUTION:The objective enzymatic reaction process uses an enzyme chemically modified with an amphiphatic polymeric polyethylene glycol in an organic solvent as a solubilized catalyst. The enzyme is separated and recovered by cooling the reaction system after the enzymatic reaction and the recovered enzyme can be reused. Various enzymatic reactions in organic solvents can be performed in high efficiency and the used enzyme can be recovered and reused by this process.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a non-aqueous enzymatic reaction method. More specifically, the present invention relates to a method for producing physiologically active substances such as esters and peptides and chemical products in the fields of producing pharmaceuticals, foods, agricultural and marine products, cosmetics and organic intermediate raw materials, and uses various hydrolases as parents. Chemically modified with polyethylene glycol (PEG), which is a water-soluble polymer,
The chemically modified enzyme is made soluble in various organic solvents within a certain reaction temperature range, and the chemically modified enzyme is used as a solubilizing catalyst for various organic syntheses. The present invention relates to an enzymatic reaction method, which comprises depositing from a reaction system, recovering it, and then repeating the reaction.

[0002]

2. Description of the Related Art Enzymes are a kind of organic catalyst produced by living cells, and can be regarded functionally as common chemical catalysts. The enzymatic reaction proceeds as follows: (1) Mild conditions (normal temperature, normal pressure). (2) The reaction proceeds in an aqueous solution. (3) The reaction has high specificity (reaction specificity, substrate specificity,
Position specificity, stereospecificity). (4) The enzyme has biodegradability, and the post-treatment of the reaction is easy. In fact, various enzymes are used, and various decomposition and synthesis reactions are performed using a buffer solution or an organic solvent-buffer solution or an organic solvent as a reaction solvent to actually produce a useful substance. ing. On the other hand, since the enzyme has a protein as a main constituent, it has various restrictions in handling due to its properties as a biocatalyst. In general, enzymes are water-soluble, and many substrates used in ordinary organic synthesis are soluble in organic solvents, and it is difficult to find a solvent that solubilizes both enzymes and substrates. Further, it is generally said that an enzyme is insoluble in an organic solvent and is unstable in a solution even if it is dissolved. For this reason, it cannot be used repeatedly like a chemical catalyst, and a large amount of catalyst is consumed for each reaction, which is disadvantageous in industrial use.

A method for improving the stability in an organic solvent, which is a problem when industrially using an enzyme, has been devised.
One is immobilization of the enzyme, and the other is solubilization and stabilization in an organic solvent by chemical modification of the enzyme. The immobilization of the enzyme is carried out by physically adsorbing and immobilizing the enzyme on various carriers in an aqueous solution or by chemically bonding it, or by encapsulating the enzyme with a crosslinkable polymer. The immobilized enzyme thus prepared has improved stability against an organic solvent as compared with a normal enzyme, and after the reaction, it is said that the immobilized enzyme can be reused by separating and recovering the immobilized enzyme. There is. However, the improvement in the stability of the enzyme with respect to the organic solvent by immobilization is a result of the motility of the enzyme molecule being suppressed, and the stability of the enzyme itself has not been essentially improved. In addition, since such an immobilized enzyme is insoluble in an organic solvent, it forms an aggregate in a solution, and it is considered that far fewer active sites than the enzyme molecule originally has as a catalyst are used for the reaction. , Its catalytic activity is very low compared to the solubilizing enzyme. On the other hand, by chemically modifying an enzyme with polyethylene glycol (PEG), which is an amphipathic polymer, the enzyme can be solubilized in various organic solvents and its stability can be improved. Such a polyethylene glycol modifying enzyme is described in the literature (Trend. Biotech., 4,190 (1986), Cancer Biochem. Biop.
hys., 7, 175 (1984)) etc., the enzyme reaction in a homogeneous system is realized because it is soluble in organic solvents, so when using immobilized enzyme or unmodified enzyme as a suspension. Compared to a heterogeneous reaction like
Much higher reactivity can be achieved in organic solvents. However, since such a polyethylene glycol modifying enzyme is soluble in both an organic solvent and an aqueous solution, it is difficult to recover the enzyme from the reaction system and the enzyme cannot be used repeatedly. ..

[0004]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The inventors of the present invention have made extensive studies on a method of utilizing the advantages of using polyethylene glycol modifying enzyme and immobilized enzyme and overcoming the drawbacks, and as a result, solubilized in an organic solvent. The present inventors have found that the above-mentioned polyethylene glycol-modifying enzyme can be used as a homogeneous catalyst at a certain reaction temperature, and that the enzyme can be recovered as a precipitate by lowering the temperature of the solution, and can be repeatedly used. As is clear from the above description, an object of the present invention is to provide an enzymatic reaction method using a chemically modified enzyme as a catalyst in an organic solvent, and a method for recovering and reusing used enzymes.

[0005]

The present invention has the following constitutions (1) to (4). (1) An enzyme reaction method characterized by using an enzyme chemically modified with an amphipathic polymer polyethylene glycol as a solubilizing catalyst in an organic solvent. (2) An enzyme reaction method, wherein the enzyme described in (1) is either a proteolytic enzyme or a lipolytic enzyme. (3) After completion of the reaction, the solubilized enzyme is precipitated by cooling the reaction solution, and separated and recovered from the reaction system, wherein the enzyme reaction method described in (1) above. (4) The enzymatic reaction method according to (1) above, wherein the recovered chemically modified enzyme is repeatedly used as a catalyst. Summarizing the above (1) to (4), the present invention provides various polyethylene glycol modifying enzymes chemically modified with an amphipathic polymer polyethylene glycol in an organic solvent. When used in the solubilizing temperature range, chemical reactions such as peptide synthesis reactions and transesterification reactions can be efficiently performed in a homogeneous system, and after the reaction is stopped, the enzyme reaction solution is cooled to separate the modified enzyme as a precipitate. It is a method of repeatedly using a polyethylene glycol-modifying enzyme, which is characterized in that the polyethylene glycol-modified enzyme is recovered and repeatedly used.

The structure and effect of the present invention will be described in detail below. Examples of the proteolytic enzyme used in the present invention include serine proteases such as chymotrypsin, subtilisin, and carboxypeptidase; thiol proteases such as papain, bromelain, and ficin;
Metallic proteases such as moricin can be mentioned,
Typical examples of the lipolytic enzyme include esterase. As the esterase, lipase is usually used, and depending on its origin and type, genus Candida, genus Mucor, genus Rhizopus, genus Aspergillus, genus Athrobacter ,,
It may be of microbial origin such as Pseudomonas, or of animal origin such as porcine pancreas or of plant origin. However, there is no particular limitation as long as it is an enzyme that can achieve the desired reaction.

The polyethylene glycol derivative used as an enzyme modifier in the present invention may be linear or branched, and its molecular weight is from several thousand to tens of thousands. Although it can be used, it can be appropriately selected depending on the enzyme to be modified and the purpose. Usually, a polyethylene glycol portion having a molecular weight of about 5,000 to 10,000 is desirable. As the organic solvent used in the present invention, hexane, toluene, benzene, ethyl acetate and the like can be used, but in selecting them, it is necessary to consider the solubility of the modifying enzyme, the substrate and the product. The reaction temperature may be a temperature at which the polyethylene glycol-modifying enzyme is solubilized, but usually 30-80 ° C is desirable.

The present invention will be described below with reference to examples. Example 1 (Preparation of polyethylene glycol-modified papain) Papain 0.05M acetate buffer suspension (manufactured by Sigma)
16 mg of 25 mg protein / ml, activity: 19 U / mg protein) in 0.05M phosphate buffer (pH 7.2)
It was dissolved in 80 ml, 9.6 g of methoxypolyethylene glycol (produced by NOF CORPORATION, molecular weight: about 5000) was added with stirring, and the mixture was further stirred at room temperature for 40 minutes. After completion of the reaction, 0.05M phosphate buffer (pH 7.2) 200
ml and pure water 500 ml were added, and the insoluble matter was filtered. Next, the filtrate was ultrafiltered using an ultrafiltration membrane (PM-10, manufactured by Amicon) under a pressure of 3.8 Kgf / cm ² , and then the solution (124 ml) remaining in the filter was removed. It was dialyzed against pure water overnight. After dialysis, freeze-dry the solution,
3.8 g of polyethylene glycol modified papain was obtained. In the obtained PEG-papain, 74% of the free amino groups in the papain molecule were modified by quantifying the amino group and measuring the hydrolytic activity, and the residual hydrolytic activity was 50%.

Example 2 (Study on recovery of polyethylene glycol-modified papain from toluene solution) 40 mg of polyethylene glycol-modified papain obtained in Example 1 was added to 4 ml of water-saturated toluene containing 3 mg / ml of dithiothreitol. Dissolve at 30 ℃ (enzyme protein amount: 0.05mg / ml), 1 of this solution
50 μl was collected, toluene was distilled off by an evaporator, the residue was dissolved in 3 ml of pure water, and the protein amount was 278
It was quantified by the absorbance in mm. (Dissolved enzyme amount) Next, the remaining solution was ice-cooled for 15 minutes to form a precipitate,
Centrifuge with a small tabletop centrifuge (10,000 rpm, 3
Then, 150 μl of this supernatant was collected, the toluene was distilled off with an evaporator, the residue was dissolved in 3 ml of pure water, and the protein amount was quantified by the absorbance at 278 mm (supernatant enzyme amount). .. The same operation was repeated twice more to quantify the dissolved enzyme amount and supernatant enzyme amount. Table 1 shows the quantitative results of the enzyme amount in the repeated operation.

[0010]

[Table 1]

From the results shown in Table 1, it was possible to recover 90% or more of polyethylene glycol-modified papain dissolved in toluene at 30 ° C. by ice cooling and centrifugation.

Example 3 (Study on recovery of polyethylene glycol modified papain from ethyl acetate solution) In Example 2, except that ethyl acetate was used in place of toluene and the melting temperature of the modifying enzyme was 37 ° C. The experiment was performed in the same manner as in Example 2. The results are shown in Table 2.

[0013]

[Table 2]

From the results shown in Table 2, the polyethylene glycol-modified papain dissolved in ethyl acetate at 37 ° C. was
It was possible to recover 97% or more by ice-cooling and centrifugation.

Example 4 (Synthesis of peptide by polyethylene glycol-modified papain) Water-saturated toluene 3 in which 9 mg of dithiothreitol was dissolved
Benzoylalanine methyl ester (Bz-Al
a-OMe) and triethylamine-neutralized leucine methyl ester hydrochloride (Leu-OMeHCl) dissolved in 48 mM and 50 mM, respectively, were used as a substrate solution. 30 mg of the polyethylene glycol-modified papain prepared in Example 1 was added to 3 ml of this substrate solution, and the reaction was started at 25 ° C. After reacting for 10 minutes, 10μ of the reaction solution
1 was collected and the oligopeptide produced was quantified by high performance liquid chromatography. At the same time, the remaining reaction solution was ice-cooled for 15 minutes to precipitate the polyethylene glycol-modified enzyme, which was then centrifuged (10,0
00 rpm, 3 minutes), the supernatant was removed by decantation, and the precipitate was washed twice with 0.6 ml of ice-cold toluene, and then the modified enzyme was recovered. The protein amount of the obtained polyethylene glycol modifying enzyme was quantified in the same manner as in Example 2. The modified enzyme recovered was added to 3 ml of the substrate solution, and the second reaction was performed. The same operation was repeated 5 times. The peptide produced by the first operation was benzoylalanyl leucine methyl ester (Bz-Ala-
Leu-OMe) and benzoylalanyl leucyl leucine methyl ester (Bz-Ala- (Leu) ₂ -O.
Me), and the peptide synthesis yield is benzoylalanine methyl ester (Bz-Ala-OMe) which is the substrate.
Against 97%. Table 3 shows the enzyme protein recovery rate and peptide synthesis yield in the repeated operation.

[0016]

[Table 3]

Comparative Example 1 (Peptide Synthesis with Unmodified Papain) Peptide synthesis was carried out under the same reaction conditions as in Example 4 except that unmodified papain was used as the enzyme instead of the polyethylene glycol modified enzyme. After reacting for 10 minutes, 10 μl of the reaction solution was sampled and the amount of oligopeptide produced was quantified by high performance liquid chromatography. result,
No product accumulation was observed.

Example 5 (Synthesis of peptide by polyethylene glycol modified papain) In Example 4, ethyl acetate was used in place of toluene as a solvent, and the enzyme was dissolved and the reaction temperature was 37 ° C.
The same operation as in Example 4 was carried out except that the above was used, and repeated use was repeated 3 times. The enzyme protein recovery rate and peptide synthesis yield in each operation are shown in Table 4.

[0019]

[Table 4]

Example 6 (Preparation of polyethylene glycol-modified lipase) Lipase F-AP derived from the genus Rhizopus (Amano Pharmaceutical Co., Ltd.) 60 m
g to 10 ml of 0.4 M borate buffer (pH 10.0)
Dissolved in activated polyethylene glycol PEG
2 (manufactured by Seikagaku Corporation, molecular weight 10,000) 0.8
g was added and reacted at room temperature with stirring. After reacting for 30 minutes, 100 ml of 0.1 M borate buffer (pH 8.0)
Was added to stop the reaction. Unreacted polyethylene glycol was removed using an ultrafiltration membrane (PM-30, manufactured by Amicon). The resulting modified enzyme solution was dialyzed against pure water for one day and then freeze-dried to obtain 0.3 g of polyethylene glycol-modified lipase. The rate of modification of the lipase with polyethylene glycol was 60% and the activity of 75% of the unmodified enzyme was retained by quantifying the amino group and measuring the hydrolysis activity.

Example 7 (Study on Recovery of Polyethylene Glycol Modified Lipase from Toluene Solution) In Example 2, the polyethylene glycol modified lipase obtained in Example 6 was used as the enzyme instead of the polyethylene glycol modified papain, and dithiothreitol was used. Is added to the solution and the amount of polyethylene glycol-modified lipase dissolved is 20 mg (concentration, 5
(mg / ml) except that the amount of enzyme protein recovered was quantified under the same conditions. The results are shown in Table 5.

[0022]

[Table 5]

Example 8 (Transesterification with polyethylene glycol-modified lipase) 20 mg of the polyethylene glycol-modified lipase prepared in Example 7 was added to 1-pentyn-3-ol (HC≡
_{C-CH (OH) -CH 2} -CH 3) 0.1mM, is added to toluene containing vinyl laurate 0.1 mM, 30
The reaction was carried out at ° C. After reacting for 24 hours, the reaction solution was ice-cooled at 0 ° C. for 15 minutes and then centrifuged to collect the polyethylene glycol-modified enzyme, and the amount of 1-pentyn-3-ol-lauric acid ester produced was quantified by gas chromatography. did. As a result, the recovery of polyethylene glycol-modified lipase was 90%, and 1-pentyn-3-ol-lauric acid ester was obtained with a reaction yield of 50%.

Comparative Example 2 (Transesterification Reaction with Unmodified Lipase) In Example 8, the reaction was carried out using 5 mg of unmodified lipase instead of the polyethylene glycol modified lipase. No progress was observed.

[0025]

EFFECTS OF THE INVENTION According to the present invention, various enzyme reactions in an organic solvent can be efficiently carried out, and the used enzyme can be easily recovered and repeatedly used.

Claims (4)

[Claims] 1. An enzyme reaction method, which comprises using an enzyme chemically modified with an amphipathic polymer polyethylene glycol as a solubilizing catalyst in an organic solvent. 2. An enzyme reaction method, wherein the enzyme according to claim 1 is either a proteolytic enzyme or a lipolytic enzyme. 3. The enzymatic reaction method according to claim 1, wherein after the completion of the reaction, the solubilized enzyme is precipitated by cooling the reaction solution, and separated and recovered from the reaction system. 4. The enzymatic reaction method according to claim 1, wherein the recovered chemically modified enzyme is repeatedly used as a catalyst.