JPH07155178A - Method for purifying modified enzyme - Google Patents

Abstract

PURPOSE:To efficiently purify a chemically modified enzyme in good yield, operability and treatment rate by dialyzing it with a hollow fiber-type dialysis tube. CONSTITUTION:An aqueous solution of an enzyme chemically modified for improving its activity, safety, stability, water solubility, etc., is dialyzed using a hollow fiber-type dialysis tube. By this treatment, binders (derivatives), decomposed products, buffer salts and other additives, as impurities, can be removed in high efficiency from the solution.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a modified enzyme in the production of the modified enzyme.

[0002]

Enzymes are widely used in various industrial fields such as detergents, scouring of textiles, decomposition of fats and oils, starch, food processing, pharmaceuticals, clinical tests, biosensors, cosmetics, and conversion / production of useful substances. Has been. One problem in measuring such utilization is that the stability of the enzyme is generally low, and the requirement is often not satisfied. That is,
Heat is applied, extremely high pH conditions and conversely low pH
Under the conditions, most enzymes are easily denatured and inactivated in the presence of a mixture of a surfactant, an organic solvent and the like, and further by long-term storage. Moreover, since the enzymes used are of different origins to the human body, their antigenicity, skin sensitization, and irritation become problems when they are applied to medicines, cosmetics, detergents and the like.

Chemical modification of enzymes has been attempted as a method for coping with these problems. For example, a method for improving clearance and antigenicity in blood by modifying uricase, asparaginase, streptokinase, etc., which are used as therapeutic enzymes, with polyethylene glycol (Japanese Patent Publication No. Sho 61).
No. 42558, JP-A-57-118789), a method of modifying superoxide dismutase with a polysaccharide or polyethylene glycol to suppress antigenicity and improve heat stability (JP-A-58-16685), Alternatively, chymotrypsin is modified to give intramolecular cross-linking, and stabilization is performed (Biochimica et Biophysica Act).
a 522, 277 to 283 (1978), ibd 485, 1 to 12 (1977)) and the like have been proposed.

When the modified enzyme thus obtained is used in combination, it is administered to the human body or comes into contact with it, especially when used for pharmaceuticals, cosmetics, detergents, etc. Is required to be purified and separated. That is, in such modification, since a highly reactive reagent is used as a binder, those which have an unfavorable effect on the human body such as these reagent derivatives and decomposition products coexist as impurities in the synthesis reaction solution. In addition to these, reaction aids and buffer salts are also included, and these need to be separated and removed. In addition, water-soluble polymers such as polysaccharides and polyalkylene glycols generally have a fairly wide molecular weight distribution, and although oligomers with low molecular weight may be included in the reaction system, they do not participate in enzyme modification. It is desirable to remove these derivatives left over from.

The inventors of the present invention also have a superoxide dismutase or protease modified with a polysaccharide or polyalkylene glycol for the purpose of stabilization and suppression of skin sensitization (Japanese Patent Laid-Open Nos. 63-245671 and 63-45671). 1-67
186, JP-A-2-219571, JP-A-2-219952, JP-A-4-27388, JP-A-4-88982) and the like,
It has been reported that a chemically modified enzyme under appropriate conditions can provide a modified enzyme excellent in physical properties such as activity, stability, safety and water solubility. Especially in the case of protease, if a polysaccharide such as dextran is used as a modifier and modification is performed by controlling various conditions, it is possible to obtain a highly stabilized modified enzyme in addition to suppressing the above-mentioned performance and autodigestion. I found it. When synthesizing this modified enzyme, a method of reacting a polysaccharide with cyanuric chloride, cyanogen bromide, etc. to obtain an activated form, and then reacting with an enzyme is used, but in the solution after the reaction, cyanuric chloride or bromide is used. It contains a decomposed product or derivative of a binder such as cyan, a borate buffer salt used for pH adjustment during the reaction, etc., and these must be removed efficiently and completely. Especially when cyanuric chloride is used, the three C-Cl sites of cyanuric chloride are involved in the binding reaction with polysaccharides and enzymes, and also undergo hydrolysis or react with glycine added as a post-treatment agent. However, since very small amounts remain as they are, various derivatives are produced.

In order to remove such impurities and purify the modified enzyme, it is desirable to purify the modified agent having an active group introduced therein before reacting with the enzyme. Further, after the binding, a method of washing with an organic solvent or a method of precipitating by separation, or a salting-out method may be considered as a purification method in some cases. Furthermore,
Some impurities have different molecular weights and have similar solubility to the modified enzyme itself.In such a case, impurities are removed by a method using a dialysis bag, or by chromatographic separation based on the principle of ultrafiltration or gel filtration. A method of removing it can be considered. However, the method using a dialysis bag is complicated in operation because it is a batch process, and is not suitable for large-scale industrial processing, and in addition, there is a problem that the amount of liquid generally expands and the burden of the post-treatment process increases. . Also,
Washing and purification by ultrafiltration require repeated treatments many times, and in order to increase the treatment capacity, large equipment is required, and in that case the amount of liquid remaining in the device is large and the product yield is high. There is a problem of lowering. Further, the method of chromatographic purification is not suitable for treating a large amount of reaction solution, and the time required is long, so that its application is limited.

[0007]

Based on the above-mentioned circumstances, the present inventors have efficiently purified a modified enzyme from a synthesis reaction solution,
As a result of earnest studies for the purpose of recovering, the present invention has been reached, and the purpose of the present invention is to improve the purification effect, yield, operability and processing speed, and to provide a low-cost modified enzyme. It is to provide a purification method of.

[0008]

The present invention is a method for purifying a modified enzyme, characterized in that when a chemically modified enzyme is purified, the enzyme is purified using a hollow fiber type dialysis tube. Is achieved.

The hollow fiber type (hollow fiber type) dialysis tube used in the present invention is appropriately prepared depending on the molecular size or molecular weight of the modifying enzyme to be purified and impurities coexisting with it, and further its chemical properties. Select materials, dialysis performance, etc. Examples of the material of the hollow fiber type dialysis tube used in the present invention include cuprophane, cellulose acetate, polymethylmethacrylate, polyacrylonitrile, polysulfone, ethylene-polyvinyl alcohol, and compounds of these series, depending on the purpose. The best one should be used. For example, the one made of cuprophan has a small molecular weight cutoff and is good in terms of yield, but the treatment efficiency is slightly low, whereas the one made of polysulfone may be disadvantageous in terms of yield but may have a purification effect. It has excellent processing capacity.

From the viewpoint of treatment efficiency and purification effect, the water permeability when evaluated by the method described below is preferably 20 ml /
(mmHg · m ² · h) or more, more preferably 50 ml / (mmHg ·
It is preferable to use those having a dialysis performance of m ² · h) or more and permeability of serum albumin of preferably 2% or less, more preferably 1% or less from the viewpoint of yield.

Method for evaluating water permeability: A physiological saline solution was passed through a dialysis tube (inside the hollow fiber) having a closed outlet at 37 ° C.,
Measure the amount of liquid that passes through the hollow fiber and comes out to the dialysate side (outside the hollow fiber). The permeated liquid amount is obtained by changing the transmembrane pressure, and the permeated liquid amount per hour for a pressure of 1 mmHg, a membrane area of 1 m ² is calculated from the relation of the permeated liquid amount to the transmembrane pressure in the vicinity of 100 mmHg.

Evaluation method of serum albumin permeability: A dialysis tube (inside the hollow fiber) is filled with physiological saline containing 1% of serum albumin, and a dialysate side (outside of the hollow fiber) is filled with physiological saline. After standing at 37 ° C. for 1 hour, the amount of albumin permeated to the dialysate side is determined, and the ratio to the amount of albumin present inside the hollow fiber first is calculated.

Since the dialysis performance is affected by the conditions such as the flow rate of the dialysate, the flow rate of the dialysate, the transmembrane pressure, and the temperature in addition to the specifications of the dialysis tube itself, it may be set to an optimum value. For example,
In order to suppress the increase in liquid volume due to dialysis treatment, facilitate the recovery of the modifying enzyme, and obtain a high purification effect, the flow rate is preferably 80 ml / (min · m ² ) or less and the transmembrane pressure is applied. The ratio of the outflow rate to the flow rate through the dialysis tube is preferably 0.3 to 1.2, more preferably 0.
It is preferable to set it to 4 to 1.0.

The purification method of the present invention may be carried out under the above conditions using the above hollow fiber type dialysis tube after the modified enzyme is appropriately produced by a conventional method. Further, when the modification reaction solution contains an impurity having low water solubility as a precipitate or a turbidity, it is preferable to remove it in advance by filtration, centrifugation or the like.

Examples of the modifying enzyme according to the present invention include:
Examples include those in which an enzyme such as a protease and a water-soluble polymer such as a polysaccharide are bound, and more specifically, those in which the enzyme and the polysaccharide are bound via a triazine ring, activated by cyanogen bromide. Examples thereof include those obtained by reacting a polysaccharide with an enzyme. In particular, for the purification of a protease modified with a polysaccharide such as dextran using cyanuric chloride as a binder or a protease modified with a polysaccharide such as dextran activated with cyanogen bromide, the purification can be suitably performed under the above conditions. The method is remarkably excellent in processing efficiency, purification effect, yield, operability and economical efficiency as compared with other methods.

The production of a polysaccharide-modifying enzyme using cyanuric chloride as a binder is generally carried out by reacting cyanuric chloride with a polysaccharide and adding the obtained activated polysaccharide to a poor solvent under acidic conditions. After separation, it is carried out by reacting with an enzyme. Further, after the reaction of cyanuric chloride with the polysaccharide, the amount of the triazine ring introduced into the polysaccharide is adjusted to 50 mol% or more of the total amount of the triazine ring present in the synthesis solution, so that the activated polysaccharide is removed from the synthesis solution. Without separation and purification,
An enzyme solution can be added directly to the synthesis solution to carry out the binding reaction. According to this method, all the steps can be simplified, a low-cost, stable and safe modified enzyme can be provided, and scale-up for industrial production can be supported, which is preferable. As a method for carrying out this production method, a polysaccharide concentration of 4.5% by weight or more in the synthesis solution is reacted with cyanuric chloride, or a synthesis solution containing an activated polysaccharide is treated in the same manner as the purification method of the present invention. Once refined,
It can be carried out by a method of removing unreacted cyanuric chloride derivative and then reacting with an enzyme.

When the purification method of the present invention is applied to the production of a polysaccharide-modifying enzyme using cyanuric chloride as a binder, the activated polysaccharide is not separated from the synthesis solution and purified as in the above-mentioned method. When a modified enzyme solution obtained by a method in which an enzyme solution is directly added to the solution to perform a binding reaction is purified, a large amount of a cyanuric chloride decomposition product or derivative coexists in the synthesis solution, but the activated polysaccharide is Purification can be performed under the same purification conditions as in the case of the production method in which the enzyme is separated and then reacted with the enzyme, and the whole process can be simplified.
Also, a synthetic solution containing an activated polysaccharide is once purified in the same manner as in the purification method of the present invention, and is applied to the purification of a modified enzyme solution prepared by a method of removing an unreacted cyanuric chloride derivative and then binding the enzyme. If so, more complete purification can be expected.

[0018]

According to the method for purifying a modified enzyme of the present invention,
It is possible to remove binders and their derivatives, degradation products, buffer salts, and other additives that coexist as impurities from the modified enzyme aqueous solution with purification efficiency, high yield, easily, and continuously with good operation efficiency. It is possible and its economic efficiency is very advantageous. Further, the activity, stability, safety, etc. of the obtained modified enzyme are also good. Further, depending on dialysis conditions, concentration can be performed at the same time. Hereinafter, the present invention will be specifically described with reference to examples.

[0019]

Example 1 To 25 l of a ⁴ % by weight dextran aqueous solution (average molecular weight 4 × 10 ⁴ ) was added at room temperature and pH of 7.5 to 9.5.
While maintaining the above, 7.5 l of an acetone solution containing 5% of cyanuric chloride was added, and then the pH was adjusted to 3. This solution was added to an acetone bath, and the precipitated activated dextran was collected by filtration. 10 l of 0.1 M borate buffer (pH 9.0) in which 450 g of this activated dextran was dissolved
50g of alkalophilic Bacillus-derived protease (Novo Nordisk, Esperase ^™ ) was added and reacted at 25 ° C for 20 hours, then 50g of glycine was added, and heat treatment was further performed at 60 ° C for 35 hours. Then, it was subjected to protease modification with dextran. After the completion of the reaction, the aqueous solution was dialyzed and purified using a polysulfone dialyzer (Kuraray Co., Ltd., PS-1.6UW, membrane area 1.6 m ² ). As treatment conditions, a dialysate (using ion exchanged water) flow rate of 1 l / min, a reaction solution flow rate of 85 ml / min, and an exhaust flow rate (outflow rate) of 63 ml / min were used.

When the treated solution was analyzed by high performance liquid chromatography (G-3000SW, manufactured by Tosoh Corporation), the decomposition product of cyanuric chloride and its glycine derivative which were impurities in the reaction solution were not detected at all. The residual amount of boric acid, which is a buffer salt, was measured for the modified enzyme collected by distilling off the solvent under reduced pressure, and the result was 0.0
It was 17%, indicating a high purification effect. The time required for this dialysis purification is about 2 hours, and it can be seen that the method of the present invention can achieve a high degree of purification extremely simply and quickly. The water permeability of the dialyzer used in this example is 1
05 ml / (mmHg · m ² · h), permeability of serum albumin is 0.
It was 6%.

[0021]

Example 2 5 wt% dextran (average molecular weight 6 × 1
P a 0 ⁴ ~9 × 10 ⁴⁾ solution 20 l with 2N-NaOH
While maintaining at H11, 2.3 l of a 10% cyanogen bromide aqueous solution was added at 10 ° C. Next, the pH was adjusted to 9, and 1 liter of a solution prepared by dissolving or suspending 125 g of a protease derived from alkalophilic Bacillus (Esperase ^™ , manufactured by Novo Nordisk) in water was added and mixed, and the mixture was mixed at 10 ° C. and pH 9
A dextran-modified protease was prepared by a method in which 100 g of glycine was added after reacting for 16 hours under the conditions of, and further reacted at room temperature for 5 hours. The obtained reaction solution was dialyzed and purified in the same manner as in Example 1. However, the dialysis conditions were a flow rate of the reaction solution of 63 ml / min and a discharge rate of 55 ml / min. With respect to the modified enzyme powder obtained by distilling off the solvent, the mixed bromine ion was quantified by fluorescent X-ray analysis and the amount of glycine was quantified by amino acid analysis.
It was 0.032%, and a high degree of purification could be performed extremely easily.

[0022]

Example 3 10 l of a 5 wt% dextran aqueous solution (average molecular weight 4 × 10 ⁴ ) was added at room temperature to a pH of 8.0 to 9.5.
While maintaining the above, 2 l of an acetone solution containing 5% of cyanuric chloride was added to synthesize activated dextran. The ratio of the triazine ring introduced into dextran for this solution was 57 mol% with respect to the total amount of the triazine ring present in the solution. Subsequently, 3 l of a 0.5 M borate buffer solution (pH 9.2) in which 50 g of an alkaliphilic Bacillus-derived protease (Novo Nordisk, Esperase ^™ ) was dissolved was added and reacted at 25 ° C. for 20 hours. After that, 50 g of glycine was added to adjust the pH to 8.3 to 8.6.
While adjusting to, further heat treatment at 60 ℃ for 30 hours,
Protease modification with dextran was performed. The aqueous solution after completion of the reaction was purified by dialysis in the same manner as in Example 1.
However, the treatment conditions were that the dialysate (using ion-exchanged water) flow rate was 1.3 l / min, the reaction solution flow rate was 60 ml / min, and the discharge flow rate was 43 ml / min. When the treated solution was analyzed by high performance liquid chromatography (G-3000SW, manufactured by Tosoh Corporation), the decomposition product of cyanuric chloride and its glycine derivative which were impurities in the reaction solution were not detected at all.
Further, the residual amount of boric acid as a buffer salt was measured with respect to the modified enzyme collected by distilling off the solvent under reduced pressure. As a result, it was 0.022% with respect to the modified enzyme, and a high purification effect was recognized. The time required for this dialysis purification was about 4 hours, and it can be seen that a high degree of purification can be achieved very simply and quickly. Further, in this example, the steps from the production of the modified enzyme to the purification were simplified, which was favorable.

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Claims (1)

[Claims] 1. When purifying a chemically modified enzyme,
A method for purifying a modified enzyme, which comprises purifying using a hollow fiber type dialysis tube.