JPS5982097A - Improved method for preparation of 6-aminopenicillanic acid using immobilized enzyme process - Google Patents

Abstract

PURPOSE:To prepare 6-aminopenicillanic acid without lowering the enzymatic activity, by treating crude aqueous solution of benzyl-penicillin with an ultrafiltration membrane having a cut-off molecular weight of >=5,000, and treating the product with immobilized penicillin acylase. CONSTITUTION:An aqueous extract of benzyl penicillin obtained by the extraction of benzyl penicillin fermentation broth, or a solution obtained by dissolving crude benzyl penicillin or its water-soluble salt separated from the benzyl penicillin fermentation broth in an aqueous medium, is adjusted to the optimum pH of penicillin acylase or thereabout, the aqueous extract of aqueous solution is subjected to the ultrafiltration treatment with a cut-off molecular weight of >=5,000, and the resultant treated liquid is made to react with an immobilized penicillin acylase.

Description

[Detailed description of the invention] 6-aminobeniclanic acid (6-APA
).

Conventionally, various chemical deacylation methods and butyric deacylation methods have been reported as methods for producing 6-APA entirely from PcG. As a chemical deacylation method,
A method of dehydrating, drying, protecting with all ester groups of iPeG lupenicillin, converting it to iminochloride, further converting it to iminoether, and then hydrolyzing is currently used.
In addition to the complexity of the process, since 6-APA is an intermediate for manufacturing pharmaceuticals, there are problems in terms of contamination with impurities.

On the other hand, in the enzymatic deacylation method, a method using immobilized penicillin acylase, in which penicillin acylase is recently immobilized, allows continuous use of penicillin acylase and continuous enzymatic reaction, making it possible to industrialize in terms of manufacturing costs. However, efforts have been made to prevent deactivation of the enzyme activity of immobilized penicillin acylase and to reduce manufacturing costs by increasing the number of continuous uses.

By the way, PcG, which is another major factor in the success or failure of reducing the production cost of 6-APA, is usually isolated and purified from the PcG fermentation process with sodium salt or potassium salt in the form of commercially available industrial raw material bulk. However, the steps of extracting PaG from the fermentation process, repeating melting, salt formation, isolation, purification, and drying are significant factors in the manufacturing cost of PcG. In the enzymatic deacylation method, the above industrial raw material bulk is reconstituted into an aqueous solution and used.

Giaeobbe et.
6-APA by the immobilized enzyme method, with the overall purpose of reducing the production cost by omitting the steps of salt formation, isolation, purification, and drying to strip PaG from glue fermentation broth. Manufacturing has been attempted ( Br + z voice e
Engineering, Vol. 4, 245-2.
52, Planllum Press.

1978).

With this Giaeobbe method, the manufacturing cost of 6-APA is lower than the conventional method using commercially available industrial bulk sound.
However, the aqueous extract of PcG obtained from the fermentation process and substantially pure PeG f were used alternately, and the method did not use only the aqueous extract obtained from the fermentation process.

Moreover, the enzymatic activity of the immobilized penicillin acylase used [7] is significantly lower than that of the conventional immobilized enzyme method for producing 6-APA, which has the disadvantage that the number of continuous uses of the immobilized enzyme is reduced. Currently, it is not possible to industrially produce 6 APA by an immobilized enzyme method using only an aqueous extract of PeG obtained from a fermentation process.

Therefore, the present inventors continued various studies in order to solve all of the above-mentioned drawbacks, and as a result, we obtained crude PeG by dissolving the extract obtained by extracting PcG fermentation broth with an organic solvent into an acidic aqueous solution.
Obtained by ultrafiltrating an aqueous solution with a molecular weight of 5000 or more;
n, 7 (When 6-APA'ifi is produced by the action of immobilized penicillin anlase on the treatment solution, it is possible to use it continuously for a long period of time without decreasing the penicillin acrase activity, reducing the production cost of 6-APA. I learned that money contributes greatly to

Furthermore, the present inventors found that the quality of commercially available industrial raw material bulks of sodium or potassium salts of PCG varies significantly depending on the manufacturer, and that some bulks with low purity contain factors that inactivate benicline acrase activity. Knowing the existence of
It has been learned that when 6-APA is manufactured, the activity of immobilized penicillin anlase decreases, resulting in a significant decrease in the number of continuous uses and an increase in the manufacturing cost of 6-APA.

However, if the bulk is made into an aqueous solution, subjected to ultrafiltration in the same manner as described above, and then subjected to an enzyme reaction, it becomes possible to use the bulk continuously for a long period of time without reducing the penicillin acylase activity. 4- I learned that it greatly contributes to reducing manufacturing costs.

The present invention has been completed based on the above findings, and is a method for enzymatically producing 6-APA by reacting PcG with immobilized penicillin acylase in an aqueous medium. A method for producing 6-APA, characterized in that an aqueous solution of a salt having a molecular weight of 5000 or more is subjected to an ultrafiltration treatment, and the resulting treated solution is treated with immobilized beni7line acylase, the method comprising: The first purpose is 1. PcG '17 Used in the production of 6-APA by the immobilized enzyme method in the form of an aqueous extract without being isolated from the PcG fermentation process, and can be used continuously for a long period of time without reducing penicillin acylase activity. ．． The second objective is to provide a production method that can reduce the production cost of 6-APA by the immobilized enzyme method using all commercially available industrial PeG bulk. To provide a manufacturing method that does not reduce penicillin acrase activity by removing factors that inactivate penicillin acrase activity in the bulk, enables continuous use for a long period of time, and thereby reduces the manufacturing cost of 6-APA. It's about doing.

Extraction with an aqueous organic solvent such as ethyl acetate, butyl acetate, methyl in butyl ketone, butanol, etc.
It is used in the form of an aqueous solution obtained by dissolving PcG from the extract into an aqueous medium. Since the aqueous solution will be used to act on the immobilized penicillin acylase later, it should be adjusted in advance to an optimum value for the penicillin acrase.

Further, PcG used in the present invention is used in the form of an aqueous solution obtained by dissolving a commercially available bulk PcG potassium salt or sodium salt for industrial raw material in an aqueous medium. The aqueous solution should be adjusted to the optimum level of penicillin acylase as before. The bulk is usually extracted with an organic solvent and transferred into an aqueous medium several times to form a potassium or sodium salt in the organic solvent solution, and then completely isolated and dried. In some cases, it is a product that is further refined, but from the viewpoint of manufacturing cost, it is not possible to use substantially pure Pc.
G does not need to be a water-soluble salt, but is a crude bulk with poor purity that contains a factor that completely inactivates penicillin acylase activity, for example, a polymeric substance such as a polypeptide or protein that easily binds to the present enzyme. That's fine.

In this way, the optimal voice of penicillin acylase is adjusted to f'
The aqueous solution of the PcG water-soluble salt has a molecular ffi: 500
The sample is passed through an ultrafiltration machine capable of filtering 0 or more polymers, and then subjected to ultrafiltration treatment. The above-mentioned ultrafiltration is carried out by a method using a known ultrafiltration membrane, and at least polymeric substances with a molecular weight of 5,000 or more and 30,000 or less should be removed.

The desired 6-
Manufactured by APA1. Ru.

As the penicillin acylase, penicillin acrase produced by microorganisms, particularly bacteria, is used.

Such penicillin acrase-producing bacteria include, for example, the genus Actipurases, the genus Arthrobacterium, the genus Bacillus, the genus Erwinia, the genus Etscherichia, the genus Flavobacterium, the genus Micrococcus, the genus Micromonospora, the genus Nocardia, and the genus Proteus. genus, Cuydomonas spp.
It is a microorganism belonging to the genus Serratia, Streptomyces, etc. Examples include: Actinoplanes utahensls
ATCC14539Arthrobacter tiv
ne++cens ATCC6947Arthro
hacter viscosug ATCC15
294 Bacillus megaterrun
ATCCI 4945ATCC14946 B-4001RM-P748 Erwinla stewsrtil ATC
C13531Esdherichla call
ATCIC9637ATCC11105 ATCC13529 MCIB8743 MCIB8743A Flavobaeterium suaveoleng
ATCC13533Micrococcus 'ca
ndldug ATCC84568- Micrococcus roseu++
ATCC416Mlcromonompora
purpureoehromogenes ATC
C13634NocardLa sp ATCC
13635Proteus rettIrerl
ATCC9250ATCC31052 Psaudomona@aarugino*a
NRRLIol 4JPssudomonas f
luoregcans NRRLBIOPse
udomonas maltophlla A
TCC17808 Serratia rubidae
a ATCC181S
treptomyces grissug
IFO3355Streptorrryces 1a
vendulae ATCC13664ATCC
13665 ATCC21138, etc., and penicillin acylase-producing bacteria belonging to the genus Bacillus and genus Etschierichia are particularly preferred. The above-mentioned penicillin acylase is produced as an intracellular enzyme or an extracellular enzyme depending on the type of microorganism, and these are collected from a culture by a known method. In the case of an intracellular enzyme, penicillin acrase may be collected from whole cells or used in the cells.

The penicillin acylase is immobilized by a known method for producing an immobilized enzyme. For example, the following method may be mentioned.

Method for enclosing cells with cellulose triacetate [JP-A No. 47-39694, J, Antibiotics
, 2J22B (1973)), method of enclosing cells in polyacrylamide [JP-A-49
No.-118892, Europe J J, Appl
, Microbiol, .

A method of entrapping cells with glycidyl methacrylate, N, d-methylene bisacrylamide and dimethylaminopropionitrile [JP-A-53-69888], a method of entrapping cells with an epoxy polymer ( Bi.

technol, Letters, Sat., 171 (
1979)], a method for covalently bonding an enzyme to DEAE cellulose by an alkylation method [: Biochem,
Biophys, Aeta, 284.

278 (1972)], a method for succinilating an enzyme and adsorbing it with DEAE-Sephadec [JP-A-47-28187], a method for copolymerizing an enzyme with acrylamide, a copolymer of N,N'-methylene-bis-acrylamide and maleic anhydride. A method for covalently bonding an enzyme to Amberlite XAD-7 using glutaraldehyde and hexamethylenediamine [JP-A-49-5]
No. 0183], a method for covalently bonding an enzyme to water-soluble Ficoll (a copolymer of sucrose toepichlorohydrin) by cyanogen bromide activation method [JP-A-49-93588], cyanogen bromide activation of an enzyme Method for covalently bonding with dextran [JP-A-49-12558725587
[Methods in Bn+cyme,
Vol.

44, 759, Academic Prass,
New York, 1976], a method for covalently bonding an enzyme to Amberlite IRC-50 using glutaraldehyde [JP-A No. 50-95479]; Covalent bonding method with copolymer with acrylate [Blot
echnol, Eioeng, , 21 * 13
17 (1979)], a method for covalently linking enzymes to 0M cellulose or Amberlite XAD 7 with glutaraldehyde [Blotechnol, Bio
Eng., 22, 735 (1980)), a method for covalently bonding enzymes to porous polyacrylonitrile partially minified with glutaraldehyde [
JP-A-55-48392, JP-A-55-39712].

The above immobilized penicillin acylase was immobilized with PcG in aqueous medium.
However, if the immobilized enzyme is insoluble in water, it can be carried out by either 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 PcG solution and stirring while adjusting the pH to a constant value. The immobilized enzyme can be recovered from the reaction solution by filtration or centrifugation and used repeatedly. Moreover, this batch method can also be carried out continuously.

12- When using the column method, fill the column with the immobilized enzyme,
The KPcG solution in the column is passed through, and in this case, it is preferable to recirculate the column at a high flow rate. That is, by limiting the deacylation reaction rate per column passage, the
It is possible to suppress the extreme decline in The reaction liquid that has passed through the column is cooled and stored in a storage tank, the pH is constantly neutralized to 6.5 to 95 with an alkali such as alkali hydroxide, and the reaction liquid is passed through a heat exchanger and recirculated to the column. It is effective in preventing deterioration of immobilized enzyme activity and decomposition of beninphosphorus due to pH decrease, and can promote deacylation reaction. In order to minimize pressure loss due to high flow rates, the column is preferably designed to have as shallow a bed as possible, and recirculation is normally carried out at a flow rate of 100 to 300 bed volumes per hour. Recirculation is more than 90 chi of PeG (i −
It is preferable to continue using APA until it becomes stable.

The above enzymatic reaction is usually carried out at a temperature in the range of 15 to 40°C. The optimum pH for the reaction varies depending on the penicillin acylase producing strain, but if it is necessary to maintain the pH at the optimum level, the reaction is usually carried out at around 6 to 9. good. The reaction time varies depending on the enzyme titer, the amount of substrate immersed, the flow rate, etc., but is usually 1 to 10 hours. Therefore, in the case of a column type, it is preferable to pass the reaction within the appropriate reaction time.

The concentration of PcG in the above enzyme reaction is 1 to 20 W/V
Although it is used within the range of
It is preferable to use it within a range of v%.

Next, 6-APA is isolated and purified from the reaction solution, which can be carried out by known methods. For example, after removing PeG and carboxylic acid remaining in the reaction solution by acidic extraction with a non-hydrophilic organic solvent such as methyl in butyl ketone, ethyl acetate, or butyl acetate, the pH is adjusted to 43 and 6-APA is isoelectrically removed. Although point precipitation may be performed, after adding a hydrophilic organic solvent such as acetone or methanol to the reaction solution,
6-APA may be directly isoelectrically precipitated at pI (4,3). In the present invention, since the substrate concentration is extremely high, it is possible to precipitate 6-APA from the reaction solution in high yield and purity without concentrating the reaction solution. 6-APA can be directly crystallized.

The conversion rate to PcG (1') 6-APA can be determined by various methods. For example, by high-performance liquid chromatography, the reaction solution was adjusted to pH 2j), and all unreacted penicillin and the produced carboxylic acid were extracted and removed using methyl in butyl ketone. 6-APA can be quantified by the iodine absorption method and simultaneously acrylated with phenylacetic acid chloride, and the antibacterial activity of the resulting PcG can be determined by bioassay using the paper disc method.

Penicillin acylase titer measurement method 0.1 M phosphate buffer (pH 7.5) 4.5 ml
, 4(W/V)% PcG potassium salt/0. IM phosphate buffer (PH7,5) 0.5 mI! Add the immobilized enzyme whose total weight has been measured in advance to M solution consisting of 3
0.5ml from 0 volume, which is reacted at 7℃ for 30 minutes! '
31 nl of a buffer solution consisting of 1 ml of 0.05 N NaOH and 2 ml of 20 thiacetic acid
%P-dimethylaminobenzylaldehyde/methano 15
- Add to 0.5 ml of 6-APA solution, react at room temperature for 10 minutes, measure absorbance at 415n+nVc, and determine the amount of 6-APA produced.

As for enzyme activity, 1 unit (IU) is defined as the activity of producing 1 μmol of 6-APAi per minute.

Next, the present invention will be specifically explained with reference to participation examples and examples, but these are not intended to limit the present invention.

Reference Example 1 120mJ of EJ diethyl ether was added to 1.5t of lithium aluminum hydride, and the polyacrylonitrile fiber with a porous structure (manufactured by Asahi Kasei Industries, acrylonitrile of 90mm or more, porous pore size of 40 to 4000A) was added to 1.5t of lithium aluminum hydride.
)1.5 f was added thereto, and the mixture was heated under reflux for 1 hour in a 50°C oil bath. After the reaction, the fibers were taken out and washed with ether, then mixed with 1N hydrochloric acid, water, 1N sodium hydroxide solution, water, 0
．． 7 tons of aminated polyacrylonitrile fibers with a porous structure were washed with 1M phosphate buffer (pH 7, 5) in this order.
(wet weight) was obtained.

Reference Example 2 16- Immobilized Penicillin Acylase The aminated polyacrylonitrile fiber having a completely porous structure obtained in Reference Example 1 was cooled and treated with i12.5% glutaraldehyde/borate buffer (pH 8,5), 24078/!
1. Add to the inside and press at 0℃ for 20 minutes. After -1ν of borate buffer (pHl'1.5), 0.1 M
-II Wash with mild phosphoric acid solution (pH 7,5), and
i, '%7, Bacillus megaterium R-400
(FERM P-748) was added to 113 ml of the acylase enzyme solution (60 U/ml), stirred at room temperature for 4 hours, and then left at 5°C overnight. (I didn't admit it).

0.1 M phosphate buffer (pH 7.5) containing 0.5 M common salt, 0.1 M phosphate buffer (pH 7.5)
7, 5) in the order of washing, and immobilized penicillin acylase (
584 U/g) 6.8 f (wet weight) total was obtained.

Example 1 PcG Potassium 474. (PcGK) (Pfizer, Lot & G23248-34008)
Dissolve in 300 ft distilled water and add f'1. Ultrafilter (manufactured by Asahi Kasei Industries, ultrafiltration module ACL)
-1010, hollow fiber membrane, molecular weight cut off 1300Q). Add 300ml of distilled water to this treatment solution.
/3 times, PcGK 281.2# (yield 93.
7%) of ultrafiltrate (1900 ml of ultrafiltrate containing PcG
K148■/me) was obtained.

Example 2 Immobilized penicillin acylase 17.0 obtained in Reference Example 2
? (wet weight) was packed into a column (3.4 x 5 cm), and the ultrafiltrate of PcGK obtained in Example 1 was placed in a storage tank.
8m1! (5ot as PcGK)'r added, O, 12
Add M2M borate buffer pH 8,4) to make a total volume of 500
−. This PcGK aqueous solution was made by Honda at 50t/
The column flow rate flowed into the column from the bottom of the column, and the column effluent was returned to the storage tank for continuous circulation. During this time, the temperature of the aqueous solution in the storage tank was maintained at 34℃ in a constant temperature bath, and
Adjust the pH using an automatic pH controller with N sodium hydroxide aqueous solution.
i 8.4 and high performance liquid chromatography (H
0 residual Pe tracked the amount of remaining PcG by PLC)
When the amount of G becomes 2.0μ/fnl or less, the circulation of the reaction solution is completely stopped, and in order to recover the reaction solution in the column, a 0.4M boric acid buffer (pH 7) for washing is added from the bottom of the column. ，
5) 100 me, and the reaction solution and washing solution in the column were discharged into the storage tank.

Add 340ml of methanol to the reaction solution in the storage tank.
While cooling, the pH was adjusted to 4.3 (/C) with 6N hydrochloric acid and left in the refrigerator overnight. The precipitate was collected, washed with cold methanol 150°C, and dried to obtain 6-APA. Washing started from the start of circulation. Process i1 patch until completion, reaction time until the amount of remaining PaG in the reaction solution becomes 2.Ofl!/d or less, dry weight of 6-APA, its yield, and residual activity rate of immobilized penicillin acylase. I asked for

Next, all the storage tanks are newly set up, the entire PcGK aqueous solution is charged, and the same process as above is continuously repeated to obtain 6-
Manufactured by APA Q. Meanwhile, the reaction time for each batch,
Dry weight of 6-APA, its yield and immobilized beni y
The residual activity rate of IJ acylase was determined.

Their results are shown in Table 1.

19- Table 1 Reference Example 3 In Example 2, pcGK (manufactured by Pfizer, LotAG23) was used instead of the ultrafiltered pcGK aqueous solution.
248-34008 ) 50 P total 0.12 M borate buffer (pH 8,4) at tow/v % concentration in ultrafiltrated aqueous solution 1500+n! ,'i were used to produce 5-APA for each batch according to the process described in Example 2.

The results are shown in Table 2.

20- From the results shown in Table 2, 6-APA' (r) is produced by the immobilized enzyme method using PeGK bulk with poor purity.
The penicillin acylase activity was already 2 in the first l batch.
0% or more, indicating not only a significant decrease in the stability of penicillin acylase activity and a prolonged reaction time with each batch increase, but also the continuous use of immobilized penicillin acylase over a long period of time was not possible. It all shows that.

On the other hand, according to the results shown in Table 1 obtained in Example 2 using an aqueous solution in which P cGK bulk with poor purity was subjected to ultrafiltration in advance, the stability of penicillin acylase activity was extremely good. This indicates that continuous use is possible over a long period of time, and this means that the present invention can produce 6-APA at a lower cost industrially than the production example in Reference Example 3. Example 3 PcG fermentation broth (Toyo Jozo Co., Ltd., Lot A 2096
) from which bacterial cells were removed using a drum filter, 10 tons of cultured tear fluid (containing 30 q/d of PcGK) was adjusted to pH 2.0 with 6N hydrochloric acid, and then extracted three times with butyl acetate It. Combine the Pnar acetate layers and add potassium carbonate-potassium acetate buffer (pl (7.2-7.3) 300
7. ml of pHk potassium acetate aqueous solution three times each time.
After adjusting the pH to 2 to 7.3, PeG was transferred to the aqueous layer. The aqueous layer was concentrated under total reduced pressure until the butyl acetate was distilled off.

900 ml of the purified aqueous extract (2 as PcGK)
(containing 57 g) in an ultrap filter (manufactured by Asahi Kasei Kogyo Co., Ltd., ultrafiltration module ACL-1010, hollow fiber membrane, molecular weight 1
Ultrap filter treatment was carried out through a fractionation of 3,000 or more. This treatment solution requires 300 m7 of distilled water! Add 3 times each, pcG
1,800 ml of ultrafiltrate (134/7 loaves of PcGK was obtained).

Example 4 In Example 2, ultrafiltration of PcGK obtained in Example 3 instead of the PCGK aqueous solution! , 373 ml (PcGK
0.12 M borate buffer (pH 8,
Example 2 was carried out using an aqueous solution prepared by adding 4) to 500%
IC6-AI”A for each batch according to the process described in
All manufactured.

The results are shown in Table 3 and fc.

Table 3-1232 Reference Example 4 In Example 2, the ultra-F: iM L * PcGK
Instead of the aqueous solution, 175 ml of the non-ultrafiltrated aqueous extract obtained in Example 3 (509 as PcGK) was added with 0.
Using an aqueous solution made up to 500ml by adding 12M borate buffer (pH 8,4), according to the steps described in Example 2,
The entire 5-APA was prepared for each batch. The result is the fourth
It was as shown in the table.

Table 4 From the results in Table 4, in the conventional immobilized enzyme method of Reference Example 4, when the aqueous extract obtained from the fermentation broth was used as PcGK, the residual rate of enzyme activity decreased significantly, and each This method cannot be called an industrial method because the reaction time in batches is significantly prolonged, and the immobilized penicillin acrase cannot be used continuously for a long period of time.

In contrast, in Example 4, according to the results in Table 3 obtained using the aqueous extract obtained from P(! It is relatively stable even when used continuously for a period of time, indicating that it can be used continuously for a long period of time.It is possible to produce 6-APA industrially compared to the production example in Example 4. It means.

Conventionally, PcGK was extracted from an aqueous extract obtained from fermentation broth.
Considering that the isolation yield is about 85 cm at most, the present invention does not require this isolation step, which leads to at least a reduction in the manufacturing cost of this step. It can be said that this is a sufficiently in-house method for producing 6-APA by the immobilized enzyme method, which is used in the form of an aqueous extract obtained from n-APA.

patent applicant Toyo Brewery Co., Ltd. A folding screen

Claims (1)

[Scope of Claims] l) A method for producing 6-aminopenicillanic acid tl-in an oxygenated manner by reacting benzylpenicillin with immobilized penicillin acylase in an aqueous medium, in which an aqueous solution of crude benzylpenicillin or a water-soluble salt thereof is 1. A method for producing 6-aminobeniclanic acid, which comprises performing an ultrafiltration treatment with a molecular weight of 5000 or more and allowing immobilized penicillin acylase to act on the resulting purified treated solution. 2) The production method according to claim 1, wherein the aqueous solution is an aqueous extract of benzylpenicillin obtained from a benzylpenicillin fermentation process by an extraction step. 3) The production method according to claim 2, wherein the aqueous extract is a bath solution whose pH is adjusted to or around the optimum pH for penicillin acylase. 4) The method according to claim 1, wherein the aqueous solution is a solution of crude benzylpenicillin or a water-soluble salt thereof isolated from a benzylpenicillin fermentation process in an entirely aqueous medium. 5. The production method according to claim 4, which is a solution adjusted to have a KpH at or near the optimum pH of 51 aaiz penicinyl acrase.