JPH10201471A - Recovery and purification of enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently separating and recovering an enzyme from an aqueous solution containing the enzyme without deactivating the enzyme. SOLUTION: An aqueous solution containing an enzyme is formulated with a polycarboxylic acid selected from the group consisting of a >=5C vinyl based hydrocarbon-maleic anhydride copolymer and a methacrylic acid ester-maleic anhydride copolymer at pH4-6 and a produced precipitate is recovered.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently separating and recovering an enzyme from an aqueous solution of an enzyme containing impurities without deactivating the enzyme.

[0002]

2. Description of the Related Art As a method for separating an enzyme from an aqueous solution containing the enzyme, conventionally, a salting-out method using inorganic salts such as sodium sulfate, ammonium sulfate and sodium chloride, and a precipitation fractionation method using a water-soluble polymer such as polyethylene glycol are known. A solvent precipitation method using an organic solvent such as ethanol, acetone, isopropyl alcohol and the like has been proposed.

[0003] However, the salting-out method and the precipitation fractionation method using a water-soluble polymer use a large amount of salts and polymers, and thus pose environmental problems.
It is known that their use is limited in terms of safety.

As a method for recovering beta-amylase, one of the enzymes, polyacrylic acid,
A polycarboxylic acid selected from the group consisting of a hydrolyzed ethylene-maleic anhydride copolymer and a hydrolyzed methyl vinyl ether-maleic anhydride copolymer was prepared at pH 3
A method is disclosed in which the mixture is mixed within a range of from 4.5 to 4.5 and the resulting precipitate is separated and recovered (Japanese Patent Application Laid-Open No. 50-5588).
However, under the strong acidity of pH 3, many enzymes lose their activity. Further, the solution containing impurities is usually colored, but the hue is not improved by this method, and there is a problem that the obtained enzyme is also colored. On the other hand, there has been reported a method in which an aqueous solution containing an enzyme is adjusted to a pH equal to or higher than the isoelectric point of the enzyme, a polymer anion electrolyte and a polymer cation electrolyte are added, and the enzyme is insolubilized and separated (Japanese Patent Application Laid-Open No. 63-163). -304983).
However, in this method, the pH of the enzyme-containing aqueous solution must be equal to or higher than the isoelectric point of the enzyme. There is a possibility that it may be deactivated below, which is not preferable.

[0005]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for efficiently separating and recovering an enzyme from a solution containing impurities without inactivating the enzyme.

[0006]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies and have found that an aqueous solution containing an enzyme is
The present inventors have found that an enzyme having an improved hue can be efficiently recovered by adding a specific polycarboxylic acid at pH 4 to 6 and separating the resulting precipitate, thereby completing the present invention.

That is, according to the present invention, an aqueous solution containing an enzyme is prepared by adding a polymer selected from the group consisting of a vinyl hydrocarbon-maleic anhydride copolymer and a methacrylic ester-maleic anhydride copolymer having 5 or more carbon atoms. Carboxylic acids, pH
The present invention also provides a method for recovering an enzyme, wherein the method comprises adding the precipitates described in 4 to 6, and recovering the resulting precipitate.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The enzyme to which the present invention is applied is not particularly limited, but an enzyme having an isoelectric point of 5 or more, particularly an alkaline enzyme used for a detergent or the like is preferable. Examples of such enzymes include alkaline protease, alkaline esterase, alkaline carbohydrase, lyase and the like. Among these, specific examples of the protease include pepsin, trypsin, chymotrypsin, collagenase, keratinase, elastase, subtilisin, papain, aminopeptidase, carboxypeptidase and the like. Also, specific examples of esterases include:
Gastric lipase, pancreatic lipase, plant lipases, phospholipases, cholinesterases, phosphatases and the like. As carbohydrase, cellulase, maltase, saccharase,
Amylase, pectinase, α- and β-glycosidase and the like. Examples of lyases include carboxy lyase, aldehyde lyase, oxoacid lyase, hydrolyase, lyase that acts on polysaccharides, and ammonia lyase.

The vinyl hydrocarbon having 5 or more carbon atoms of the vinyl hydrocarbon having 5 or more carbon atoms / maleic anhydride used in the present invention includes hexene, diisobutylene, styrene, 1-octene, 2- Octene, 1-decene, 1-dodecene, 1-tetradecene, and the like. Examples of the methacrylate of the methacrylate-maleic anhydride copolymer include methyl methacrylate, ethyl methacrylate, hydroxyethyl methacrylate, and methacrylic acid. Examples thereof include butyl, benzyl methacrylate, and dodecyl methacrylate. Of these, diisobutylene and styrene are preferred. The average molecular weight of these polycarboxylic acids is preferably in the range of 1,000 to 100,000, particularly preferably in the range of 2000 to 50,000. These polycarboxylic acids may be used singly or as a mixture of two or more kinds. The amount of the polycarboxylic acids is preferably 20% by weight or more, particularly preferably 50 to 100% by weight, based on the enzyme. As the amount of addition increases, the recovery rate increases, but the concentration of the enzyme in the recovered product is undesirably low.

As described above, when a specific polycarboxylic acid is added and the pH is adjusted to 4 to 6, the enzyme having a higher isoelectric point becomes a cationic polymer, to which the polycarboxylic acid is adsorbed and immobilized. It is believed that precipitation occurs. On the other hand, the impurities in the solution are likely to remain in the solution without precipitating because many impurities are anionic.

The pH of the solution is from 4 to 6, preferably from more than 4.5 to 6 or less, particularly preferably from 5 to 6. The pH
Is less than 4, the enzyme is severely deactivated, whereas 4
Within the range of ~ 6, it is relatively stable. Further, when the color of the enzyme solution containing impurities is in the range of pH 4 to 6, especially 5 to 6, the color shifts from the enzyme precipitate side to the residual solution side, so that an enzyme with an improved hue can be obtained.

The pH is automatically adjusted by the addition of the above-mentioned polycarboxylic acid.
A pH adjuster such as acetic acid, citric acid, lactic acid, butyric acid, and phosphoric acid may be added.

When a polycarboxylic acid is added to adjust the pH to 4 to 6, precipitation occurs gradually. The precipitate thus obtained can be easily separated by means such as centrifugation or simple filtration, which can be used as it is because it has enzyme activity in suspension.

If the separated precipitate is dissolved in an alkaline solution, polycarboxylic acids are released, so that only the enzyme can be separated and purified. Where the pH of the alkaline solution
Is preferably 8 or more, particularly preferably 8 to 10. As a specific method for separating the enzyme, ion exchange chromatography, gel filtration chromatography, dialysis, membrane separation and the like are easy and preferable.

[0015]

According to the present invention, an enzyme having an improved hue can be efficiently separated and recovered from an aqueous solution containing the enzyme without deactivating the enzyme.

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.
In the following examples and test examples, diisobutylene,
As the maleic anhydride copolymer, Demol EP manufactured by Kao Corporation was used.

Example 1 (1) As an aqueous enzyme solution, an aqueous solution containing a crude enzyme having protease activity (derived from Bacillus sp. KSM-K16 (FERM P-3367), isoelectric point 10.6) (enzyme protein concentration: 1) 0.5% by weight). To this aqueous solution, a diisobutylene-maleic anhydride copolymer (abbreviated as A in Table 1) was added as an anionic polymer so that the weight ratio was 0.3 to 1 times the weight of the enzyme protein in the aqueous solution. Then, the pH was adjusted to 5 with citric acid to prepare a sample. (2) Since a precipitate was formed in the solution, the precipitate was separated into a precipitate and a supernatant by centrifugation, and the respective enzyme activities were measured to determine the activity yield. Table 1 shows the results.

[0018]

Table 1 Sample (additive, enzyme protein mass: additive amount (weight ratio)) Activity yield (%) Control 0 Sample 1 (A, 1: 0.3) 68 Sample 2 (A, 1: 0. 6) 90 Sample 3 (A, 1: 0.9) 90

Example 2 (1) As an aqueous enzyme solution, an aqueous solution containing a crude enzyme having protease activity (derived from Bacillus sp. KSM-K16 (FERM P-3367)) (enzyme protein concentration: 1.5% by weight) was used. Was.
In this aqueous solution, a styrene-maleic anhydride copolymer (manufactured by Kao Corporation) was used as an anionic polymer so that the weight ratio was 0.1 to 0.5 times the mass of the enzyme protein in the aqueous solution.
Demol ST) (abbreviated as A in Table 2) was added, the pH was adjusted to 5 with citric acid, and a sample was prepared. (2) Since a precipitate was formed in the solution, the precipitate was separated into a precipitate and a supernatant by centrifugation, and the respective enzyme activities were measured to determine the activity yield. Table 2 shows the results.

[0020]

Table 2 Sample (additive, enzyme protein mass: additive amount (weight ratio) Activity yield (%) Control 0 Sample 1 (A, 1: 0.1) 46 Sample 2 (A, 1: 0.3) ) 80 Sample 3 (A, 1: 0.5) 80

Example 3 (1) As an enzyme aqueous solution, an aqueous solution (enzyme protein concentration: 1.5% by weight) containing a crude enzyme having protease activity (derived from Bacillus sp. KSM-K16 (FERM P-3367)) was used. Was.
In this aqueous solution, 1% as an anionic polymer was added such that the weight ratio was 0.2 to 1 times the mass of the enzyme protein in the aqueous solution.
-Octene-maleic anhydride copolymer ^* (abbreviated as A in Table 3) was added, the pH was adjusted to 5 with citric acid, and a sample was prepared. * The 1-octene-maleic anhydride copolymer used was a copolymer synthesized under the following conditions. 1-octene / maleic anhydride = 1: 1 weight ratio Benzoyl peroxide: 0.01% by weight based on the total amount of 1-octene and maleic anhydride Reaction temperature: 80 to 85 ° C. Reaction time: 4 hours (2) In liquid Since a precipitate was formed, the precipitate was separated into a precipitate and a supernatant by centrifugation, and the respective enzyme activities were measured to determine the activity yield. Table 3 shows the results.

[0022]

[Table 3] Sample (additive, enzyme protein mass: additive amount (weight ratio)) Activity yield (%) Control 0 Sample 1 (A, 1: 0.5) 62 Sample 2 (A, 1: 1) 73

Example 4 (1) As an aqueous enzyme solution, an aqueous solution containing a crude enzyme having amylase activity (derived from Bacillus sp. KSM-AP1378 (FERM BP-3048), isoelectric point 9.2) (enzyme protein concentration: 0) 0.3% by weight). In this aqueous solution, a diisobutylene-maleic anhydride copolymer (abbreviated as A in Table 4) as an anionic polymer so that the weight ratio becomes 0.2 to 0.5 times the weight of the enzyme protein in the aqueous solution. And add pH 5 with citric acid.
And a sample was prepared. (2) Since a precipitate was formed in the solution, the precipitate was separated into a precipitate and a supernatant by centrifugation, and the respective enzyme activities were measured to determine the activity yield. Table 4 shows the results.

[0024]

Table 4 Sample (additive, enzyme protein mass: additive amount (weight ratio)) Activity yield (%) Control 0 Sample 1 (A, 1: 0.2) 75 Sample 2 (A, 1: 0. 4) 80

Example 5 100 ml of an aqueous solution of a crude protease enzyme (1.5% by weight)
, Diisobutylene-maleic anhydride (weight ratio 0.5
After addition, the pH was adjusted to 5 with citric acid, and a precipitate was obtained by centrifugation. The precipitate was recovered, and 100 ml of 100 mM glycine NaOH buffer (pH 9.5) was added to dissolve the precipitate. As a result, 76% of the protease activity was recovered and 70% of the dye component was removed.

Test Example 1 As the enzyme aqueous solution, a crude enzyme having protease activity (obtained in Example 1) was used. In this aqueous solution,
Diisobutylene-maleic anhydride copolymer was added in a weight ratio of 0.5 times the amount of the enzyme protein in the aqueous solution, the pH was adjusted to 3 to 5 with citric acid, and a precipitate was obtained. Separated from aqueous solution. 100 mM glycine-sodium hydroxide buffer (pH 9.
5), and the absorbance (ABS500) of the supernatant was measured. Table 5 shows the results.

[0027]

Table 5 pH supernatant precipitate 5 0.732 0.135 4 0.655 0.315 3 0.405 0.384

From these results, it can be seen that if the pH during the reaction is lowered, the colored impurities move to the precipitation side. Therefore,
Enzymes recovered in the pH range of the present invention have improved hue.

Test Example 2 As an aqueous enzyme solution, a crude enzyme having protease activity (obtained in Example 1) was mixed with a diisobutylene-maleic anhydride copolymer (Demol EP manufactured by Kao Corporation) in an amount of 0.3%. % (Weight / volume), and the residual activity after heat treatment at 50 ° C. for 10 minutes was measured. Table 6 shows the results. The pH was adjusted with citric acid.

[0030]

[Table 6] pH added No added 570 55 4.5 50 10 4 250

Test Example 3 As an aqueous enzyme solution, a crude enzyme having protease activity (obtained in Example 1) was mixed with a diisobutylene-maleic anhydride copolymer (Demol EP manufactured by Kao Corporation) in an amount of 0.3%. % (Weight / volume), and the residual activity after storage at the following temperature for 3 days was measured. Table 7 shows the results. The pH was adjusted with citric acid.

[0032]

[Table 7]

From the above Test Examples 2 and 3, it can be seen that the precipitate obtained by the method of the present invention can be used as a stable enzyme without separating polycarboxylic acids.

Claims (4)

[Claims] 1. A polycarboxylic acid selected from the group consisting of a vinyl hydrocarbon-maleic anhydride copolymer and a methacrylate-maleic anhydride copolymer having 5 or more carbon atoms is added to an enzyme-containing aqueous solution. A method for recovering an enzyme, comprising adding the precipitate at pH 4 to 6 and recovering the resulting precipitate. 2. The method according to claim 1, wherein the pH is more than 4.5 and not more than 6.
The collection method described. 3. The method according to claim 1, wherein the pH is 5-6. 4. The method according to claim 1, wherein the enzyme is selected from alkaline protease, alkaline esterase, alkaline carbohydrase and lyase.