JP3126578B2 - Purification method of modified enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

BACKGROUND ART Enzymes are widely used in various industrial fields such as detergents, scouring of textile products, decomposition of fats and oils, starch, etc., food processing, pharmaceuticals, clinical tests, biosensors, cosmetics, and conversion and production of useful substances. Have been. One problem in measuring such utilization is that the stability of enzymes is generally low and often unsatisfactory for their requirements. That is,
Heated or extremely high pH conditions or conversely low pH
Under the conditions, most enzymes are easily denatured and inactivated by coexistence of a mixture of a surfactant, an organic solvent, and the like, and further by long-term storage. In addition, since the enzymes used are of different origins for the human body, when applied to pharmaceuticals, cosmetics, detergents, etc., their antigenicity, skin sensitization and irritation become problems.

[0003] As a method for addressing these problems, chemical modification of enzymes has been attempted. For example, uricase, asparaginase, streptokinase, and the like used as therapeutic enzymes are modified with polyethylene glycol to improve clearance in blood and antigenicity (Japanese Patent Publication No. Sho 61).
JP-A-42558, JP-A-57-118789), a method of modifying superoxide dismutase with a polysaccharide and polyethylene glycol to suppress antigenicity and improve heat stability (JP-A-58-16885). Alternatively, chymotrypsin is modified to give intramolecular cross-links, and the stability is measured (Biochimica et Biophysica Act
a 522, 277-283 (1978), ibd 485, 1-12 (1977)) and the like.

When the modified enzyme thus obtained is incorporated and used, particularly when used in medicines, cosmetics, detergents, etc., the modified enzyme is completely administered to the human body or comes into contact with it. Must be purified and separated. In other words, in such modifications, since highly reactive reagents are used as binders, derivatives of these reagents and decomposed products that have an undesirable effect on the human body coexist in the synthesis reaction solution as impurities. In addition, it also contains a reaction aid and a buffer salt, which need to be separated and removed. In addition, water-soluble polymers such as polysaccharides and polyalkylene glycols generally have a fairly broad molecular weight distribution, and may contain low molecular weight oligomers. It is desirable to also remove these derivatives remaining in the above.

[0005] The present inventors have also proposed superoxide dismutase or protease modified with polysaccharides or polyalkylene glycols for the purpose of stabilization and suppression of skin sensitization (JP-A-63-245671, JP-A-63-245661). 1-67
186, JP-A-2-219571, JP-A-2-219572, JP-A-4-27388, JP-A-4-88982), and the like.
It is reported that a chemical modification of an enzyme under appropriate conditions can provide a modified enzyme having excellent properties such as activity, stability, safety and water solubility. In particular, in the case of protease, when polysaccharides such as dextran are used as modifiers and modification is performed by controlling various conditions, in addition to the above-mentioned performance, autolysis is also suppressed, and a highly stabilized modified enzyme can be obtained. I found it. When synthesizing this modified enzyme, a method is used in which a polysaccharide is reacted with cyanuric chloride, cyanogen bromide, or the like to form an activated form, and then reacted with the enzyme.However, cyanuric chloride or bromide is contained in the solution after the reaction. It contains decomposition products and derivatives of a binder such as cyanide, borate buffer salts used for pH adjustment during the reaction, and the like, and these need to be efficiently and completely removed. In particular, when cyanuric chloride is used, the three C-Cl sites of cyanuric chloride participate in the binding reaction with polysaccharides and enzymes, undergo hydrolysis, and react with glycine added as a post-treatment agent. Since a very small amount remains as it is, various derivatives are formed.

In order to remove such impurities and purify the modifying enzyme, it is desirable to purify the modifying agent into which the active group has been introduced before reacting with the enzyme. Further, after the binding, a method of washing with an organic solvent, or a method of separating and precipitating, and in some cases, a salting-out method can be considered as a purification method. Furthermore,
Some impurities have similar solubility properties to the modified enzyme itself except for the molecular weight.In such cases, the impurities are removed by a method using a dialysis bag, or by chromatographic separation based on the principle of ultrafiltration or gel filtration. There is a method of removing it. However, the method using a dialysis bag is a batch process, which complicates the operation, is unsuitable for industrial large-scale processing, and has a problem that the burden on the post-processing step increases because the liquid volume generally expands. . Also,
Washing and purification by ultrafiltration requires a number of repetitive treatments.In order to increase the processing capacity, large equipment is required, and in that case, the amount of liquid remaining in the apparatus 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 its application is limited due to the long required time.

[0007]

SUMMARY OF THE INVENTION Based on the above circumstances, the present inventors efficiently purify a modified enzyme from a synthesis reaction solution,
As a result of intensive studies for the purpose of recovering, the present invention has been achieved, and an object of the present invention is to provide a modified enzyme which is excellent in purification effect, yield, operability and processing speed, and is low in cost. To provide a method for continuous purification of

[0008]

The present invention provides a method for purifying a chemically modified enzyme comprising the steps of: a) using a hollow fiber type dialysis tube; b) passing a dialyzed liquid inside the hollow fiber; D) A method for continuously purifying a modified enzyme, characterized by adjusting a liquid flow rate and / or a transmembrane pressure by passing a dialysate to the outside, which achieves the above object.

The hollow fiber type (hollow fiber type) dialysis tubing used in the present invention can be appropriately prepared according to the molecular size or molecular weight of the coexisting impurities with the modifying enzyme to be purified, and furthermore, its chemical properties. Select the material, dialysis performance, etc. Examples of the material of the hollow fiber type dialysis tube used in the present invention include those comprising cuprophan, cellulose acetate, polymethyl methacrylate, polyacrylonitrile, polysulfone, ethylene-polyvinyl alcohol and their series compounds, depending on the purpose. The best one may be used. For example, those made of cuprophan have a small molecular weight cut-off and are good in terms of yield, but the processing efficiency is rather low, whereas those made of polysulfone may be disadvantageous in terms of yield, but the purification effect, Excellent in processing capacity.

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

Evaluation method of water permeability: A physiological saline solution was passed through a dialysis tube (inside the hollow fiber) having a discharge port at 37 ° C.
The amount of liquid passing through the hollow fiber and coming out of the dialysate (outside of the hollow fiber) is measured. The permeate volume is determined by changing the transmembrane pressure, and the permeate volume per hour is calculated based on the relationship between the permeate volume and the transmembrane pressure in the vicinity of 100 mmHg at a pressure of 1 mmHg, a membrane area of 1 m ² , and one hour.

Evaluation method of serum albumin permeability: A dialysis tube (inside the hollow fiber) is filled with a physiological saline containing 1% serum albumin, and a dialysate side (the outside of the hollow fiber) is filled with physiological saline. After leaving 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 initially present inside the hollow fiber is calculated.

The dialysis performance is affected by conditions such as the flow rate of the dialysis tube itself, the flow rate of the dialysate, the flow rate of the dialysate, the transmembrane pressure, and the temperature. For example,
In order to suppress the increase in the liquid volume due to the dialysis treatment, to facilitate the recovery of the modified enzyme, and to obtain a high purification effect, the flow rate of the liquid is preferably 80 ml / (min · m ² ) or less, and The ratio of the outflow to the flow through the dialysis tube is preferably 0.3 to 1.2, and more preferably 0.1 to 1.2.
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-mentioned conditions using the hollow fiber type dialysis tube after the modified enzyme is appropriately produced by a conventional method. When the modification reaction solution contains low water-soluble impurities as precipitates or turbidity, it is preferable to remove them by filtration, centrifugation or the like in advance.

Examples of the modifying enzyme according to the present invention include:
Examples include those in which an enzyme such as a protease is bound to a water-soluble polymer such as a polysaccharide, and more specifically, those in which an enzyme and a polysaccharide are bound via a triazine ring, and activated by cyanogen bromide. And those obtained by reacting a polysaccharide with an enzyme. In particular, when purifying a protease modified with a polysaccharide such as dextran or a protease modified with a polysaccharide such as dextran activated with cyanogen bromide using cyanuric chloride as a binding agent, purification can be suitably performed under the above conditions. Compared with other methods, the method is far superior in terms of processing efficiency, purification effect, yield, operability and economic efficiency.

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 resulting activated polysaccharide to a poor solvent under acidic conditions. After separation, the reaction is performed by reacting with an enzyme. Also, after the reaction of cyanuric chloride with the polysaccharide, if the amount of triazine rings introduced into the polysaccharide is adjusted to 50 mol% or more of the total amount of triazine rings present in the synthesis solution, the activated polysaccharide can be converted from the synthesis solution. Without separation and purification
A binding reaction can be performed by directly adding an enzyme solution to the synthesis solution. According to this method, the entire process can be simplified, a stable and safe modified enzyme can be provided at low cost, and the scale-up for industrial production can be supported, which is preferable. As a method for carrying out this production method, a polysaccharide concentration is adjusted to 4.5% by weight or more in the synthesis solution to react with cyanuric chloride, or a synthesis solution containing an activated polysaccharide is prepared in the same manner as the purification method of the present invention. Once purified,
The reaction can be carried out by, for example, removing unreacted cyanuric chloride derivative and then reacting with the 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 and purified from the synthesis solution once as described above. When purifying the modified enzyme solution obtained by directly adding the enzyme solution to the solution and performing the binding reaction, a large amount of the cyanuric chloride hydrolyzate or derivative coexists in the synthesis solution. Purification can be performed under the same purification conditions as in the case of the production method of reacting with an enzyme after separation, and the entire process can be simplified.
Further, the synthesis solution containing the activated polysaccharide is purified once in the same manner as the purification method of the present invention, and is applied to the purification of a modified enzyme solution prepared by a method in which unreacted cyanuric chloride derivative is removed and then bound to 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 easily remove the binder and its derivatives, decomposed products, buffer salts, and other additives that coexist as impurities from the aqueous enzyme solution subjected to the modification reaction, with good purification efficiency, high yield, and continuously, with good operation efficiency. It is very economical. In addition, the activity, stability, safety and the like of the obtained modified enzyme are also good. Further, depending on the dialysis conditions, concentration can be performed at the same time. Hereinafter, the present invention will be described specifically with reference to examples.

[0019]

Example 1 25 l of a ⁴ % by weight aqueous solution of dextran (average molecular weight 4 × 10 ⁴ ) was added at room temperature to a pH of 7.5 to 9.5.
While maintaining the pH at 7.5, 7.5 l of an acetone solution containing 5% of cyanuric chloride was added, and the pH was adjusted to 3. This solution was added to an acetone bath, and the precipitate of activated dextran that precipitated was collected by filtration. 10 l of a 0.1 M borate buffer (pH 9.0) in which 450 g of the activated dextran was dissolved.
50 g of an alkaliphilic Bacillus-derived protease (Novo Nordisk, Esperase ^™ ) was added and reacted at 25 ° C. for 20 hours. Then, 50 g of glycine was added, and the mixture was further heated at 60 ° C. for 35 hours. And dextran-modified protease. After completion of the reaction, the aqueous solution was purified by dialysis using a polysulfone dialyzer (Kuraray Co., Ltd., PS-1.6UW, membrane area 1.6 m ² ). The processing conditions were a dialysate (using ion-exchanged water) at a flow rate of 1 l / min, a flow rate of the reaction liquid of 85 ml / min, and a discharge flow rate (outflow rate) of 63 ml / min.

When the treated solution was analyzed by high performance liquid chromatography (G-3000SW, manufactured by Tosoh Corporation), no decomposed cyanuric chloride and its glycine derivative were detected as impurities in the reaction solution. The residual amount of boric acid as a buffer salt was measured for the modified enzyme collected by evaporating the solvent under reduced pressure.
17%, indicating a high purification effect. The time required for this dialysis purification is about 2 hours, which indicates that a high degree of purification can be achieved very simply and quickly by the method of the present invention. Note that the water permeability of the dialyzer used in this example is 1
05 ml / (mmHg · m ² · h), permeability of serum albumin is 0.
6%.

[0021]

Example 2 5% by weight dextran (average molecular weight 6 × 1)
0 ⁴ -9 × 10 ⁴ ) 20 l of aqueous solution is p-poured with 2N-NaOH.
While maintaining H11, 2.3 l of a 10% aqueous solution of cyanogen bromide was added at 10 ° C. Then, the pH was adjusted to 9, and 1 liter of a solution in which 125 g of an alkaliphilic bacterium-derived protease (Novo Nordisk, Esperase ^™ ) was dissolved or suspended in water was added and mixed.
After 16 hours of reaction, dextran-modified protease was prepared by adding 100 g of glycine and further reacting at room temperature for 5 hours. The obtained reaction solution was purified by dialysis 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 flow rate of 55 ml / min. As for the modified enzyme powder obtained by distilling off the solvent, the amount of bromide ion was determined by X-ray fluorescence analysis, and the amount of glycine was determined by amino acid analysis.
It was 0.032%, and a high degree of purification could be performed very easily.

[0022]

EXAMPLE 3 10 l of a 5% by weight aqueous dextran solution (average molecular weight: 4 × 10 ⁴ ) was added at room temperature to a pH of 8.0 to 9.5.
While maintaining the temperature, 2 l of an acetone solution containing 5% of cyanuric chloride was added to synthesize activated dextran. In this solution, the ratio of the triazine ring introduced into the dextran to the total amount of the triazine ring present in the solution was 57 mol%. Subsequently, 3 l of a 0.5 M borate buffer (pH 9.2) in which 50 g of an alkaliphilic Bacillus-derived protease (Esperase ^™ manufactured by Novo Nordisk) was dissolved, and the mixture was reacted at 25 ° C. for 20 hours. Thereafter, 50 g of glycine was added and the pH was adjusted to 8.3-8.6.
While further adjusting the heat treatment at 60 ℃ for 30 hours,
Dextran protease modification was performed. After completion of the reaction, the aqueous solution was purified by dialysis in the same manner as in Example 1.
However, the processing conditions were a dialysate (using ion-exchanged water) flow rate of 1.3 l / min, a reaction liquid flow rate of 60 ml / min, and a discharge flow rate of 43 ml / min. When the treated solution was analyzed by high performance liquid chromatography (G-3000SW, manufactured by Tosoh Corporation), no decomposed cyanuric chloride and its glycine derivative were detected as impurities in the reaction solution.
The residual amount of boric acid as a buffer salt was measured for the modified enzyme collected by distilling off the solvent under reduced pressure. As a result, it was 0.022% of the modified enzyme, indicating a high purification effect. The time required for this dialysis purification is about 4 hours, which indicates that a high degree of purification can be achieved very simply and quickly. In this example, the steps from production of the modified enzyme to purification were simplified, which was favorable.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 9/00 101 A61K 7/00 A61K 38/46 B01D 61/28 C11D 3/386

Claims (1)

(57) [Claims] When purifying a chemically modified enzyme, a) a hollow fiber type dialysis tube is used, b) a dialysate is passed through the hollow fiber, c) a dialysate is passed through the hollow fiber, and d) a liquid flow rate. And / or a method for continuously purifying a modified enzyme, which comprises adjusting the transmembrane pressure .