JPH0476673B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel modified enzymes. More specifically, lysyl endopeptidase (Achromobacter protease) (hereinafter abbreviated as PLP) produced from Achromobacter lyticus is intermolecularly crosslinked and polymerized using polyalkylene glycol as a crosslinking agent. The present invention relates to the novel modified enzyme obtained. Oxygen reactions are widely used in general, but enzymes are usually used dissolved in an aqueous solution, so it is extremely difficult to recover the enzymes after the reaction is completed.
Separation and purification of reaction products is also not easy. Furthermore, enzymes are by no means stable, and are normally used in the neutral range at room temperature and under normal pressure, but they are subject to significant loss during recovery. Therefore, the requirements common to all enzymes are to be stable over a wide temperature range and pH range, to be usable in various solvents, and to be easily recovered. It was a natural request. PLP is an extrabacterial enzyme produced by Achromobacter lyticus M497-1 isolated from soil.
Discovered by Soejima, Masaki et al., agricultural and biological chemistry,
Volume 42, page 1443 (1978), and Japanese Patent Publication No. 119085 (1978)
It is the same enzyme as Achromobacter protease described in No. The optimum temperature is 45℃, the optimum pH for protease activity is 8.5 to 10.5 when casein is used as a substrate, and the molecular weight by gel filtration method is
27000, isoelectric point PH6.9, strongly inhibited by diisopropylphosphochloride and tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, ethylenediaminetetraacetate, orthophenanthroline and It is a serine enzyme that is not inhibited by p-chloromercury benzoate, and specifically hydrolyzes the ester and amide bonds in the carboxyl group of L-lysine, thus preventing enzymatic degradation of peptides and lysyl-lysine during amino acid sequencing. It is well known that enzymes are useful in the decomposition and synthesis of peptides. In recent years, human insulin has been synthesized from porcine insulin using this enzyme (see JP-A-54-135789 by Soejima and Masaki et al. and Biochemical and Biophysical Research by Morihara and Oka et al. Comic Union,
(Vol. 92, p. 362 (1980)). Since all of these methods are liquid-phase reactions that use a water-soluble enzyme alone, it is necessary to separate and purify the obtained human insulin and PLP, which is a foreign protein. If the purification is insufficient, the immunological side effect of producing antibodies due to foreign proteins cannot be detected when administered to the human body as a drug, and especially when administered continuously over a long period of time, this can lead to extremely serious consequences. There is a risk of causing Furthermore, it goes without saying that separation and purification requires a great deal of effort and time, and since the enzyme after separation is inactivated, it cannot be reused. The economic loss will be huge. Therefore, it leads to some kind of inducibility of this enzyme,
It is clear that if it is used in a form that increases stability and can be easily separated from the reaction solution, it will be advantageous in terms of improving the quality of the target product and economically. In general, many methods are known for deriving enzymes into derivatives in order to achieve this purpose. Among these methods, the most representative methods are: (1) converting enzymes into water-insoluble derivatives; (2) method of ionically bonding the enzyme to a water-insoluble carrier; (3) method of adsorbing the enzyme to a water-insoluble carrier; (4) method of cross-linking the enzyme with a cross-linking agent to make it insoluble in water. (5) polymerizing a carrier to entrap the enzyme and making it insoluble in water. However, since the so-called solidified enzyme obtained by the above method is insoluble in water, when it is used in various reactions in the batch method, the reaction solution may become a non-uniform suspension state or the specific gravity may vary. Immobilized enzymes with a high concentration precipitate quickly, so if you want the reaction to proceed quickly, it is best to stir the reaction solution as much as possible or rotate the reaction vessel to make the suspension as uniform as possible. It is necessary to create a turbid state, prolong the contact time between the immobilized enzyme and the reactant, or increase the frequency of contact. Therefore, immobilized enzymes may not be used in reactions where the substances to be reacted are extremely unstable and easily oxidized by oxygen in the air, and where vigorous mechanical stirring or rotation must be avoided. . In addition, in reactions using immobilized enzymes that are insoluble in water, naturally the reaction is heterogeneous between the solid phase and the liquid phase, so compared to reactions in the liquid phase, it is inevitable that It is an undeniable fact that the reaction rate is slow. Furthermore, in the column method and batch method, when a water-insoluble immobilized enzyme is used in the reaction, the substrate is dissolved in the solvent, but the reaction products and/or by-products generated during the reaction are Tomoga,
When the enzyme is poorly soluble or insoluble in the solvent used, it becomes extremely difficult to separate it from the immobilized enzyme. Many methods have been tried to separate the reaction product produced as a precipitate from the immobilized enzyme, but for example, methods using organic solvents that do not cause precipitates, Depending on the type of organic solvent, the immobilized enzyme may sometimes be significantly deactivated, resulting in a decrease in the number of times of repeated use and poor economic efficiency. Furthermore, even if the reaction product is dissolved in the reaction solvent, during repeated use, the reaction product may cause nonspecific adsorption to the water-insoluble immobilized enzyme, resulting in the recovery of the target product. There is also the drawback that the rate is poor. Additionally, as the immobilized enzyme is repeatedly used,
Since the specific activity of the carrier gradually decreases, it is naturally necessary to replenish new immobilized enzyme to increase the reaction rate. However, when carrying out the reaction in a batch method, the solvent In some cases, the amount cannot be increased, and in such cases, the reaction is actually impossible. In order to solve these problems with immobilized enzymes, the present inventors have conducted intensive research on modified enzymes that are soluble in water and can be easily separated from raw materials and obtained products. Alkylene glycol (hereinafter abbreviated as PAG) is used as a crosslinking agent,
We have discovered that by intermolecular cross-linking polymerization of PLP, a novel modified enzyme can be obtained that is stable, maintains insolubility, and is easy to separate from raw materials and reaction products without reducing enzyme activity. , we have completed the present invention. Until now, in the field of enzyme therapy, the aim of reducing the immunogenicity of enzyme therapy is to chemically bond non-immunogenic polymers to amino acid residues present on the surface of the enzyme molecule, thereby determining the antigen present on the surface of the enzyme molecule. In order to cover the site and reduce or eliminate its ability to bind to antibodies, the enzyme protein is coated with synthetic polymers,
For example, there have been attempts to chemically modify the enzyme protein with monovalent polyethylene glycol such as monomethoxypolyethylene glycol, but in this case, the purpose was to reduce the immunogenicity of the enzyme protein. For this purpose, the surrounding area is covered with monovalent polyethylene glycol, that is, monomethoxypolyethylene glycol. However, as in the present invention, the enzyme is further polymerized while remaining insoluble, and the substrate used in the enzyme reaction and the reaction To date, there has been no example of cross-linking and polymerizing enzymes using divalent polyalkylene glycol as a cross-linking agent for the purpose of facilitating separation from the product, and the present inventors have developed this method based on their own knowledge. This is a completed new invention. The modified enzyme of the present invention, namely polyalkylene glycol-lysyl endopeptidase polymer (hereinafter referred to as
It is abbreviated as PAG-PLP polymer. ), after activating the hydroxyl group in the PAG molecule with an activating agent,
Manufactured by reacting with PLP. The polyalkylene glycol used in the present invention includes polyethylene glycol (hereinafter referred to as
It is abbreviated as PEG. ), polypropylene glycol,
Examples include polybutylene glycol, and divalent ones in which the hydroxyl groups at both ends are not substituted with methoxy groups are used. Further, in the case of PEG, those with a molecular weight of 200 to 20,000 are usually used, but those with a molecular weight of 6,000 to 20,000 are preferable. The method for obtaining the modified enzyme of the present invention preferably includes two
It is done in the process. First, PAG is activated by reacting with an activator. As the activator, for example, 1,1'-carbonyldiimidazole, cyanuric chloride, etc. are used, and these
It is preferable to use 1 to 10 times the amount. The reaction is generally carried out in an organic solvent such as dioxane or benzene, preferably at a temperature of 0 to 50°C. It is preferable to perform the second reaction after the PAG activated in this manner is once isolated and purified from the reaction solution. Isolation of activated PAG is carried out after the reaction.
Good results can be obtained by removing unreacted low molecular weight substances by dialysis, gel filtration, ultrafiltration, or precipitation using an organic solvent. In the second step, activated PAG and PLP are reacted in an aqueous solution, preferably under slightly alkaline conditions. The amount of activated PAG is 1 to 1000 times the number of amino groups in the PLP molecule. The reaction was carried out at 0-40°C and for 1-5 hours using cyanuric chloride activated PAG.
When PAG activated with 1,1'-carbonyldiimidazole is used, the process is completed in 1 to 5 days.
For separation and purification after the reaction, use methods commonly used to purify water-soluble polymer compounds, especially proteins, such as dialysis, gel filtration, ultrafiltration, precipitation with organic solvents, etc., as appropriate. I can do that. The final concentration of PLP in the reaction solution is usually 0.02-20
It is adjusted to % by weight, more preferably 0.1 to 15% by weight. If the enzyme concentration is too low, the yield of the target water-soluble PAG-PLP polymer will be low, making it impractical. Furthermore, if the enzyme concentration is too high, the resulting polymer may become water-insoluble. The activated PAG used in the present invention may be any one having a difunctional group or more, but it is usually activated with the above-mentioned 1,1'-carbonyldiimidazole or cyanuric chloride. An activated PAG having a difunctional group or a trifunctional group is used. The PAG-PLP of the present invention has the same substrate specificity as PLP before polymerization, and also has an optimum pH, an optimum temperature,
Regarding Michaelis constant (Km value) and maximum velocity Vmax, there was no change from the value of PLP before polymerization. For example, the substrate specificity of PEG-PLP is shown in Table 1.

[Table] The activity of the enzymes mentioned above was measured as follows. That is, 0.2M2-amino-2-methyl-1,
Add 2.6 ml of 3-propanediol buffer (PH9.5),
Add 0.3ml of 2.5mM each substrate solution, prewarm at 30℃, add 0.1ml of enzyme solution, and adjust according to each substrate.
The reaction was carried out at 30°C for 10 minutes to 20 hours. After the reaction, 10 ml of 45% acetic acid aqueous solution was added to stop the reaction, and the absorbance was determined by colorimetric measurement at 405 nm. The enzyme unit is per minute at 30°C.
The amount of enzyme that produces 1 μmole of p-nitroaniline was defined as 1 unit. The enzyme titer was calculated according to the following formula. (The molecular extinction coefficient of p-nitroaniline is
It is 9.62×10 ³ l・mole ^-1・cm ^-1 . ) Activity (u/ml) = ΔO.D./min×1/9.62×4.0/0.1・
Dilution factor As is clear from Table 1, the PEG-
PLP polymer strongly acts on the amide bond of the carboxyl group of lysine, has almost no effect on arginine, and does not hydrolyze the amide bond of other amino acid residues, and is similar to PLP before polymerization. They exhibit the same substrate specificity. One of the major features of the enzyme derivative of the present invention is that, due to its large molecular weight, the desired reaction product and enzyme can be separated and recovered very easily by gel filtration. The advantage of the polymer is that it can be used repeatedly, as there is no loss in its enzymatic activity. That is, PLP
has a molecular weight of 27,000 to 30,000, so it is not easy to completely separate it from peptides or proteins with a molecular weight of 100,000 or less by gel filtration, and other, more complicated methods must be used. Derivatives are extremely high molecules with a molecular weight of 400,000 or more, so they can be easily separated, and are extremely useful in the decomposition of proteins and peptides, and in the separation and purification of synthetic products and enzymes. As described above, the modified enzyme of the present invention has the same substrate specificity, optimum pH, optimum temperature, Michaelis constant (Km value), and maximum velocity Vmax as the enzyme before modification, does not reduce enzyme activity, and is water soluble. Since it is polymerized while maintaining its properties, it is stable and easy to separate from the reaction solution, so there is no risk of it getting mixed into the reaction product and causing problems, and the enzyme itself can be easily reused repeatedly. In addition, an unexpected effect of the modified enzyme of the present invention is that the solubility in organic solvents is significantly increased as a result of modification. As an indication of this effect, Table 2 shows the solubility data of the PEG-PLP polymer of the present invention in various organic solvents.

【table】

[Table] In addition, the amidase activity of the solution of this product dissolved in the organic solvent shown in Table 2 was heated at 30°C for 200 minutes, and the activity was compared with that dissolved in water. Table 3 shows the results of the comparison.

【table】

[Table] As is clear from Table 3, PLP before polymerization dissolves only in dimethyl sulfoxide and benzene, and even after dissolution, almost no activity remains; on the other hand, PEG-PLP polymer is not inactivated in acetone, but rather has increased activity in PEG ₆ -PLP, and retains considerable activity in dioxane, benzene, chloroform and tetrahydrofuran. In this way, the PAG-PLP polymer can be used for reactions not only in hydrogen but also in organic solvents. Conventionally, it was common knowledge that enzymatic reactions were carried out in the presence of water due to the solubility of enzymes, and that they could not be carried out in organic solutions. For example, we can expect new developments in various technological fields that utilize enzyme reactions. For example, peptide synthesis by chemical methods generally has the disadvantage that racemization easily occurs due to the application of heat and pressure, resulting in a decrease in optical purity.Additionally, when pressure is applied, special equipment is required. On the other hand, in peptide synthesis using enzymes, racemization is difficult to occur because the reaction is generally carried out at room temperature and pressure, and the substrate specificity of the enzyme is high, so even if racemic raw materials are used, either one of them will not occur. Since it reacts only with raw materials and is easy to optically resolve, products with high optical purity can be obtained. In general, peptide synthesis is preferably carried out in an organic solvent from the viewpoint of the reaction mechanism and the solubility of the raw materials and reaction products used, but in the case of enzymatic methods, the solubility of the enzyme used in the reaction In order to express activity, the reaction must be carried out in an aqueous solution, and even when using an organic solvent that is miscible with water, it is said that the concentration of the organic solvent should be as low as possible; Used at concentrations below 65%. Therefore, in peptide synthesis using enzymes, it is recommended to use organic solvents at high concentrations from the reaction mechanism of peptide synthesis, but on the other hand, in order to use enzymes as catalysts, organic solvents should be used at as low concentrations as possible. They are subject to various constraints arising from the trade-off between the two. However, the present inventors focused on the solubility of the enzyme derivative of the present invention, that is, the PAG-PLP polymer, in organic solvents, and used the PEG-PLP polymer in an organic solvent at a high concentration of 95% by volume. As a result of trying to synthesize peptides, they discovered the surprising fact that peptide synthesis proceeds with high yield. That is,
20 mg (3.5 mM) of desalanine insulin and 20 mg of threonine insulin in the presence of 0.95 units of PEG ₆ -PLP polymer in 85 vol. % dimethylformamide, 10 vol. % ethanol, and 5 vol. % 0.5 M Tris-HCl buffer (PH 6.5). 90mg (340mM) of tertiary butyl ester
The reaction was carried out at 37° C. for 15 hours, and the reaction solution was analyzed by high performance liquid chromatography, and it was confirmed that B ³⁰ -threonine tertiary butyl ester insulin was obtained at a conversion rate of 81%. In this case, dimethylformamide and ethanol are used as organic solvents, and as shown in Table 3, the PEG-PLP polymer dissolved in these solvents has the following effects in the hydrolysis of peptides:
Although it did not have much activity remaining, it unexpectedly showed enzymatic activity in peptide synthesis, and the recovered PEG ₆ -PLP polymer
There was no loss in the enzyme activity. PAG in organic solvent systems with high solubility
- It is obvious that the use of PLP polymers can be an effective means for the synthesis and hydrolysis of lysine-containing peptides. The modified enzyme of the present invention, that is, the PAG-PLP polymer, is water-soluble, so it can be reacted in a homogeneous system, and it is extremely stable, so it can be recovered and used repeatedly without loss of enzyme activity. Enzyme reactions can be carried out in high yields with water alone or with a wide range of water-miscible organic solvents.
Furthermore, since the PAG-PLP polymer of the present invention has a high molecular weight, it can be easily removed from the reaction system by, for example, molecular sieve gel filtration. Since the recovery of lysyl peptides is also extremely advantageous, the utility value of the present invention is extremely high. Specifically, it can be used for a wide range of purposes such as enzymatic decomposition of peptides during amino acid sequencing, and decomposition and synthesis of lysyl peptides. ,
There is a tremendous amount to contribute to this industry. The present invention will be explained in more detail below with reference to Reference Examples and Examples, but it goes without saying that the present invention is not limited thereto. Reference example 1 PEG6000 (average molecular weight
7500, manufactured by Lion Oil) in 50 ml of dioxane, 1,1'-carbonyldiimidazole 4.05
After adding and dissolving g, the mixture was reacted at 37°C for 2 hours. After the reaction, the reaction solution was placed in a dialysis tube (Spectrapore 6, molecular weight cutoff 3500, manufactured by Spectrum Medical Industries), dialyzed against water overnight, and the dialyzed solution was freeze-dried and activated.
PEG6000 (hereinafter abbreviated as activated PEG ₆ )
I got 14.5g. Reference Example 2 5.5 g (0.03 mole) of cyanuric chloride was dissolved in 400 ml of anhydrous benzene containing 10 g of anhydrous sodium carbonate, and 100 g (0.005 mole) of PEG20000 (average molecular weight 20000±5000, manufactured by NOF) was added, and the mixture was heated to room temperature. After reacting overnight, the reaction mixture was filtered, and 600 ml of petroleum ether was slowly added to the solution with stirring.
After removing the precipitate, redissolve the precipitate in 400 ml of benzene, slowly add 600 ml of petroleum ether with stirring, and remove the precipitate. Similarly, repeat the precipitation step and overstep several times to activate
PEG20000 (hereinafter abbreviated as activated PEG ₂₀ )
80g was obtained. Example 1 3.6 units of PLP in 10mM borate buffer (PH8.5)
After dissolving in 0.5ml, activated PEG ₆ obtained in Reference Example 1
338 mg was added and dissolved, and the mixture was stirred and reacted at 4°C for 72 hours. After the reaction, transfer the reaction solution to Sephadex G-100.
(manufactured by Pharmacia Co., Ltd.) column (φ1.5 x 85 cm), and the column volume ratio was 0.36.
2.92 units (81.1% yield) of PEG ₆ -PLP polymer were obtained. The properties of the polymer obtained here are as follows. (1) Molecular weight 400,000 or more (by gel filtration method using Sephadex G-200) (2) PH stability Britton-Robinson buffer (PH
As a result of measuring the activity of this polymer after holding it at 30℃ for 1 hour using
11 and was found to be stable. (See Figure 1) (3) Thermal stability After heating at each temperature for 1 hour using 0.2M 2-amino-2-methyl-1,3-propanediol buffer (PH9.5), this The polymer was found to be stable up to 40°C. (See Figure 2) (4) Michaelis constant Benzoyl lysine-p-nitroanilide was used as a substrate in 0.2M 2-amino-2-methyl-1,3-propanediol buffer (PH9.5) at 30°C. In this case, the Km value of this polymer was 0.17mM. Moreover, the maximum speed Vmax was 0.19 μmole/min/ml. (5) Optimal PH As a result of measuring the activity of this polymer at each PH at 30°C using Britton-Robinson buffer (PH 3 to 12), the optimal PH of this polymer was 9.5. It was hot. (6) Optimum temperature Measure the activity at each temperature using benzoyl lysine-p-nitroanilide as a substrate in 0.2M 2-amino-2-methyl-1,3-propanediol buffer (PH9.5). As a result, the optimum temperature for this polymer was 50°C. (7) Storage stability In 10mM borate buffer (PH8.5) at 4℃
As a result of checking the activity after storage for 40 days, there was no decrease in activity, and no decomposition of the polymer was observed by gel filtration using a Sephadex G-100 column. Example 2 PLP2.8 units and activation obtained in Reference Example 2
300mg of PEG ₂₀ and 100mM borate buffer (PH9.2)
After dissolving in 0.5 ml and reacting with stirring at 4°C for 1 hour, the same treatment as in Example 1 was carried out to obtain PEG ₂₀ -
1.58 units of PLP polymer (yield 56.4%) were obtained.
The properties of the obtained polymer are shown below.
(The measurement method was the same as in Example 1.) (1) Molecular weight: 400,000 or more. (2) PH stability: Stable at PH5-11 (see Figure 1). (3) Thermal stability: Stable up to 40℃ (see Figure 2) (4) Michaelis constant (Km value): 0.18mM Maximum speed Vmax: 0.18μmole/min/ml (5) Optimal PH: PH9.5~ 10.0 (6) Optimum temperature: 50℃ (7) Storage stability: No deactivation or decomposition

[Brief explanation of drawings]

FIG. 1 shows the curves showing the PH stability of the PEG ₆ -PLP polymer and PEG ₂₀ -PLP polymer of the present invention in Example 1 and Example 2, and FIG. represents a curve showing

Claims (1)

[Scope of Claims] 1. A modified enzyme obtained by cross-linking and polymerizing lysyl endopeptidase produced by Achromobacter reticus (Achromobacter protease) with polyalkylene glycol. 2. The modified enzyme according to claim 1, wherein the polyalkylene glycol is a polyalkylene glycol activated with 1,1'-carbonyldiimidazole. 3. The modified enzyme according to claim 1, wherein the polyalkylene glycol is a polyalkylene glycol activated with cyanuric chloride. 4. The modified enzyme according to claim 1, 2 or 3, wherein the polyalkylene glycol is polyethylene glycol. 5 The molecular weight of polyethylene glycol is from 200
20,000.