U.S. patent application number 10/629549 was filed with the patent office on 2004-06-10 for method for preparing bioreactor.
This patent application is currently assigned to Fujisawa Pharmaceutical Co. Ltd.. Invention is credited to Asai, Katsushi.
Application Number | 20040110264 10/629549 |
Document ID | / |
Family ID | 26519191 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110264 |
Kind Code |
A1 |
Asai, Katsushi |
June 10, 2004 |
Method for preparing bioreactor
Abstract
The present invention is a method for purifying an enzyme,
particularly an acylase, comprising causing selective aggregation
and precipitation of a contaminant enzyme, particularly
deacetylase, by the use of a surfactant, particularly a cationic
surfactant. The present invention is also a method for regenerating
an immobilized enzyme carrier, particularly a synthetic adsorbent
or ion exchange resin, comprising allowing a protease to act on the
immobilized enzyme to remove the enzyme from the carrier, said
immobilized enzyme being prepared by binding the enzyme with the
carrier and optionally crosslinking the enzymes by the use of a
crosslinking agent after binding. Particularly, the present
invention is the above-mentioned carrier regeneration method,
wherein the carrier has fine pores, and the above-mentioned carrier
regeneration method, wherein the enzyme is cephalosporin C acylase.
According to the enzyme purification method of the present
invention, undesirable contaminant enzyme for the objective enzyme,
which cannot be removed by a conventional purification method, can
be selectively separated and removed. Therefore, the inventive
method is useful for the production of a standard enzyme product
having a higher purity. In addition, the method for regenerating an
immobilized enzyme carrier of the present invention based on
degradation of the enzyme by a protease can remove the enzyme from
the carrier, particularly a carrier having fine pores, extremely
efficiently, so that repeated recycled use of the carrier is
realized, which is beneficial in terms of environmental and
economical aspects.
Inventors: |
Asai, Katsushi; (Osaka,
JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Fujisawa Pharmaceutical Co.
Ltd.
4-7, Doshomachi 3-chome, Chuo-ku, Osaka-shi
Osaka
JP
541-8514
|
Family ID: |
26519191 |
Appl. No.: |
10/629549 |
Filed: |
July 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10629549 |
Jul 30, 2003 |
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09566721 |
May 9, 2000 |
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6630338 |
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09566721 |
May 9, 2000 |
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09147678 |
Feb 12, 1999 |
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6165758 |
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09147678 |
Feb 12, 1999 |
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PCT/JP97/02663 |
Jul 30, 1997 |
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Current U.S.
Class: |
435/196 ;
435/198 |
Current CPC
Class: |
C12N 11/02 20130101;
C12N 11/089 20200101; Y10S 435/816 20130101; C12N 9/80
20130101 |
Class at
Publication: |
435/196 ;
435/198 |
International
Class: |
C12N 009/16; C12N
009/20 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 1996 |
JP |
212383/1996 |
Aug 12, 1996 |
JP |
212387/1996 |
Claims
What is claimed is:
1. A method for purifying an enzyme, comprising causing selective
aggregation and precipitation of a contaminant enzyme by the use of
a surfactant.
2. The method of claim 1, wherein the surfactant is cationic.
3. A method for purifying acylase, comprising causing selective
aggregation and precipitation of contaminant enzyme by the use of a
cationic surfactant.
4. The method of claim 3, wherein the acylase is cephalosporin C
acylase, and the contaminant enzyme is deacetylase.
5. The method of claim 3 or claim 4, wherein the cationic
surfactant is of a methyl type or a benzyl type.
6. The method of claim 5, wherein the cationic surfactant is
alkyl(palm)dimethylbenzyl ammonium chloride.
7. The method of any of claims 3 to 6, wherein the cationic
surfactant is used in a concentration of 0.1-0.6%.
8. A method for regenerating an immobilized enzyme carrier,
comprising allowing a protease to act on an immobilized enzyme to
remove the enzyme from the carrier, said immobilized enzyme being
prepared by binding the enzyme with the carrier and optionally
crosslinking the enzymes by the use of a crosslinking agent after
binding.
9. The method of claim 8, wherein the carrier is a synthetic
adsorbent or an ion exchange resin.
10. The method of claim 8 or claim 9, wherein the carrier has fine
pores.
11. The method of any of claims 8 to 10, wherein the enzyme is
cephalosporin C acylase.
12. The method of any of claims 8 to 11, wherein the protease is an
acidic protease.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for preparing a
bioreactor. More particularly, the present invention relates to a
method for purifying an enzyme by the use of a surfactant and a
method for regenerating an immobilized enzyme carrier by the use of
a protease.
BACKGROUND ART
[0002] A bioreactor is a system technology to reproduce a
biochemical reaction in an artificial container, and what is meant
by this term has been gradually expanding. As used herein, however,
a bioreactor means a reactor wherein an enzyme itself is used as a
catalyst. For this end, the enzyme should be immobilized in some
way for an economical use of a catalyst. Thus, an immobilized
enzyme plays the principal part of a bioreactor. One of the
important factors that determine the superiority of an immobilized
enzyme is the purity of the enzyme used.
[0003] An enzyme protein is purified from a biological sample or
cell culture supernatant by appropriately combining the
conventionally-known various protein separation techniques
according to the properties of the objective enzyme and contaminant
protein. At present, predominant separation techniques include, for
example, a method utilizing difference in solubilities, such as
salting out, solvent precipitation method and the like; a method
utilizing difference in molecular weights, such as dialysis,
ultrafiltration, gel filtration, polyacrylamide electrophoresis and
the like; a method utilizing electric charges, such as ion exchange
chromatography and the like; a method utilizing specific affinity,
such as affinity chromatography and the like; a method utilizing
difference in hydrophobicities, such as reversed phase high
performance liquid chromatography and the like; and a method
utilizing difference in isoelectric points, such as isoelectric
point electrophoresis and the like.
[0004] In not a few cases in practice, however, contaminant protein
cannot be removed completely from the objective enzyme. This is
attributable to the absence of noticeable difference between the
objective enzyme and contaminant protein in various
physico-chemical properties that the conventional protein
separation methods utilize. For example, cephalosporin C acylase
derived from Pseudomonas [enzyme that converts cephalosporin C and
glutaryl 7-aminocephalosporanic acid (GL7-ACA) to
7-aminocephalosporanic acid (7-ACA); hereinafter to be abbreviated
as CC acylase] can be purified up to an approximately 95% purity by
repeatedly separating a crude cell extract solution as a starting
material by steps such as dialysis, ammonium sulfate fractionation,
anion exchange chromatography and the like (Japanese Patent
Unexamined Publication No. 5-84078). The contaminant deacetylase,
however, cannot be removed by a conventional method because it
shows nearly the same behavior on anion exchange resin column as CC
acylase. A deacetylase deactylates 7-ACA to produce deacetyl 7-ACA,
causing less yield of 7-ACA. Thus, there remains a strong demand
for a novel enzyme purification method capable of separating and
removing such undesirable proteins.
[0005] Another factor determining the superiority of an immobilized
enzyme is the life of an immobilized enzyme carrier. In general
terms, enzymes tend to be unstable to heat, strong acid, strong
alkali, organic solvent and the like, and easily lose activity even
under the conditions preferable for enzyme reactions. An
immobilized enzyme shows decreasing enzyme activities with the
repeated use thereof, thereby lowering the production efficiency of
the objective substance. A degraded immobilized enzyme is generally
disposed, but when an ion exchange resin is used as a carrier, a
recycled use of the carrier is desirable from environmental and
economical considerations.
[0006] Conventional methods for regenerating an immobilized enzyme
carrier include use of a strong acid or strong alkali to liberate
and remove the enzyme from the carrier. However, the enzyme cannot
be removed completely from the carrier by this method, which causes
drastic decrease in the activity of the immobilized enzyme upon
repeated regenerations of carrier and reimmobilizations of enzyme.
Particularly when the carrier has fine pores, the enzyme dogs in
the fine pores, resulting in appreciable degradation due to
regeneration and reimmobilization.
DISCLOSURE OF THE INVENTION
[0007] It is therefore an object of the present invention to
provide a method for selectively separating and removing a
contaminant enzyme which is undesirable for the objective enzyme
and which cannot be removed by conventional separation techniques,
thereby to enable production of a highly pure objective enzyme.
Another object of the present invention is to provide a method for
regenerating an immobilized enzyme carrier, which is capable of
efficiently removing an enzyme from a carrier.
[0008] The present inventors have conducted intensive studies in an
attempt to achieve the above-mentioned objects and had a conception
to utilize selective aggregation and precipitation of protein
caused by surfactants. Thus, they investigated from various aspects
using the system of CC acylase solutions contaminated by
deacetylase. The results revealed that the addition of a
surfactant, particularly a cationic surfactant, leads to selective
aggregation and precipitation of deacetylase, and using this
action, the present inventors have succeeded in preparing a
standard CC acylase product having a high purity and a high
production efficiency of 7-ACA.
[0009] The present inventors have also succeeded in removing CC
acylase efficiently by allowing a protease to act on an immobilized
enzyme obtained by adsorbing CC acylase onto an ion exchange resin
carrier and crosslinking CC acylase with glutaraldehyde. Moreover,
they have found that, in case of an immobilized enzyme using the
above-mentioned enzyme and crosslinking agent, the use of an acidic
protease is particularly effective for the removal of enzymes,
which resulted in the completion of the present invention.
[0010] Accordingly, the present invention provides a method for
purifying an enzyme, comprising causing selective aggregation and
precipitation of contaminant enzyme by the use of a surfactant. The
present invention also provides the above-mentioned method for
purifying an enzyme particularly acylase, wherein the surfactant is
cationic. The present invention also provides a method for
purifying the above-mentioned acylase, wherein the acylase is
cephalosporin C acylase and the contaminant enzyme is deacetylase.
The present invention further provides a method for purifying the
above-mentioned acylase, wherein the surfactant is a methyl type or
benzyl type cationic surfactant, particularly,
alkyl(palm)dimethylbenzyl ammonium chloride. In addition, the
present invention provides a method for purifying the
above-mentioned acylase, wherein the cationic surfactant is used in
a concentration of 0.1-0.6%.
[0011] The present invention moreover provides a method for
regenerating an immobilized enzyme carrier, comprising allowing a
protease to act on an immobilized enzyme to remove the enzyme from
the carrier, said immobilized enzyme being prepared by binding the
enzyme with the carrier, such as a synthetic adsorbent and an ion
exchange resin, and optionally crosslinking the enzymes by the use
of a crosslinking agent after binding. Particularly, the present
invention provides a method for regenerating the above-mentioned
carrier, wherein said carrier has fine pores. Moreover, the present
invention provides a method for regenerating the above-mentioned
carrier, wherein the enzyme is CC acylase. Furthermore, the present
invention provides a method for regenerating the above-mentioned
carrier,wherein said protease is an acidic protease.
BRIEF DESCRIPTION OF THE DRAWING
[0012] FIG. 1 shows the effect of regeneration conditions on the
activity of immobilized CC acylase after regeneration and
reimmobilization.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The enzyme applicable to the purification method of the
present invention is not subject to any particular limitation as
long as it shows less aggregation and less precipitation than other
contaminant enzymes with respect to at least one surfactant
Preferably, not less than 70% of the enzyme remains in a solution
under the conditions wherein not less than 95% of the contaminant
enzyme aggregates and precipitates. Examples of preferable enzyme
include acylase, particularly CC acylase.
[0014] The contaminant enzyme to be removed by the purification
method of the present invention should have property to allow
easier aggregation and precipitation than the objective enzyme with
respect to at least one surfactant. For example, when the objective
enzyme is acylase, the contaminant enzyme is exemplified by
deacetylase and the like. The contaminant enzyme may be contained
in plurality. When each contaminant enzyme is aggregated and
precipitated by different kinds of surfactants, one surfactant is
added first to cause aggregation and precipitation of a first
contaminant enzyme, followed by removal of the precipitates by
centrifugation or filtration, and then, a different surfactant is
added to cause aggregation and precipitation of another contaminant
enzyme.
[0015] The surfactant to be used in the present invention is not
particularly limited and can be selected from among cationic,
anionic and nonionic surfactants, in accordance with the property
of the objective enzyme and contaminant enzyme. When deacetylase,
which contaminates an acylase solution, is to be removed, for
example, preferably a cationic surfactant, more preferably a methyl
type or benzyl type cationic surfactant, particularly methyl and
benzyl type cationic surfactants [e.g., alkyl(palm)dimethylbenzyl
ammonium chloride and the like], are used.
[0016] The surfactant can be used in a concentration that varies
depending on the kind of surfactant and the combination of the
objective enzyme and contaminant enzyme. The desirable
concentration is such that 95% or more of the contaminant enzyme
aggregates and precipitates, and 70% or more of the objective
enzyme remains in the solution. When the objective enzyme is
acylase and the contaminant enzyme is deacetylase, for example, the
surfactant is added to the concentration of 0.1-0.6%.
[0017] The carrier that can be applied to the inventive method for
carrier regeneration is subject to no particular limitation as long
as it can be used for immobilization of enzyme by typical carrier
binding methods. Examples thereof include natural adsorbents such
as active charcoal, acidic clay, kaolinite, celite, alumina,
bentonite, silica gel, titanium oxide, chitin, tannin and the like;
synthetic adsorbents such as polystyrene resin having a hydrophobic
residue (e.g., propyl, butyl, hexyl, phenyl and the like)
introduced thereinto, polyacrylamide gel and the like;
polysaccharide gel having an ion exchange group; ion exchange
resin; and the like. Preferred are synthetic adsorbents and ion
exchange resin. The kind of carrier can be appropriately changed
according to the property of the enzyme to be immobilized and the
like.
[0018] The inventive carrier regeneration method is particularly
effective when the carrier has fine pores (the fine pores in the
present invention mean those having a diameter of 100 nm or less).
When an enzyme has been immobilized on a carrier by a carrier
binding method and a crosslinking method in combination, for
example, a conventional method using a strong acid or a strong
alkali fails to dissolve the enzyme insolubilized by forming
crosslinks. The enzyme then cloggs in the fine pores to cause
striking degradation of the carrier. In contrast, the inventive
regeneration method using a protease provides efficient removal of
the insolubilized enzyme from the carrier, since the insolubilized
enzyme is degraded by the action of the protease, resulting in
redissolution of the enzyme.
[0019] The enzyme to be immobilized on a carrier is subject to no
particular limitation as long as it can be bound with the carrier
without losing its activity by physical adsorption, ionic bonding
and the like, and various enzymes derived from microorganisms,
plants and animals can be used. Industrialized enzymes include
acylase, amino acylase, invertase, nitrile hydratase, sugar
isomerase and the like.
[0020] An enzyme can be immobilized onto a carrier by a
conventional carrier binding method (an immobilizing method wherein
an enzyme is bound with a water insoluble carrier, including a
binding method by physical adsorption in the present invention).
Particularly, an enzyme is desirably bound with a carrier by ionic
bonding, hydrophobic bonding or physical adsorption. When an ion
exchange resin is used as a carrier, for example, an equilibrated
resin is packed in a column and a suitable buffer containing an
enzyme is passed through the column (400-1000 units/mL column),
thereby allowing the enzyme to bind with the resin.
[0021] When the carrier binding alone is not sufficient to prevent
enzyme leakage, enzymes after binding with the carrier are
crosslinked with a crosslinking agent and immobilized. Examples of
the crosslinking agent include glutaraldehyde, hexamethylene
diisocyanate and the like. The crosslinking reaction is performed
by washing the immobilized enzyme obtained by the above-mentioned
method and circulating a crosslinking agent dissolved in a suitable
buffer through a column for approximately 0.5-2 hours. The
unreacted functional group in the crosslinking agent needs to be
inactivated with an inactivating agent, such as glycine, which is
circulated through the column for approximately 0.5-1 hour.
[0022] An immobilized enzyme carrier can be regenerated as in the
following. An immobilized enzyme prepared by the above-mentioned
immobilization method is repeatedly subjected to known enzyme
reaction until it loses half the enzyme activity, and a solution
(pH 3-5 or pH 9-11) containing 3-50 units/mL of an acidic protease
(or alkaline protease) is passed through the column containing said
immobilized enzyme, in an about 5-15-fold volume of the column and
at a flow rate of 5-20 column volume/hour (hereinafter to be
indicated as SV 5-20) for about 1 to 5 hours. Regeneration reaction
temperature is 20-60.degree. C., preferably 30-50.degree. C.
[0023] The enzyme can be reimmobilized onto a regenerated
immobilized carrier in the same manner as in the first
immobilization.
EXAMPLES
[0024] The present invention is explained in more detail in the
following byway of Examples that are for exemplification only, and
do not limit the present invention in any way.
Example 1
Separation and Removal of Deacetylase from CC Acylase Solution by
Various Surfactants
[0025] (1) Culture of CC Acylase Producing Bacteria and
Purification of Crude Extract Solution
[0026] Mutant CC acylase N-176 producing E. coli JM109 (PCCN 176-2)
FERM BP-3047 was cultured according to the method disclosed in
Example 3 of Japanese Patent Unexamined Publication No. 5-84078 and
the cells were harvested by centrifugation. The obtained cell
pellets were disrupted with a homogenizer and soluble protein
component was extracted with water, which was followed by
centrifugation to remove debris, whereby a crude extract solution
was obtained.
[0027] (2) Removal of Contaminant Deacetylase by Surfactant
[0028] Polyethyleneimine was added to the crude extract solution
obtained in the above-mentioned (1) to allow precipitation of the
nucleic acid, which was followed by centrifugation to recover
supernatant. This supernatant was applied to DEAE column
equilibrated with phosphate buffer (pH 7.0), washed with water and
eluted with 0.2M-NaCl. (By this step, .beta.-lactamase, which is
the other contaminant enzyme, is selectively recovered and removed
in nonadsorbed Traction.) To the obtained eluate was added a
cationic surfactant: alkyl(palm)dimethylbenzyl ammonium chloride
[trademark : Cation F2-50; manufactured by NOF Corporation
(hereinafter the same)], alkyl(palm)trimethyl ammonium chloride
(trademark: Cation FB) or dodecyltrimethyl ammonium chloride
[trademark: Cation BB], an anionic surfactant: alkyl glycine
[trademark: Nissan Anon LG] or a nonionic surfactant:
polyoxyethylene octylphenyl ether (trademark : Nonion HS-210) or
polyoxyethylenesorbitan monooleate (trademark: Nonion OT-221) to
the final concentrations of 0.1, 0.3 and 0.5%, and the mixtures
were stood still at room temperature for 16 hours. The activities
of CC acylase and deacetylase in each sample before and after the
addition of the surfactant were measured and the changes thereof
were examined. The enzyme activity was calculated by quantitatively
determining 7-ACA and deacetyl 7-ACA produced from GL7-ACA as a
substrate. The results are shown in Table 1. Cation F2-50 showed
90% or more of CC acylase residual activity at the concentration of
0.1-0.3% and reduced the activity of contaminant deacetylase to not
more than 5%. At the concentration of 0.5%, deacetylase was removed
nearly completely. The other two cationic surfactants also showed
selective removal of deacetylase at the concentration of 0.5%. When
an anionic or nonionic surfactant was used, deacetylase could not
be removed completely in the concentration range examined, but the
deacetylase activity decreased to nearly half or less almost
without decrease of the CC acylase activity.
1TABLE 1 Effect on selective removal of contaminant deacetylase by
the addition of various surfactants residual activity.sup.1) (CC
acylase/deacetylase) Surfactant Addition concentration (%)
(trademark) 0 0.1 0.3 0.5 Cation F2-50 100/100 95/5 92/1 70/0
Cation FB 100/100 100/55 100/40 80/1 Cation BB 100/100 100/55
100/45 85/5 Nissan Anon LG 100/100 100/60 100/50 95/30 Nonion
HS-210 100/100 100/50 100/50 100/45 Nonion OT-221 100/100 100/50
100/50 100/45 .sup.1)The residual activity is expressed in relative
activity based on the enzyme activity before addition of surfactant
(addition concentration 0%) as 100.
Example 2
Dissolution of Crosslinked CC Acylase Precipitate by Protease
[0029] To 10 mM Tris HCl buffer (pH 8.0, 5 mL) containing 67
units/mL of CC acylase N176 (how to obtain this enzyme is disclosed
in Japanese Patent Unexamined Publication No.5-84078) was added 150
mM Tris HCl buffer (pH 8.7, 5 mL) containing 20% glutaraldehyde and
the mixture was stirred for 1 hour to allow reaction, whereby a
crosslinked product was precipitated. The supernatant was removed
by centrifugation and the precipitate was recovered. To the
precipitate was added 2N--NaOH, 0.1N--NaOH, 0.1N--NaOH+alkaline
protease [Bioprase AL-15 (30 units); manufactured by Nagase
Biochemicals Ltd.], 2N--HCl, 0.1N--HCl or 0.1 N--HCl+acidic
protease [Denapsin (30 units); manufactured by Nagase Biochemicals
Ltd.] by 3 mL and the precipitate was immersed therein for 2 hours
at 40.degree. C. As a result, addition of NaOH or HCl alone did not
cause change in the precipitate, but addition of HCl+acidic
protease resulted in the complete dissolution of the precipitate
and a clear solution was obtained. While the addition of
NaOH+alkaline protease also resulted in nearly complete dissolution
of the precipitate, the solution was cloudy and complete
dissolution was not achieved. From the above results it was shown
that CC acylase insolubilized by crosslinking g treatment is
effectively redissolved by the use of protease, particularly an
acidic protease.
Example 3
Regeneration of Immobilized CC Acylase Carrier by Protease
[0030] (1) Preparation of Immobilization Carrier
[0031] A brand new styrene-divinyl benzene highly porous strong
basic ion exchange resin [80 mL, HPA 25 (having fine pores of
diameter 10-100 nm); manufactured by Mitsubishi Chemical
Corporation] was immersed in 1N--NaOH:methanol=1:1 for one day and
packed in a column (350 mm.times.15 cm). Then, 3-fold volume of
2N--HCl was passed through the column at SV3 and the column was
washed with 5-fold volume of water (SV3), whereafter 5-fold volume
of 0.1N--HCl was passed through the column at SV3 and the column
was washed with water at SV3 until the pH at the outlet of the
column became 4 or above.
[0032] (2) Immobilization of CC Acylase
[0033] HPA 25 (80 mL) prepared in the above-mentioned (1) was
packed in a column and 10 mM Tris HCl buffer (pH 8.0, 240 mL)
containing CC acylase N176 (67 units/mL was passed through the
column at SV3. After washing the column with 240 mL of water (SV3),
60 mM phosphate buffer (pH 8.0, 300 mL) containing 2%
glutaraldehyde was circulated at SV20 for one hour while adjusting
the pH to 7.5. After crosslinking, the column was washed with 400
mL of water (SV3). Then, 60 mM phosphate buffer (pH 8.0, 300 mL)
containing 0.2M glycine was circulated at SV10 for one hour to
inactivate unreacted aldehyde groups. Using water (400 mL or more),
the column was washed at SV5 to give 82 mL of acylase immobilized
on HPA 25.
[0034] (3) Production of 7-ACA by Immobilized CC Acylase
[0035] CC Acylase immobilized on HPA 25 (20 mL) and prepared in the
above-mentioned (2) was packed in a column with a jacket, and 6.0
mg/mL glutaryl 7-aminocephalosporanic acid (GL7-ACA) solution (100
mL) was circulated for one hour at 20.degree. C. at circulation
flow rate of about 70 mL/min while adjusting the pH to 7.75 with
NaOH, whereby 7-ACA was obtained. After the reaction, the reaction
mixture in the column was pushed out with water and 7-ACA was
recovered. The above-mentioned reaction was repeated 100 times
(continuous batch reaction), which was taken as one cycle.
[0036] (4) Regeneration of Immobilization Carrier
[0037] Using 2N--NaOH, 0.1N--NaOH+Bioprase (3000 ppm, 10 units/mL)
or 0.1N--HCl+Denapsin (3000 ppm, 10 units/mL) as a regeneration
agent, HPA25 was regenerated every one cycle. The regeneration
agent (10-fold volume of carrier) was circulated at 40.degree. C.,
SV about 20 for 5 hours.
[0038] After the regeneration reaction, the above-mentioned steps
(1)-(4) were repeated 2 to 4 times, and the CC acylase activity
after reimmobilization (2) was determined. The changes of the
activities were compared.
[0039] The results are shown in FIG. 1. When 2N--NaOH was used as a
regeneration agent, the activity of the immobilized CC acylase
decreased in nearly half at every cycle, whereas the use of
0.1N--HCl and Denapsin (acidic protease) did not result in decrease
of the enzyme activity even after.3 cycles. When 0.1N--NaOH and
Bioprase (alkaline protease) were used, the activity of CC acylase
decreased at every cycle but in a smaller magnitude as compared to
the single use of NaOH, and not less than 50% of the original
activity was expressed even after 4 cycles.
[0040] The enzyme purification method of the present invention is
characterized in that undesirable contaminant enzyme for the
objective enzyme, which cannot be removed by a conventional
purification method, is separated and removed by utilizing the
selective aggregation and precipitation of protein by a surfactant.
According to the present invention, therefore, a standard enzyme
product having a higher purity than the conventional products can
be produced. In addition, the method for regenerating an
immobilized enzyme carrier of the present invention based on the
action of protease on enzyme can remove enzyme from a carrier
extremely efficiently, so that a long term continuous recycled use
of the carrier is realized. Consequently, the present invention
greatly contributes to the reduction of the production cost of a
bioreactor and prevention of environmental destruction associated
with the waste disposal of carrier.
[0041] This application is based on application Nos. 212383/1996
and 212387/1996 filed in Japan, the contents of which are
incorporated hereinto by reference. All publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically described herein.
* * * * *