JPS6022920B2 - Production method of 6-aminopenicillanic acid by immobilized enzyme method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 6-aminobenicillanic acid (6-APA) by an immobilized iQ enzyme method.

Conventionally, various chemical deacylation methods and enzymatic deacylation methods have been reported as methods for producing 6-APA from penicillin.

Chemical deacylation methods, especially methods in which dehydrated and dried natural penicillin is protected with an ester group, followed by iminochloride and iminoetherization, and then hydrolyzed, are widely used, but the process is not only complicated but also difficult. ,6
Since APA is an intermediate for pharmaceutical manufacturing,
There was a problem with the contamination of impurities. On the other hand, in the conventional enzymatic deacylation method, in order to expect a high reaction yield, natural penicillin, which is a substrate, must be used at a low concentration of about 0.1 to IW/V%. 6-APA is obtained as a dilute solution, so in order to recover 6-APA from it, the reaction solution must be concentrated, even though 6-APA is a chemically unstable substance. It had a serious drawback: As a result of continuing various studies regarding the production method of 6-APA using the immobilized enzyme method, the present inventor found that
In immobilizing penicillin acylase, an acrylonitrile polymer with a porous structure containing free amino groups, which the present inventor found to be an enzyme support, was used to immobilize penicillin acylase with extremely high specific activity. By using the immobilized penicillin acylase described above, even when natural penicillin in a highly concentrated solution of 3 to 12 W/V% or more is used as a substrate, 6 to APA can be obtained with a high reaction rate of 95% or more. What you get,
Moreover, 6-APA can be recovered with high purity and high yield by simply adjusting the pH of the reaction solution to the same dew point as 6-APA without requiring a concentration operation for the obtained reaction solution. Since the immobilized penicillin acylase can withstand repeated reuse for an extremely long period of time, the proportion of the immobilized enzyme in the manufacturing cost of 6-APA can be almost ignored, and furthermore, the penicillin fermentation It can be used not only at the stage where penicillin is isolated and purified in the form of a salt from a liquid, but also at an extract obtained by dissolving penicillin in an aqueous solution, and has advantages such as being able to produce 6-APA at a lower cost. I saw the facts and published them. The present invention has been completed based on the above-mentioned various findings, and includes a method for enzymatically producing 6-APA by allowing immobilized penicillin acylase to act on penicillin. 6-A characterized by being immobilized on an acrylonitrile-based polymer
This is a method for producing PA.

The method of the present invention will be explained in detail below.

The penicillin used in the present invention is a known natural penicillin or a water-soluble salt thereof that is affected by penicillin acylase.

As the natural penicillin, for example, penicillin G and penicillin V are preferable, and penicillin G is particularly preferable. The water lacquer salt may be any known salt as long as it does not adversely affect penicillin acylase activity and is water-soluble, but alkali metal salts, such as sodium salts and potassium salts, are usually preferred. In the present invention, not only penicillin isolated and purified in the form of an alkali metal salt from a penicillin fermentation solution, but also penicillin extracted from a penicillin fermentation solution with a non-hydrophilic organic solvent and transferred from this organic layer to an aqueous solution can be used. It is also possible to use it in the form of a penicillin extract in the extraction process. The penicillin acylase used in the present invention uses penicillin as a substrate and converts it into 6-AP.
It is a known enzyme that has the action of hydrolyzing A.

The present enzyme is preferably an enzyme preparation collected from cultured cells of known penicillin acylase-producing bacteria or culture furnace fluid. The above enzymes are collected by known methods and partially or fully purified. The above penicillin acylase producing bacteria include, for example, Actinoplanes, Arthrobacter, Bacillus, Erwinia, Etscherichia, Flavobacterium, Micrococcus, Micromonospora, Nocardia, Proteus, Pseudomonas, It is a microorganism belonging to the genus Serratia or Streptomyces. An example is Actinoo
lanes unehensis ATCCI4539A
JthrobaCtertumeSCe ship ATCC6
947AJthrobaCterVISCos female AT
CCI5294Bacmus megateriumA
TCCI4945 ATCCI494
6〃B-40valent ERM-P748Enw
iniaS ni Wareji ATCCI3531Esdh
erichiacoli ATCC9637〃
ATCCIII05 ATCCI3
529 〃 NCIB8743 〃 NCIB8743A F1avobacteriumsuaveolensA
TCCI3533MiCroCoCCoCandid
uS ATCC8456MiCroCoCCo
eFemale ATCC416MiCr.

m. nOSp. ra purpu grasp. Chr. m.
genesATCCI3634Nocardis sp
．． ATCCI3635Proteus ret bag ri
ATCC9250 ATCC31052P
seMomonasaemgnosaNRRLIOIyamaPSeudomonaS fluoreSCeship NR
RLBIOPseudomonas maltophi
laATCCI7808Sematialubidae
aATCCI81SueptomyCeSQ-Se female
IF.

3355S 口eptomyCeS 1avendula
e ATCCI3664〃 ATCC
Examples include I3665 ATCC21138.

Of course, other penicillin acylase producing bacteria can also be used. Also, Advances in A
pplied Microbiology Vol. 1
7. P, 311-369, 1974 (ed. E.J.V.
an ship mmee i. Penicillin acylase produced by the fungi described in ) may also be used. In this case, it is preferable to use penicillin V as the natural penicillin. In the present invention, the support used for immobilizing penicillin acylase is a water-impossible acrylonitrile-based polymer having free amino groups, and is a support for acrylonitrile-based monomers such as acrylonitrile, methacrylonitrile, Q-chloroacrylonitrile, and cinnamitrile. It is a homopolymer, a copolymer, or a copolymer of this with other comonomers containing ethylenic double bonds.

Copolymers with other comonomers mentioned above include, for example, styrenic monomers M such as styrene, methylstyrene, ethylstyrene, nitrostyrene, chlorostyrene, bromostyretho, chloromethylstyrene, methyl (meth)acrylate, ethyl (meth)acrylate, etc. , butyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, (
Acrylic acid ester monomers such as polyethylene glycol meth)acrylate, conjugated dienes such as butadiene and isoprene, halogenated olefins such as vinyl chloride and vinylidene chloride, vinyl ether monomers such as ethyl vinyl ether and butyl vinyl ether, methyl vinyl ketone , vinyl ketone monomers such as ethyl isoprobenyl ketone, vinyl acetate, vinyl ester monomers such as pinyl benzoate, (meth)acrylamide, N,N-dimethyl (meth)acrylamide,
N-vinylpyrrolidone, N-vinylsuccinimide, N
-amide vinyl monomers such as vinyl phthalimide,
Basic monomers such as vinylpyridine, methylvinylpyridine, vinylimidazole, N,N-diethylaminoethyl (meth)acrylate, polyfunctional monomers such as dipinylbenzeso and divinyltoluene, and acrylonitrile monomers are combined with a crosslinking polymerization agent as necessary. , for example using divinylbenzene, divinyltoluene,
It is a copolymerized product. In order to obtain the above-mentioned support, it is preferable that the acrylonitrile-based polymer has a porous structure, and in order to form this porous structure, for example, acrylonitrile is homo- or copolymerized.
The monomer is subjected to suspension polymerization or emulsion polymerization in an aqueous system, or the polymer is dissolved in a solubilizing solvent such as dimethylformamide, dimethyl sulfoxide, concentrated nitric acid, an aqueous solution of Rodan's salt, an aqueous solution of zinc chloride, etc. Drop an acetone-containing aqueous solution, an ethanol-containing aqueous solution, a dimethylformamide aqueous solution, etc. into a coagulation bath and extrude it in the form of a thread or film to form particles, fibers, etc. with a porous structure.
It may be formed into a film.

Furthermore, it may be formed into a hollow fiber-like fiber. The porous structure obtained in this way probably has large pores that have the role of allowing the biologically active substance, namely penicillin acylase, to penetrate into its interior, and a role that has the role of trapping the biologically active substance on its surface and inside. It is estimated that the pores have a small pore size of about 100 to 2,000 pores, which allows biologically active substances to bind.
The pore size is distributed around A. Next, a free amino group is introduced into the above acrylonitrile polymer to obtain an aminated acrylonitrile polymer, the so-called support of the present invention. Since the present invention is carried out in an aqueous medium, the introduction of amino groups should proceed within the range in which the resulting support is water-insoluble. Various known reactions can be used as the method. For example, there is a method of reducing a nitrile group in an acrylonitrile polymer to an amino group. When the polymer already has a porous structure, this reduction is preferably carried out in an inert nonsolvent that does not dissolve the polymer so as not to destroy the porous structure. A preferred reducing agent for such a reduction reaction is lithium aluminum hydride. Inert non-solvents include organic media such as diethyl ether, dioxane, tetrahydrofuran and the like. The above reaction is carried out at room temperature to the boiling point of the nonsolvent used. The reaction time varies depending on the reaction temperature, the type, shape, structure, degree of polymerization, etc. of the polymer used, but it is usually 1.
This is done as appropriate between ~4 hours. As a result of the above reaction, the nitrile groups in the polymer are partially reduced to amino groups, but the degree of amination varies from 20 Fm per ton of free amino groups to the desired support until the support is substantially water soluble. It ends as long as it doesn't become sexual. The support obtained in this way may be washed with water, an acid aqueous solution, an alkaline aqueous solution, etc., as necessary. An alternative method for reducing nitrile groups to amino groups is to carry out catalytic reduction of acrylonitrile-based polymers in an autoclave in the presence of an organic solvent capable of dissolving the polymer; however, this reaction leads to excessive amination and This is not a preferred method because the product may become water-soluble and it also involves a step of forming a porous structure.

Furthermore, another method for obtaining an aminated acrylonitrile polymer is to react an acrylonitrile polymer having a halogen atomic group, particularly a chlorine atomic group, with ammonia to replace the chlorine atomic group with a free amine/group. It can also be carried out by reacting an acrylonitrile polymer having an epoxy group with lysine or alkylene diamine, such as hexamethylene diamine or dodecamethylene diamine. Furthermore, it can also be obtained by Hofmann decomposition of a copolymer of an acrylonitrile monomer and an amide vinyl monomer, that is, an acrylonitrile polymer having an amino group.
As another method, if a copolymer of an aromatic pinyl monomer such as a styrene monomer, divinylbenzene, or pinylethylbenzene and an acrylonitrile monomer does not have a nitro group, it is nitrated in a concentrated nitric acid-monosulfuric acid mixture. This is done by reducing the nitro groups of an acrylonitrile-based polymer with a sodium hydrosulfite-potassium hydroxide solution. Furthermore, it can also be obtained by treating a copolymer of an acrylonitrile monomer and methyl vinyl ketone with ammonia.
The support thus obtained preferably has a porous structure and has a fibrous, granular, membrane or hollow fiber shape.

This porous structure has large pores that allow biologically active substances, such as penicillin acylase, to penetrate into its interior, and small pores that serve to trap biologically active substances on its surface and inside. It is presumed that the biologically active substance is captured and bound by this. The pores are distributed in a pore size of about 100 to 2000 △. Penicillin acylase can be immobilized on the support by a physical adsorption method or a covalent bonding method (carrier crosslinking method).

These immobilization methods are described, for example, in "Immobilized Enzymes J.
This can be carried out as appropriate by widely known conventional methods such as those described in Kodansha Publishing. for example,
Crosslinkers such as glutaraldehyde, dialdehyde starch, hexamethylene diisocyanate, hexamethylene diisothiocyanate, toluene diisocyanate, xylene diisocyanate, dimethyl adipimidate, dimethyl suberimidate, dimethyl-3,3'-dithiobispropionimidate, etc. Either the amino groups of the support and penicillin acylase are bonded directly or via a baser such as lysine or hexamethylene diamine using a binding agent, or the amino groups of the support are first bonded with a carboxyl group-introducing agent such as succinic anhydride. After the reaction, this carboxyl group and penicillin acylase are bonded by a known peptide bonding method, or the support and penicillin acylase are directly bonded with a condensing agent such as 1-ethyl-3-
(3-dimethylaminopropyl)-carbodiimide is obtained by bonding using a lagoonal condensing agent.

When using the thus obtained support on which penicillin acylase is immobilized, an immobilized enzyme with a higher enzyme activity per support can generally be obtained when using a physical adsorption method than when using a covalent bonding method. However, by immobilizing using a covalent bonding method, the bond becomes strong, which prevents enzyme elution due to long-term continuous use, and also improves stability against external factors such as heat and water. In some cases, it is advantageous.

The above immobilized penicillin acylase is allowed to act on penicillin in an aqueous medium, and since the immobilized enzyme is insoluble in water, it can be carried out either by a batch method or a column method.

For example, in the case of a batch method, the reaction can proceed by suspending the immobilized enzyme in a penicillin solution and sailing the suspension while adjusting the pH at a constant level. The immobilized enzyme can be recovered from the reaction solution by filtration or centrifugation and used again. Moreover, this batch method can also be carried out continuously. When using a column method, the immobilized enzyme is packed into a column and the penicillin solution is passed through the column. In this case, it is preferable to recirculate the column at a high flow rate.

That is, by limiting the deacylation reaction rate in one column passage, it is possible to suppress an extreme drop in pH due to the generated carboxylic acid. The reaction solution that has passed through the column is cooled and stored in a storage tank, the pH is constantly neutralized to 6.5 to 9.5 with an alkali such as alkali hydroxide, and the solution is recycled to the column via a heat exchanger. This is effective in preventing deterioration of immobilized enzyme activity and decomposition of penicillin due to pH decrease, and can promote deacylation reaction. The column is designed to minimize pressure loss due to high flow rates.
It is preferable to use beds as shallow as possible with appropriate design, and recirculation is normally carried out at a flow rate of 100 to 300 bed volumes per hour. More than 90% of penicillin is 6-A recirculated.
It is preferable to continue until conversion to PA. The above enzymatic reaction is usually carried out in a range of 15 to 40 degrees centigrade.

The optimum pH for the reaction varies depending on the penicillin acylase producing strain, but if it is necessary to maintain the pH at a constant level, the reaction may normally be carried out at around 6 to 9. The reaction time varies depending on the enzyme titer, substrate liquid volume, flow rate, etc., but is usually 1 to 1 field hour. Therefore,
In the case of a column type, it is preferable to pass the reaction within the appropriate reaction time. The concentration of penicillin in the above enzymatic reaction is used in the range of 1 to 20 W/V%, but it is usually 3% in terms of high reaction rate, reuse rate, and prevention of activity deterioration of the immobilized enzyme.
It is preferable to use it within a range of 12 W/V%. Next, 6-APA is isolated and purified from the reaction solution.
This can be done by a known method.

For example, after removing penicillin and carboxylic acid remaining in the reaction solution by extraction under acidic conditions with a non-hydrophilic organic solvent such as methyl isobutyl ketone, ethyl acetate, or butyl acetate, the pH is adjusted to 4.3 and 6-APA is added. Alternatively, after adding a hydrophilic organic solvent such as acetone or methanol to the reaction solution, 6-APA may be precipitated to an isoelectric point directly at pH 4.3. In the present invention, since the substrate concentration is extremely high, 6-APA can be directly crystallized from the reaction solution in high yield and purity without concentrating the reaction solution. The conversion rate of penicillin to 6-APA can be determined by various methods.

For example, it can be determined from high performance liquid chromatography, the amount of alkali consumed by carboxylic acid production, and the pH of the reaction solution can be adjusted to
2.0, unreacted penicillin and the produced carboxylic acid were extracted and removed with methyl isobutyl ketone, and 6-A in the aqueous layer was removed.
PA can be quantitatively determined by the iodine absorption method and simultaneously acylated with phenylacetic acid chloride, and the antibacterial activity of the resulting penicillin G can be determined by the paper disc method. Penicillin acylase titration method.

1 For enzyme solution: 0.5' of enzyme solution, 0.1M phosphate buffer (pH 7.5)
) 4.0', 4 (W/V)% PcG potassium salt/0.
After reacting the reaction solution consisting of 1M phosphate buffer (pH 7.5) for 370 minutes, 0.5 minutes was added.
5', 0.0% NaOHI, 20% acetic acid 2
0.5 (W/V)% P-dimethylaminobenzaldehydonomethanol solution was added to 3 volumes of a buffer solution consisting of 1,000 ml of P-dimethylaminobenzaldehyde no methanol solution, and reacted for 10 minutes at room temperature, and the absorbance at the 41 law m was measured. Find the amount of 6-APA.

Regarding enzyme activity, 1 unit (IU) is defined as the activity that produces 1 mmol 6-APA per minute.

2 For immobilized enzyme, 0.1M phosphate buffer (pH 7.5) 4.5M, 4(W
/V)% PcG potassium salt/0.1M phosphate buffer (p
H7.5) Add the immobilized enzyme whose weight was measured in advance to a solution consisting of 0.5' and react for 30 minutes at 37q0.

6-AP produced in the same manner as in the case of the above enzyme solution.
Find the amount of A. Next, the present invention will be specifically explained by giving participation examples and examples, but the present invention is not limited thereby.

Reference Example 1 Production of polyacrylonitrile having a porous structure A 3-necked flask of 500ml was immersed in constant overflowing water at about 35°C, purged with nitrogen for about 15 minutes, and then 120ml of distilled water was placed in the flask. was added, and further added sodium alkylsulfonate for 2 nights, acrylonitrile for 80 nights, sodium persulfate for 0.1 night, and sodium hydrogen sulfite for 0.0 nights.
The emulsion was obtained by adding 33 tsp of water and stirring for about 3 hours, and then pouring it into about 500 ml of water, and adding salt to solidify it to precipitate the product. After washing with water and drying with ventilation, polyacrylonitrile (0.5%, logarithmic viscosity in dimethylformamide at 30 qC is approximately 10.5
) was obtained.

Next, 10% of this polyacrylonitrile was dissolved in 150% of dimethylformamide, and this was formed into a thread in a water bath containing 20% dimethylformamide to form polyacrylonitrile fibers having a porous structure (thickness: 20 to 35%).
Mikuoso) was obtained. Similarly, polyacrylonitrile 10
The solution was dropped into a water bath containing 20% dimethylformamide using an atomizer cup to obtain granular polyacrylonitrile having a porous structure (average particle size: 100 mesh). Reference Example 2 Preparation of support for immobilized enzyme Add 120 dry diethyl ether to 1.5 liters of lithium aluminum hydride, and add polyacrylonitrile (90% or more acrylonitrile) fiber having a porous structure obtained in Reference Example 1 to this. Add 1.5 hours of water, attach a reflux condenser, and heat under reflux in a 50qo oil bath for 10 minutes to 3 hours.

After the reaction, the fibers were taken out and washed with dry jetyl ether, followed by IN hydrochloric acid, water, IN aqueous sodium hydroxide solution,
The fibers are thoroughly washed with water to obtain aluminized polyacrylonitrile fibers (thickness: 20 to 35 microns) having a porous structure. Unreacted lithium aluminum hydride is decomposed by dropping a small amount of water. The physicochemical properties of the aminated polyacrylonitrile fiber obtained are as follows.

Color: Yellowish (more than the raw material polyacrylonitrile fiber).

As the reduction time increases, the yellow color becomes stronger. viscosity;
Unknown (cannot be measured because it does not dissolve in the following solvents)
Solubility in solvent; Aminated polyacrylic acrylonitrile Fiber Fiber DMS○ Partially dissolved* Dissolved DMF
〃 〃 Concentrated nitric acid Partially dissolved Dissolved 65% KSCN solution (60℃) Chloroform Insoluble Insoluble Acetonitrile pyridine ``Nitromethanecyclohexane'' 〃 Diethyl Able ``'' Dioxane Tetrahydrofuran 30% NaOH solution ``'' *Fiber thickness hardly changes It remains in the form of fibers and does not dissolve completely.

When water is added to the solvent, it becomes cloudy, and a subsequent experiment shows that the weight decreases, indicating that some of the solvent has been dissolved.
Aminated polyacrylonitrile fibers obtained at various reduction times 3
The fibers were placed in DMF5' and heated at 6,000 for 3 minutes.The fibers were then taken out of the furnace, washed with water, and the dry weight was measured.The following results were obtained. Reduction time Weight loss rate 5 minutes 31.3% 1 hour 29.0% 5 hours 27.0% Coloring property against sodium 2,4,6-trinitrobenzenesulfonate; polyacrylonitrile fibers do not color, but aminated Polyacrylonitrile fibers turn yellow,
It was confirmed that it has an amino group.

Amino group content: The results obtained using the following test method for the immobilized protein and estimated amino group content of the obtained support are shown in Tables 1 to 3.
Shown in the table.

i Immobilized protein 2 of the support 18 at each reaction time obtained above is 12.
5% glutaraldehydonoborate buffer (Book 8.5) 1
Add to the kite and heat for 2 minutes at 0℃.

The fibers were filtered, and boric acid buffer (pH 8.5), 0.1M
After thorough washing with phosphate buffer (pH 7.5), immediately add 0.3% bovine serum albumin or Bacillus megaterium B-400 penicillin acylase (140 U/)/0.1 M phosphate buffer (pH 7). .5) After shaking at 30°C for 1 hour, the contents of bovine serum albumin and penicillin acylase remaining in the mother liquor were determined by the LoMy method [0.5)]. Day. LoMy, J. Biol. C
hem. , 193, 265 (1951)] to determine the amount of bovine serum albumin or penicillin acylase bound to the support. Alternatively, the support was directly placed in a bovine serum albumin solution or penicillin acylase solution without being treated with glutaraldehyde, and after shaking at 30 oo for 1 hour, bovine serum albumin or penicillin acylase was added to the support in the same manner as above. The amount of adsorption was determined.

ii Estimated amino group content 9 of support 18 at each reaction time obtained above was
After treating with 12.5% glutaraldehyde solution in the same manner as above, this was treated with 0.3% concentration of glutamic acid (GIu
), threonine (Thr), 7-aminodes3cetoxycephalosporanic acid (7-ADCA) each amino acid/0
．． 1M phosphate buffer (pH 7.5)
Shake at 0oo for 1 hour.

The reaction mixture was separated into a furnace, and the amount of amino acids in the supernatant solution was determined using an automatic amino acid analyzer, and the amino group content of the support was determined as the amino acid binding capacity. In carrying out this test, it was confirmed by a test using amino acid hydratide in place of the amino acid that the amino acid used had no adsorption properties to the support. When 7-ADCA was used as the amino acid, the 7-ADCA content in the supernatant solution was determined from the ultraviolet absorption value at 254 nm using high-performance liquid chromatography, and the binding capacity of 7-ADCA to the support was determined. .
Estimated amino group content by titration 80 females of the support at each reduction time were treated with 15' of 2mM hydrochloric acid.
Soak for 1 hour with 30 ml of distilled water.

Excess hydrochloric acid is titrated with a phantom M aqueous sodium hydroxide solution, and the amide/group content in the sample support is determined as the amount of hydrochloric acid consumed as the hydrochloride. iv Test results Table 1 Protein binding amount Table 2 Amino acid binding capacity Table 3 Hydrochloric acid consumption by titration Reduction time Amino group content rM/5 minutes 175 1 hour 263 5 hours 300 8 hours 275 Adsorbed enzyme (penicillin acylase) activity ; In the preceding item i, Bacillus megaterium B-400 (FERM-P
Instead of determining the amount of penicillin acylase produced by No. 400), the penicillin acylase activity was determined by the penicillin acylase activity measurement method described above.

The results are shown in Table 4. Table 4 Reference example of adsorbed penicillin acylase activity 3 Preparation of support for immobilized entrapment enzyme Drying 1.5 mm of polyacrylonitrile (90% or more acrylonitrile) fiber having a porous structure containing 1.5 mm of lithium aluminum hydride Add ether 120 [into] and heat under reflux for 1 hour in 5,000 ml of oil.
water, INNaOH, water, 0.1M phosphate buffer (pH 7)
．． The product was washed in the order of 5) to obtain fibrous aminated polyacrylonitrile (wet weight) having a porous structure.

Reference Example 4 Preparation of Support for Immobilized Enzymes Granular polyacrylonitrile copolymer with a porous structure consisting of 55 parts of acrylonitrile, 25 parts of divinylbenzene, and 2 parts of vinylethylbenzene (approximately 100 parts of polyacrylonitrile)
) 2 nights in a 47% nitric acid-sulfuric acid mixture solution.
After nitration for two hours, it was washed with water, 6% hydrosulfite soda/was reduced in KOH solution at 60oo for 2 hours, and the product was thoroughly washed with water. .5 evening (wet weight)
get.

The physicochemical properties of the obtained aminated copolymer granules are as follows. Color: Pale yellow, yellowish color becomes stronger as reduction time increases.

Viscosity: unknown (cannot be measured because it does not dissolve in the following solvents) Solubility in solvents: DMS○, DMF, concentrated nitric acid, 65% KSCN (6000), chloroform, acetonitrile, pyridine, nitromethane, cyclohexaso,
Content of amino groups insoluble in each solvent: diethyl ether and 30% NaOH solution; When the content of amino groups was measured by the hydrochloric acid titration method described in Reference Example 2, the estimated amino group content of the support at each reduction time was as follows: .

Reduction time Amino acid content 1M/1 minute 545 1 hour 738 8 hours 780 Reference example 5 ■ A 500-liter three-bottle flask with a nitrogen introduction tube, a fly frame machine,
A reflux condenser was attached, and the flask was immersed in a hot water bath of about 40 qo and purged with nitrogen for 15 minutes.

Next, 120 ml of distilled water in which sodium alkylsulfonate had been dissolved was added to the flask, and then 70 ml of acrylonitrile without the inhibitor and 10 ml of styrene were added to emulsify. Then, 0.1 ml of potassium persulfate was added to this and polymerized for 20 to 2 hours to obtain an emulsion with a particle size of 0.1 to 1.0 ml, which was then added to 500 ml of water. Pour and stir while adding salt to solidify, and this product is acrylic. The nitrile-styrene copolymer was boiled, washed with water, dried, and then vacuumed to remove unreacted monomers to obtain a copolymer with an intrinsic viscosity of 1.3 in dimethylformamide. was dissolved in 200 ml of dimethylformamide, and added dropwise into a 20% acetone water bath using an atomizer cup. .5 to 1 skin). @ Using the same equipment as in ■ above, two-day Triton 720 (manufactured by Rohm & Hass) and two-day Delgitol (Terg) were used.
Mol) (manufactured by Union-Bido) was dissolved in 300 g of distilled water, 0.2 g of potassium persulfate was added thereto, and then 80 g of acrylonitrile from which the inhibitor had been removed and 20 g of acrylonitrile was added. Add the distilled methyl acrylate in the evening and polymerize by heating to 40 to 50 qo. After about 3 powders, polymerize to a reflux temperature of 90 CO. After the reaction, the suspension is steam distilled for 15 to 2 powders. to remove the unreacted monomer, and then add salt in the same manner as in ①.
After ventilation drying, the intrinsic viscosity in dimethylformamide was 1.
An acrylonitrile-methyl acrylate copolymer having 2 was obtained.

Next, this copolymer 100% was dissolved on dimethyl sulfoxide 100% and added dropwise into a 20% dimethyl sulfoxide water bath using an atomizer cup to form a hydrogel of the copolymer. A granular material (diameter 0.5 to 1 side) having a porous structure was obtained. ■ In place of the styrene in (■) above, divinylbenzene (1 M) was used, and the same procedure as (2) was repeated.
1 Yanagi) was obtained.

Using two portions of each of the granular copolymers having a porous structure obtained in steps (1) and (2) above, reductions were carried out as follows.

That is, 2.59 g of lithium aluminum hydride was added to a three-necked flask, and dried ether was added and evaporated. Two portions of the above copolymer were added thereto, and the mixture was heated under reflux at a constant temperature of 5,000 ℃ for 1 hour. After the reaction, water, IN
Treatment with HCI followed by IN HC1, water, INNaO
The support is washed with H, water, and 0.1 M phosphate buffer (pH 7.5) in this order to obtain a granular support having a porous structure and having amino groups and nitrile groups. Reference Example 6 Add 2.5 grams of lithium aluminum hydride to a three-necked flask, add 100 grams of dry ether, and add 2 grams of granular polyacrylonitrile having a porous structure obtained in Reference Example 1. In addition, the mixture was heated under reflux for 1 hour at 50°C, and after the reaction, water was added dropwise under ice to decompose the unreacted lithium aluminum hydride, and then INHCI was added dropwise to dissolve the decomposed product. was separated into a furnace to recover aminated polyacrylonitrile, and then IN
A granular support having a porous structure is obtained by washing in the order of HC1, water, IN HaOH, water, and 0.1M phosphate buffer (pH 7.5).

Example 1 1 Preparation of penicillin acylase concentrate A medium (pH 7.0) containing 1% polybeptone, 1% meat extract, and 0.5% salt was dispensed into 500 Erlenmeyer flasks and heated at 120°C for 20°C. After sterilization for minutes, Bacillus megaterium B-400 (FERM-pNo. 748)
was inoculated and cultured in a shaking moat for 30 oo for 48 hours. After culturing, the bacterial cells were removed and the resulting furnace solution was adjusted to pH 7.5, and Celite (JohnsN) was added to each furnace solution.
No. manufactured by es. 560) After adding 1 evening, worship the candle for about 30 minutes, and then separate the serite from the hearth.

The resulting Celite had an activity of approximately 2 skins/night. 400 bottles of Celite 80M obtained in this way were suspended in water, and this was packed into a column (diameter 9.5 x 80).
SVO. with 24% ammonium sulfate/0.1M trishydroxymethylaminomethane solution (pH 8.5). A fraction of 210' (30 U/secretion) with an absorption of 28 blood was obtained under conditions of 5. Next, 120 ml of this eluate was added to Biogel-p-10 (50-10
0 mesh) and 0.1M phosphate buffer (pH 7).
．． 5) was charged into a column (3.2 x 41 mm) and passed through a gel furnace to obtain a fraction of 120 mm with an absorption of 28 mm and no salt (21.6 U/ upon),
Further, this solution was filled into a dialysis tube and concentrated to 24' in 30% carbo wax (average molecular weight approximately 20,000)/0.1M phosphate buffer (pH 7.5), and then further 0.1M phosphate buffer. A concentrated solution of penicillin acylase produced by Bacillus megaterium B-400 (23') was obtained by dialysis in a solution (pH 7.5) overnight.
U/').

ii. Preparation of immobilized penicillin acylase. Add the aminated polyacrylonitrile fibers having a polygonal structure obtained in Reference Example 3 to a cooled 12.5% glutaraldehyde/borate buffer (pH 8.5) at 240 °C. ,000,2
Concentrate for 0 minutes, remove from the oven, and add boric acid buffer (pH 8).
．． 5) Wash with 0.1M phosphate buffer (pH 7.5) in this order, and add this to Bacillus megaterium B-400.
In addition to the acylase enzyme solution (60 U/) produced by 113 racks, it was heated at room temperature for 4 hours, further left at 5°C overnight, and then taken out in the furnace (no acylase activity was observed in the furnace solution). ).

Furthermore, this was added to 0.8M salt/0.1M phosphate buffer (p
H7.5), and 0.1M phosphate buffer (pH7.5)
) to obtain immobilized penicillin acylase (4 U/unit) of 6.8 units (wet weight). iii Production of 6-APA The above immobilized enzyme was placed in a jacketed column (diameter 1.6 x 6
(fox) and hold it at 370.

Fifty kites were placed on a magnetic stirrer in a cooling tank for cooling the storage tank at ice level, and 4.455 kg of penicillin G potassium salt was added to the storage tank at 0.02 kg.
In addition to M phosphate buffer (pH 7.5), add 50% of a solution adjusted to pH 8.0 with IN sodium hydroxide aqueous solution, and introduce this solution into the column through a 370 heat exchange jacket at a flow rate of 21%. Run in minutes. The effluent from the column is circulated to a storage tank by a pump, and the pH value is further added to this storage tank.
A meter is installed and the pH fluctuation due to the inflow of the effluent is adjusted to 7.8-8.2 using an aqueous sodium hydroxide solution. The circulation of this solution was continued for 3 hours, collecting about 10 ml of solution in the storage tank and column washing solution, and adding 3 ml of acetone to this solution.
Add 0' and adjust to 4.3 with isohydrochloric acid. Leave at 5°C overnight, remove the resulting precipitate, wash with acetone,
Dry to obtain 6-APA2.16 (purity 96%).

Furthermore, as a result of repeating the above operation 10 times, the obtained 6
No decrease in the yield of APA was observed.

Example 2 9 (wet weight) of the support 90 obtained in Reference Example 4 was 12.
5% glutaraldehydonoborate buffer (pH 8.5)
The solution was incubated for 0.000,000 minutes, and then washed with 0.1M phosphate buffer (pH 7.5). [) In addition to 3', 30q0, lit for 60 minutes, removed from the furnace, and further added 0.9M salt/0.1M
Phosphate buffer (pH 7.5), 0.1M phosphate buffer (
A fixing material (117 U/unit) in which the granules and acylase were bound was obtained.

Example 3 The granular support having a porous structure obtained in Reference Examples 5 and 6 (wet weight) was cooled with ice to 12.5
% glutaraldehyde/boric acid buffer (pH 8.5) 5
In addition to 0 secretion, 000 was incubated for 2 minutes, then taken out in the oven, and washed with 0.1M phosphate buffer (7.5).
Bacillus megaterium B was placed in an L-shaped test tube.
-400 produced acylase enzyme solution (72U/【)
3 skin [Add to the inside, heat for 60 minutes at 30 qo, then remove from the oven, and add 0.9M salt/0.1M phosphate buffer (pH
7.5) and washed with 0.1M phosphate buffer (pH 7.5) to obtain immobilized penicillin acylase.

As a result, when the copolymer obtained in Reference Example 5■ was used, the acylase activity in the support was 162U.
The acylase activity in the support when using the copolymer obtained in / evening and @ was 216 U/night, the acylase activity of the support when using the copolymer obtained in ■ was 192 U/night, and in Reference Example 6. The acylase activity in the obtained granular support having a porous structure was 252 U/night. Example 4 Granular support 1.5 having a porous structure obtained in Reference Example 6
Soak the water in 25 parts of acetone, add 1 part of triethylamine, and dropwise add 1 part of succinic anhydride in acetone solution over about 5 minutes. It was taken out of the oven, washed with water, washed with INHCI, washed with water, and dried.

One portion of this reaction product was then added to a 50' Erlenmeyer flask fitted with a calcium chloride tube, and 3' of dioxane,
Furthermore, 1 night of N-hydroxysuccinimide was added, cooled on ice, and 2 parts of dioxane in which 1 night of N,N'-dicyclohexylcarbodimide was dissolved was added dropwise over about 1 minute. After the reaction, it was taken out in a furnace and washed by decantation using 2 volumes of dioxane to remove insoluble matter, further washed with dioxane, dried under reduced pressure, and then The reaction product 9 of 30 containing this active ester was sufficiently swollen with 0.1M phosphate buffer (pH 7.5), taken in a furnace, and then added to an acylase enzyme solution (72U) produced by Bacillus megaterium B-400. /I) 5, stirred for 4 hours at pH 7.5 in an ice bath, removed from the furnace after reaction, and 0.1M
After washing with phosphate buffer (pH 7.5), unreacted active ester groups were removed by soaking in IM glycine/0.1M phosphate buffer (pH 7.5) at room temperature for 60 minutes. This was then separated into a furnace, and the solid phase was mixed with 0.9 of common salt/0.1
Wash with M phosphate buffer (pH 7.5) and further add 0.1
840 females (green weight) of immobilized penicillin acylase (178 U/) were obtained by washing with M phosphate buffer (pH 7.5). Example 51 Preparation of penicillin acylase concentrate 1% polybebutone, 1% meat extract, A medium containing 0.5% salt (pH 7.0) was placed in 30-capacity jar fermenters, and after steam sterilization at 12,000 ℃ for 2 minutes,
200 seed cultures of Bacillus megaterium B-400, which had been cultured in advance at 30°C for two lifetimes, were aseptically transplanted into a medium with the same composition as above, and the aeration rate was 2/min.
01, 30 while crossing the ocean at 300 revolutions per minute and ventilating.
Incubate for 7 hours at ℃.

After culturing, 5 bottles of culture were collected and the bacterial cells were removed using a Westfaria centrifuge, and Celite (Jo) was added to the culture solution.
No. manufactured by hnsManvmeSales. 560)90
After worshiping the light for about 30 minutes including 0 evenings, it is lit at the hearth. Approximately 1.8 kg of the obtained Celite (having a penicillin acylase activity of approximately 20 U/day) was suspended in water, packed into a column (6.0 x 67 mm in diameter), and mixed with 24% ammonium sulfate/0. .SVO. with 1M trishydroxymethylaminomethane solution (pH 8.5). 5, yielding a fraction 390 [(78 U/') with an absorption of 28 hA. Next, the eluate 760' obtained in the same manner as the above treatment method was injected into biogel (Bio bag 1) P-10 (5
0 to 100 mesh), charged to a column (light 5.5 x 8.7 arc) isotonic with 0.1 M phosphate buffer (pH 7.5), and filtered through a gel furnace. Enzyme solution 890' (55U/
') get.

Next, the enzyme solution 2. obtained in the same manner as the above treatment method.
Fill the dialysis tube with 30% carbowax (
average molecular weight approximately 20,000)/0.1M phosphate buffer (pH 7.
5) After concentrating to about 2 times in 20 mm, the mixture is further dialyzed overnight in 0.1 M phosphate buffer (pH 7.5) to obtain 1.1 mm of penicillin acylase concentrate (120 U/ship).

ii Preparation of immobilized penicillin acylase The fibrous polyacrylonitrile having a porous structure obtained in Reference Example 3 was dissolved in 12.5% glutaraldehyde/boric acid buffer (pH 8.5) at 200 ml. Add and incubate at 0°C for 20 minutes.

After the reaction, the support was removed from the furnace and diluted with boric acid buffer (pH 8.5).
After washing with 0.1M phosphate buffer (pH 7.5), add the penicillin acylase concentrate obtained in step i above to 240 pieces of kite and incubate at room temperature for 4 hours. After standing at 5°C overnight, it was taken out of the oven and diluted with 0.9M NaCl/0.1M phosphate buffer (pH
7.5) and 0.1M phosphate buffer (pH 7.5) to form a fibrous immobilized penicillin acylase 11.2.
Wet weight (865 U/unit) is obtained.

6. Preparation of APA 7.4 μm (wet weight) of the immobilized penicillin acylase obtained in step ii above was packed into a jacketed column (diameter 2.6 x 2.7 cm) kept at 25 q0, and penicillin G was added to it. Potassium salt was dissolved in 0.02 M phosphate buffer (pH 7.5) and a solution of 150 ml, adjusted to pH 9.0, was passed through the column at a flow rate of 2.6 m/hr, and the column effluent was stored in a cooled container. P in a tank with sodium hydroxide aqueous solution.
Adjust H to 8.7-9.3 and recirculate to the column through a heat exchanger.

This operation was continued for 3 hours, and when the pH drop due to column outflow was hardly observed, the circulating fluid and the column washing solution 180 were collected, 90% of acetone was added thereto, and the pH was raised to 4 with cooled hydrochloric acid. Step 3: Leave it in the refrigerator overnight. The separated precipitate was collected in a furnace and washed with acetone.
Dry to obtain 6-APA 5.3 days (yield 91%, purity 98%)
, penicillin GO. 5% or less and phenylacetic acid (0.1% or less). When the above operation was carried out in 80 batches, the titer of the immobilized enzyme in the column decreased to about 50% of the initial value, but no decrease in the yield or purity of 6-APA was observed.

Example 6 PcG fermentation liquid 41 [(4000 mountains/secretion) furnace liquid 6 songs' (2000 skin/mine) by drum filter
was extracted at pH 2.0 using butyl acetate 22g, and Pc
Butyl acetate extract of G 21.7 women (5740 blood/secretion)
get.

Add 12.5% of 0.1M phosphate buffer (pH 6.5) to this, set the bait to 6.5% with 10% KoH, drop PcG into sodium bicarbonate, and add 6 (W/V)% PcG/0. .1M phosphate buffer 12.6 yields (9600 skin/me). 10% of this
The pH was adjusted to 7.5 with KoH and used for the following reaction.

Immobilized penicillin acylase (913 U/night) obtained in the same manner as described in Example 5 on day i (wet weight) was
Jacketed column (diameter 2.6 x 2.
7 skin) Fill it with the penicillin G solution 15 obtained above.
The column effluent was adjusted to pH 8.7-9.3 with a small aqueous NaOH solution in a cooled storage tank.
and recirculated to the column through a heat exchanger.

This operation is continued for 3 hours, and when the pH decrease due to passage through the column is almost no longer observed, 180 g of the circulating fluid and column washing solution are collected. This is extracted twice at pH 2.0 using 50% methylisobuketone to remove phenylacetic acid. Then, return the pH to 7.0 and add acetone 9.
After cooling, adjust the pH to 4.3 with hydrochloric acid and leave in the refrigerator overnight. The deposited precipitate is collected in a furnace, washed with cold acetone, and dried to obtain 6-APA5.0 (yield: 86%, purity: 96%). Example 7 2% corn stave liquor, 0.2% phenylacetic acid
Medium 160Z (pH 7.0 with potassium hydroxide aqueous solution)
0) into the fermentation tank, 120q
After steam sterilizing at O for 30 minutes, add 400% of the seed culture of Escherichia coli ATCCIII05, which had been previously cultured for 1 hour at 30QO in a medium with the same composition as above.
The cells were transplanted aseptically and cultured at an aeration rate of 150 revolutions per minute, an aeration rate of 150 revolutions per minute, and an aeration rate of 30 revolutions per minute for one hour.

The resulting culture is collected using a Westflier centrifuge. The obtained bacterial cells (3.5k9 (wet weight)) were transferred to E. Lage
Methods such as r16f [Methods mEm mol
Purified penicillin acylase solution (312 U/) 380
get me The fibrous aminated polyacrylonitrile having a porous structure obtained in Reference Example 3 was added to 200ml of 12.5% glutaraldehyde/borate buffer (pH 8.5) and incubated at 0°C for 20 minutes. .

After the reaction, the support was removed from the furnace and added to a boric acid buffer (pH 8.5).
After washing with 0.1M phosphate buffer (pH 7.5), add to a solution prepared by adding 140' of 0.1M phosphate buffer (FH7.5) to the penicillin acylase concentrate loo. Let stand at room temperature for 4 hours. After leaving it at 5°C overnight, it was taken out of the oven and diluted with 0.9M NaC/0.1M phosphate buffer (
pH 7.5) and 0.1M phosphate buffer (pH 7.5) to form fibrous immobilized penicillin acylase 11.
．． 5 units (wet weight) (780 U/) were obtained. As a result of producing 6-APA according to the method described in Example 5 using the above immobilized penicillin acylase, 5.2 units of 6-APA (yield 89.3%, purity of 98%).

Claims (1)

[Scope of Claims] 1. A method for enzymatically producing 6-aminopenicillanic acid by allowing immobilized penicillin acylase to act on penicillin, characterized by immobilizing penicillin acylase on a water-insoluble aminated acrylonitrile polymer. A method for producing 6-aminopenicillanic acid. 2. The manufacturing method according to claim 1, wherein the penicillin is natural penicillin or a water-soluble salt thereof. 3 Natural penicillin is Penicillin G or Penicillin V
The manufacturing method according to claim 2. 4. The manufacturing method according to claim 2, wherein the water-soluble salt is a sodium salt or a potassium salt. 5. The method according to claim 4, wherein the water-soluble salt is an aqueous solution having a concentration in the range of 3 to 12 W/V%. 6. The production method according to claim 1, wherein the penicillin acylase is an enzyme preparation collected from cultured cells or culture filtrate of penicillin acylase-producing bacteria. 7 The penicillin acylase-producing bacteria are of the genus Actinoplanes, Arthrobacter, Bacillus, Erwinia, Etzierichia, Flavobacterium, Micrococcus, Micromonospora, Nocardia, Proteus, Pseudomonas, Serratia, or Streptococcus. The manufacturing method according to claim 6, which is a microorganism belonging to the genus Mycetes. 8 The microorganism is Actinoplanes utahensis ATCC
14539, Arthrobacter tumesetsense ATC
C6947, Arthrobacter viscosus ATCC1
5294, Bacillus megaterium ATCC14945
, Bacillus megaterium ATCC 14946, Bacillus megaterium B-400FERM-P748, Erwinia stewarti ATCC 13531, E. coli ATCC 9637, E. coli ATCC
CC11105, E. coli ATCC135
29, E. coli NCIB 8743, E. coli NCIB 8743A, Flavobacterium suaveolens ATCC 13533, Micrococcus candidus ATCC 8456, Micrococcus roseus ATCC 416, Micromonospora perbreochromogenes ATCC 13634, Nocardia
Species ATCC13635, Proteus letgeri ATCC9250, Proteus letgeri ATCC3
1052, Pseudomonas aeruginosa NRRL10
14J, Pseudomonas fluorescens NRRLB
10, Pseudomonas maltophila ATCC1780
8, Serratia rubidaea ATCC181, Streptomyces griseus IFO3355, Streptomyces
The method according to claim 7, which is Streptomyces lavengillae ATCC 13664, Streptomyces lavengillae ATCC 13665, or Streptomyces lavengillae ATCC 21138. 9. The production method according to claim 1, wherein the water-insoluble aminated acrylonitrile polymer is a fibrous, granular, membrane-like or hollow fiber support having a porous structure. 10. The method according to claim 9, wherein the support is an acrylonitrile polymer having free amino groups ranging from 20 μM per gram of support to a point where the support becomes substantially insoluble in water. 11. The method according to claim 10, wherein the acrylonitrile-based polymer is a homopolymer or copolymer of an acrylonitrile-based monomer, or a copolymer of this and another comonomer containing an ethylenic double bond. 12. The method according to claim 11, wherein the acrylonitrile monomer is acrylonitrile, methacrylonitrile, α-chloroacrylonitrile, or cinnamitrile. 13. Patent claims in which the comonomer is a styrene monomer, an acrylic ester monomer, a conjugated diene, a halogenated olefin, a vinyl ether monomer, a vinyl ketone monomer, a vinyl ester monomer, an amide vinyl monomer, a basic monomer, or a polyfunctional monomer. The manufacturing method according to item 12. 14 Styrenic monomers include styrene, methylstyrene, ethylstyrene, nitrostyrene, chlorostyrene,
14. The method according to claim 13, wherein bromostyrene or chloromethylstyrene is used. 15 A patent claim in which the acrylic acid ester monomer is methyl (meth)acrylate, ethyl (meth)acrylate, butyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, or polyethylene glycol (meth)acrylate The manufacturing method according to scope item 13. 16. The production method according to claim 13, wherein the conjugated diene is butadiene or isoprene. 17. The production method according to claim 13, wherein the halogenated olefin is vinyl chloride or vinylidene chloride. 18. The production method according to claim 13, wherein the vinyl ether monomer is ethyl vinyl ether or butyl vinyl ether. 19. The production method according to claim 13, wherein the vinyl ketone monomer is methyl vinyl ketone or ethyl isopropenyl ketone. 20. The production method according to claim 13, wherein the vinyl ester monomer is vinyl acetate or vinyl benzoate. 21 The amide vinyl monomer is (meth)acrylamide, N,N-dimethyl(meth)acrylamide, N-vinylpyrrolidone, N-vinylsuccinimide, or N-
The manufacturing method according to claim 13, which is vinyl phthalimide. 22. The method according to claim 13, wherein the basic monomer is vinylpyridine, methylvinylpyridine, vinylimidazole or N,N-diethylaminoethyl (meth)acrylate. 23. The method according to claim 13, wherein the polyfunctional monomer is divinylbenzene, divinyltoluene, or divinylethylbenzene. 24. The manufacturing method according to claim 10, wherein the acrylonitrile polymer has a porous structure. 25. The production method according to claim 24, wherein the acrylonitrile polymer is a fibrous, granular or film-like molded product. 26. The manufacturing method according to claim 10, wherein the acrylonitrile polymer has a porous structure and is a fiber, granular or film-like molded product of polyacrylonitrile containing 90% or more of acrylonitrile. 27. The acrylonitrile-based polymer has a porous structure and is a fibrous, granular or film-like molded product of a polyacrylonitrile copolymer consisting of 55% acrylonitrile, 25 parts divinylbenzene and 20 parts vinylethylbenzene. Manufacturing method. 28 Amination is a reaction of reducing a nitrile group from an acrylonitrile polymer to an amino group, a substitution reaction of an acrylonitrile polymer having a halogen atomic group to an amino group of the halogen atomic group, and a reaction of reducing the nitrile group from an acrylonitrile polymer having a nitro group to an amino group. Claim 1, which is carried out by a reduction reaction of a group to an amino group, a Hofmann decomposition reaction of the amide group from an acrylonitrile polymer having an amide group, or a reaction of the epoxy group with a diamine from an acrylonitrile polymer having an epoxy group. Manufacturing method described. 29 Claim 28, in which the reduction reaction of a nitrile group to an amino group is carried out using lithium aluminum hydride.
Manufacturing method described in section. 30. The production method according to claim 29, wherein the reduction reaction is carried out in an inert non-solvent of the acrylonitrile polymer. 31. The method according to claim 30, wherein the non-solvent is diethyl ether, dioxane or tetrahydrofuran.