CS259996B1 - Method of 6-aminopenicillane acid's enzyme production from phenoxymethylpenicilline by means of stabilized cells - Google Patents

Abstract

The present invention relates to a process for the preparation of 6-aminopenioil acid (6-APK) from phenoxymethyl piperidine (V-penloillin) stabilized yeast cells that are labeled by enzymatic hydrolysis being carried out by suspension of yeasts containing V-peniollin amidase, particularly a yeast cell suspension Cryptooooous sp. CCY 17-22-1; stabilized agents such as dicarboxylic acid dialdehydes acids, preferably glutaraldehyde, and pre-washed with inorganic solutions salts of strong acids and / or bases, preferably ammonium sulphate, and using solution of phenoxymethylpenicillin in concentration From 1 to 10%, preferably from 4 to 7% by weight / volume, at pH 7.0 and 8.5, preferably 8.0. at temperature 10 to 45 ° C, preferably 37 ° C, in stirred reactor and after hydrolysis and cooling the reaction mixture at 0-5 ° C the suspension stabilized and washed yeast is separated and used for hydrolysis fresh phenoxymethylpenicillin solution batch and from the supernatant, optionally from the parent the caustic is recovered after adjusting the pH and temperature 6-aminopenicoliic acid, optionally regenerates phenoxytoic acid, wherein is enzymatic hydrolysis of phenoxymethylpenicillin and isolation of 6-aminophenioilic acid, optionally regenerating phenoxytoic acid at least twice.

Description

The invention relates to the enzymatic production of 6-aminopenicillanic acid from phenoxymethylpenicillin by stabilized yeast cells.

6-Aminopenicillanic acid (hereinafter 6-APK) serves as an intermediate of pharmaceutical production for the preparation of so-called semi-synthetic penicillins, in particular ampicillin. 6-APK can be prepared either chemically or enzymatically. However, the enzyme preparation of 6-APK is significantly more economically advantageous. Many pharmaceutical companies use natural penicillins, G-penicillin (benzylpenicillin) or V-penicillin (phenoxymethylpenicillin) using G- or V-penicillin amidases (penicillin acylases), which are to some extent substrate specific , depending on the side chain type of natural penicillins (N-phenylacetyl and N-phenoxyacetyl-6-APK, respectively). The source of these enzymes is usually microbial cells, especially bacteria, imperfect fungi, (fungi) and some higher fungi (basidiomycetes).

Most pharmaceutical companies use primarily benzylpenicillin as a substrate for the enzymatic production of 6-APK using either native (intact) in most cases bacterial cells of the genus Escherichia or Proteus, or a soluble, more preferably immobilized enzyme, previously isolated from producing, highly active bacterial strains.

The Czechoslovak pharmaceutical industry uses the so-called semi-continuous method of enzymatic production of 6-APK from benzylpenicillin based on chemically stabilized individual cells of the bacteria Escherichia coli according to MS. Certificate No. 201 621.

The cited method consists in separating the bacterial cells of Ei coli after their cultivation in a fermentation tank on thickening centrifuges, the concentrated aqueous suspension of the bacterial cells is stabilized with glutaraldehyde, the suspension is washed with water and the stabilized cells are permeabilized in sodium chloride in the presence of the amide, followed by washing with aqueous 2599.96

-2Suspension containing individually cross-linked and permeabilized cells is contacted with the potassium or sodium salt of benzylpenicillin (G-penicillin) in a stirred reactor and after the enzymatic hydrolysis is complete, the stabilized cell suspension is separated by centrifugation on thickening centrifuges and 6-APK is recovered from the supernatant. acid. The separated, concentrated aqueous suspension of stabilized bacterial cells, containing part of the reaction mixture from the previous cycle of use, is pumped back into the reactor, diluted with water, another portion of a new G-penicillin feed is added, and another enzyme hydrolysis cycle is started.

However, the use of this suspension biocatalyst is limited only to the production of 6-APK from benzylpenicillin (G-penicillin). However, the second type of natural penicillin, phenoxymethylpenicillin (V-penicillin), also produced in Czechoslovakia, cannot be used as a raw material for the production of 6-APK, since the substrate specificity of E. coli penicillin amidase is more than an order of magnitude shifted to benzylpenicillin hydrolysis.

Phenoxymethylpenicillin (V-penicillin) has a number of advantages over benzylpenicillin (G-penicillin); in particular the higher stability of the 3-lactam ring of this natural antibiotic in aqueous solutions depending on pH and temperature. For these reasons, V-penicillin can be administered orally in human and veterinary medicine, as opposed to exclusive perenteral re-administration of G-penicillin. This results in a further advantage for the production of 6-APK, which is a significantly lower presence of degradation products of V-penicillin, in particular phenoxymethylpenicillanic acid, resulting in better quality of 6-APK and higher recovery yield. Another advantage of producing 6-APK from V-penicillin is the fact that the second enzyme hydrolysis product, phenoxyacetic acid, has a significantly lower odor compared to phenylacetic acid, which is not negligible in social and laborious production of 6-APK factor.

In MS. No. 240 247 and No. 250 495 discloses a method for selecting microorganisms, particularly yeast, and a high-production mutant strain of Cryptococcus sp. CCY 17-22-1, including a method for its submersible fed-batch cultivation in a fermentation tank with high V-penicillinamidase activity that only hydrolyzes V-penicillin and p-hydroxy-V-penicillin, whereas G-penicillin or ampicillin is not hydrolyzed at all. The existence of this strain and a method for producing the enzyme active biomass of this 259996

-3-yeast strain and evidence of non-pathogenicity of this strain, based on hygienic-epidemiological expertise, allows us to propose a solution of enzyme production of 6-APK from V-penicillin using stabilized cells.

The method for the enzymatic production of 6-aminopenicillanic acid from phenoxymethylpenicillin stabilized yeast cells according to the present invention is characterized in that the enzymatic hydrolysis is carried out by a suspension of yeast containing the enzyme V-penicillin amidase, in particular by a suspension of yeast cells Cryptococcus sp. CCY 17-22-1, pre-chemically stabilized by the action of a bifunctional agent _t such as dicarboxylic acid dialdehydes, preferably glutaraldehyde, and pre-washed solutions of inorganic salts of strong acids and / or bases, preferably ammonium sulfate, and using a solution of phenoxymethylpenicillin at 1 0 to 10% w / v, preferably 4 to 7% w / v, at pH 7.0 to 8.5, preferably 8.0 at a temperature of 10 to 45 ° C, preferably 37 ° C in a stirred After the hydrolysis and cooling of the reaction mixture to 0-5 ° C, the suspension of the stabilized and washed yeast is separated and used to hydrolyze a fresh batch of phenoxymethylpenicillin solution and 6-aminopenicillanic acid is isolated from the supernatant or the mother liquor after adjusting the pH and temperature. , optionally regenerating phenoxyacetic acid, wherein enzymatic hydrolysis of phenoxymethylpenicillin and isolation of 6-aminopenicillanic acid, optionally generation of phenoxyacetic acid at least twice.

An acidic solution of phenoxymethylpenicillin obtained from the treatment of fermented broth after cultivation, or of intermediates obtained from the various phases of isolation and purification of phenoxymethylpenicillin, can be used in the production process.

The stabilization of the aqueous yeast suspension and washing thereof according to the present invention is carried out with stirring at temperatures of 10 to 40 ° C in the glutaraldehyde concentration range of 0.5 to 10% v / v, preferably 2% v / v, at a cell concentration of 1 to 15% The dry yeast suspension is diluted with water, the cells are separated, resuspended in water and treated with the addition of solid sulfate under stirring. ammonium in the range of its concentrations in the aqueous suspension to 0.1 _(5.0 mol. ¹ l, preferably 2.0 mol. ¹ liter, for 1 to 10 ho259996

For 4 hours, preferably 2 hours, the suspension is diluted with water, the yeast is separated and washed at least twice with water.

In addition to the technical forms of phenoxymethylpenicillin, solutions of pure phenoxymethylpenicillin in the form of an ammonium, potassium or sodium salt can be used in the process of the present invention.

The storage, storage and transport of the stabilized washed and washed yeast prior to use is carried out by either storing or suspending the aqueous suspension of cells in a refrigerated container at temperatures ranging from 1 to 30 ° C, preferably 5 to 15 ° C. the concentrated suspension is briefly treated with heavily supercooled acetone or ethanol in a ratio of 1 volume of cell suspension to 2-5 volumes of acetone or ethanol under stirring, then the cells are separated by centrifugation or filtration, the wet cake of stabilized cells is homogenized and dried at 10 to 45 ° C, preferably 30 ° C, the dry mass of the stabilized cells is mechanically homogenized and stored in well sealed containers at temperatures of 1 to 45 ° C, preferably 15 to 20 ° C before use.

Said solution of the technology of enzymatic production of 6-aminopenicillanic acid from phenoxymethylpenicillin by stabilized yeast cells has, in comparison with the known and hitherto used process of enzymatic production of this acid from benzylpenicillin by stabilized bacteria, described in US Pat. Certificate No. 201 621, the following advantages:

a) it is based on a much more stable raw material of a natural antibiotic,

b) allows to increase the quality of 6-APK, cj allows to increase the yield of 6-APK,

d) enables long-term storage, storage and transport of stabilized cells in dry form,

e) eliminates the risk of infestation of cultures with bacteriophage;

f) allows to reduce the time lag between cycles in a semi-continuous way, because yeast has better sedimentation properties than bacteria,

g) reduce unpleasant social, labor and social problems associated with 6-APK production,

h) allows for a flexible transition to alternative enzyme technology options for the production of semi-synthetic 3-lactam series antibiotics.

A certain disadvantage of the present technology in comparison with the process of production of 6-APK from benzylpenicillin, according to MS. No. 201 621, is the production phase of yeast enzyme active biomass, 259996 ί,

-5podle čs. No. 250,495, resulting in a lower yeast growth rate than bacteria, resulting in more than twice the culture time, thereby increasing the risk of secondary contamination under operating conditions. Another disadvantage is in MS. availability of phenylpropionic acid, which serves as the only source of carbon and energy at a particular stage in the yeast growth of the production strain.

The dry biocatalyst of stabilized yeast consists of a beige to brown amorphous powder containing individual dry yeast cells and artificial aggregates ”resulting from drying and incomplete homogenization in the form of irregular lumps. The dry matter content ranges from 80 to 95%. During multiple reuse, lumps and artificial aggregates disintegrate into individual cells. The sedimentation rate of the material after its swelling in water does not significantly differ from intact cells. The coarser fraction, which is about 20% of the dry mass in the course of the swelling, settles very rapidly after swelling. The 10% suspension of the swollen biocatalyst of stabilized cells results in quantitative sedimentation by centrifugation at a relative centrifugal force of 5000 g for 10 to 15 minutes. A dry biocatalyst based on stabilized yeast cells can be sieved dry. After swelling in water (24 hours) it is not possible to filter, just centrifuge. A well swollen biocatalyst after centrifugation occupies more than twice the volume of its dry form. The specific enzyme activity of the biocatalyst ranges from 60 to 140 U. g * dry matter. The biocatalyst is stable in the dry state, i.e. its specific enzyme activity does not change for at least 1 year when stored in tightly closed containers, for example sealed in polyethylene bags, at temperatures ranging from 1 to 20 ° C.

One unit (U) of the enzyme activity of V-penicillin amidase is the amount of enzyme that catalyses the formation of one yumol

6-APK from a solution of V-penicillin K-salt per minute, at * C and pH 8.0 in a 0.05 M phosphate buffer medium in the region of zero-order enzyme reaction kinetics. Dimension of activity, umol -1-1

6-APK. min. ml. The specific activity is expressed as U. g ^-1 dry cell mass.

The spectrophotometric determination of 6-APK by its reaction with p-dimethylaminobenzaldehyde according to the art. No. 116,959 _e ,

The degree of theoretical conversion of the substrate to the product was monitored both by spectrophotometry and by high pressure liquid chromatography (HPLC). Measurement conditions: column Separon SGX _NH2 7 microns x 250 mm, mobile phase: CH? CN: H ₂ O (0.006 M NaH ₂ PC> _2.4 »Ο ₂₎ = 40: 60 Flow 'flow 1 ml. min-2, temperature 25 ° C, pressure 72 bar, detection at 215 nm, AUFS = 0.02. Injection loop volume 10 µl. Both standards and real samples were dissolved in the mobile phase. The HPLC method was also used to evaluate the purity and content of the active ingredient in the product (6-APK) after isolation, as well as to measure the concentrations of the second product, phenoxyacetic acid and substrate, V-penicillin.

The criterion of usability of stabilized cells is not their specific activity, but the efficiency and quantitative degree of conversion of V-penicillin to 6-APK per time unit. The recommended analytical standard of applicability of a wet or dry biocatalyst for the preparation of 6-APK from V-penicillin is as follows: achievement of quantitative conversion of the K-salt of V-penicillin at its initial concentration of min.

6.0% w / v and biocatalyst concentration min. 70 g dry matter. ". liter at 120 minutes under conditions temperature 37 ° C, pH continuously maintained with dilute ammonia water (1: 1) in the range of values

7.9 to 8.0, continuous stirring of the reaction mixture.

The method of enzymatic production of 6-aminopenicillanic acid from phenoxymethylpenicillin by stabilized yeast cells is further exemplified in the examples, including the raw material and mechanical technology of the production.

Examples

Example 1

Raw materials for production of stabilized yeast

Unwashed cell paste (15 to 20% dry matter) or concentrated aqueous yeast cell suspension (80 to 100 g dry matter, liter ^). Glutaraldehyde (25% aqueous solution).

Ammonium sulphate (pure).

Acetone (pure).

Dry ice.

Potassium hydroxide (pure). .

Drinking water.

Distilled water.

- Specification of machinery for the production of stabilized yeast

2 · Reaction pressure boiler 500 liters, 1 pc, AKV, equipped with agitator with variable mixing frequency 100 to 1000 revolutions. min ” ¹ . Boiler fitted with a volume measuring device.

2 · Mixing pressure boiler 2 m ³ , 1 pc, equipped with stirrer, mixing frequency 200 revolutions. min ” ¹ .

2 · Sedimentation centrifuge, 1 piece, with partial blasting.

j4. Mixing boiler for acetone 500 liters, 1 pcs, AKV, duplicator, brine cooling, high-speed propeller stirrer 500 to 1000 revolutions. min, ¹ , boiler equipped with volume measuring device. Explosion-proof design.

£. Separation resp. filter device, 1 pc, 4 alternative devices can be used.

5/1. Vacuum sucker, 1 pc, with densely perforated sieve, AKV or plastic with inserted textile material - filter cloth, high vacuum.

5/2. Filter press, 1 piece,> fitted with either aspirocellulose plates or filter press.

AND

5/3. Rotary vacuum filter, 1 pc, drum covered with filter cloth, high vacuum.

5.4. Filter centrifuge, 1 pc, AKV, drum coated with filter press, explosion-proof design.

6. Plastic trays, 100 pcs, measuring 50 x 100 cm, placed one above the other in metal structures that are transportable by a forklift truck.

2 · Fine mill sieves, 20 pcs.

2 · Fluid dryer, 1 pc, explosion-proof.

9. Ball mill, 1 pc.

and

Process of production of stabilized yeast

Concentrated cell suspension of intact yeast in amounts

100 liters, which was obtained by submersive cultivation and separation according to the procedure of Art. of the certificate No. 250 495, is pumped into the boiler 2 · The suspension contains 70 to 100 g of cell dry matter. liter ^"first, aqueous cell suspension device 2 in a perfectly homogenized frekvencí.míchání increased, during vigorous stirring, to 100 liters of thickened suspension is gradually added to 8 liters of 25% aqueous solution of glutaraldehyde. Actual concentration of glutaraldehyde 259996

-8 in the reaction mixture is 2% v / v. The suspension is allowed to stir vigorously for about 15 minutes, then the pH is measured and adjusted with a 40% (w / v) aqueous solution of KOH ranging from 7.5 to 7.8. After adjusting the pH, the stirring intensity is reduced to 250 to 300 rpm. min < ^-1 & gt ^; and is stirred continuously for 3 hours. The pH is then measured, the suspension is diluted with water to the maximum and pumped into the mixing boiler 2. Here, it is again diluted to a maximum with water, the stirred suspension is stirred in this apparatus for about 30 minutes and then pumped onto a centrifuge 3. The suspension is concentrated to a volume of 100 liters with a dry matter content of 70 to 100 g.

. liter 1. Centrifuge supernatant is slightly opalescent but must not contain individual yeast cells (centrifuge adjustment). The centrifuge sediment is pumped back to the boiler 1.

To 100 liters of the aqueous suspension of stabilized yeast presented in the boiler 11, solid ammonium sulphate in a total amount of 20.5 kg is gradually added with stirring. The current concentration of ammonium sulfate in the suspension is 265 g. liter, i.e. 2.0 mol. liter * ^. The suspension is stirred vigorously for about 1 hour, then the stirring is slowed to about 200 to 300 rpm. min ”^ and stirring at this intensity.

for 2 hours. After this time, the stirred suspension is maximally diluted with water and pumped to the mixing boiler 12. Here it is again diluted with water as much as possible, stirred for about 30 minutes and then pumped to a 2L centrifuge. The sediment is concentrated to the original volume of 100 liters. The centrifugation supernatant is cloudy and opalescent, but must not contain individual yeast (centrifuge adjustment). Dilution of the sediment with water and further centrifugation should be repeated in the case of a very cloudy supernatant.

In the washing phase of the stabilized cells, greater cell mass loss occurs. About 75 to 80% of the dry matter of the stabilized, washed and washed cells, based on the amount of dry matter in the intact yeast starting suspension, is obtained. The stabilized yeast can be stored by pumping into stored chilled sedimentation cauldrons or used as an aqueous suspension for immediate preparation of 6-APK from V-penicillin. A second possibility is to treat the cell suspension with acetone or ethanol and to dry and store the cells) or to use them in the dry state, respectively. The enzymatic activity of yeast is sufficiently stable in both wet and dry form. However, the dry form of the biocatalyst is preferred for the transport and long-term storage of the biocatalyst.

Optional processing of the suspension of stabilized, washed and washed cells into a dry form is carried out as follows. 259996

-9100 liters of cooled suspension (5 ° C), containing 80 to 100 g dry matter. ¹ liter of stabilized and washed yeast is pumped from the storage cauldron into a mixing boiler 4, where 300 liters of heavily supercooled acetone has been introduced beforehand. Acetone is optionally further cooled in the apparatus 6 by introducing dry ice so that its temperature is at least about -10 ° C. When pumping the cell suspension from the storage kettle into the boiler 6, the present acetone is vigorously stirred at 500 to 1000 rpm. min ” ¹ . The pumping of the suspension into the present subcooled acetone must be rapid, stirring at pumping even after pumping intensive. The stabilized and washed cells are dehydrated with acetone with vigorous stirring at low temperature for a maximum of 5 minutes. The acetone cell suspension is immediately discharged to the filter device after that time. Excess acetone is suctioned off rapidly and sharply under vacuum and the wet cake is washed with a small amount of subcooled acetone and sucked off again. Acetone is recovered by distillation. The well aspirated mass is decomposed in portions into a series of trays placed in metal structures one above the other, a device <5, and the mass is spread on the trays in a thin, about 1 cm layer. The material is then dried for 24 hours at 20-30 ° in an iB oven. The dried material is sieved through fine milling sieves χ, and the remaining material in the form of amorphous baked lumps is mechanically homogenized on a χ device. The two portions are combined, weighed and homogenized. Dry, stabilized yeast cells are placed in well sealed polyethylene bottles or sealed in polyethylene bags and stored at 10-15 ° C after analysis. The dry material homogenate, as well as the aqueous suspension of stabilized yeast, were analyzed for dry matter content, specific V-penicillinamidase activity, and the efficiency and degree of conversion of the V-penicillin K-salt by the methods described herein.

Approximately 0.8 kg of stabilized, thickened, aqueous cell suspension, or about 0.7 kg of dry stabilized cell biocatalyst with a specific activity in the range of 60 to 140 U is obtained from 1 kg dry matter of intact yeast after fermentation and separation. g ” ¹ . The dry matter content of the dry biocatalyst is usually greater than β »90%. The physical properties of the dry material are set forth in the description of the present invention.

-10Example 2

Raw materials for production of 6-APK from V-penicillin using stabilized yeast

Biocatalyst (concentrated aqueous suspension, or dry form of stabilized cells), the preparation process of which was given in Example 1. V-Penicillin K-salt (min. 1500 IU / mg).

Ammonia water (pure).

Sulfuric acid (pure).

Sodium hydroxide (pure).

Sedipur CL 930, BASF, Ludwigshafen, Germany

Butyl acetate or ethyl acetate (pure) may also be used by regenerated distillation.

Acetone (pure), can also be used by regeneration by distillation.

Ethyl alcohol (pure), recovered by distillation.

Ammonium sulphate (pure).

Demineralized water.

Distilled water.

Machinery specifications for the production of 6-APK from V-penicillin using stabilized yeast

JL. Cell suspension storage tank, 5 m ³ , 2 pcs, duplicator, brine cooling, stirring, 200 rpm. min ^-1 , six-blade stirrers on common axis.

2. Dosing pump, 1 pc. h

2 · Sedimentation centrifuge, 1 piece, with partial blasting.

4 ^. Cell sedimentation kettle, 250 liters, 4 pcs, duplicator, brine cooling, mixing.

J5. Cell sediment tank, 500 liters, 4 pcs, duplicator, brine cooling, mixing.

£. Pressure fission reactor, 3 m ³ , 1 pc, duplicator, water cooling, heating, thermoregulation, mixing 180 to 250 revolutions. min ^-1 , 2 six-blade agitators mounted on a common axis, built-in pH electrode with hydrostatic fluid pressure compensation, overloaded with continuous pH adjusting device and dispensing device * for ammonia water.

2 · Dilute ammonia water tank, 400 liters, 1 pc, agitator.

-11 <8. Plate cooler, 2 pcs,. brine about 150,000 kcal. h ” ¹ ; ie = 38 ° C, t ₂ = 5 ° c.

2 · Pressure crystallization boiler, 3500 liters, 4 pcs, enamel, duplicator, brine cooling, stirrer.

10. Measuring cup and dilution boiler for sulfuric acid, 200 liters, 1 pc, enamel, duplicator, water cooling, stirrer.

11. Pressure filter, 1 pc, Variox filter with 40 x 40 cm filter bins made of asbestos cellulose, bulkheads C 10.

12. Filter centrifuge, 2 pcs, explosion-proof, diameter 0.9 m.

13. Suspension cauldron for wet product, 600 liters, 1 pc, AKV, explosion-proof design, duplicator, brine cooling, propeller stirrer.

14. Vacuum dryer, 5 pcs, non-explosive, 4 kPa, 60 kg load.

15. Mother liquor tank, 2500 liters, 2 pcs, explosion-proof design, duplicator, water cooling, stirrer, sight glass. ·

16. Extraction boiler, 1 m ³ , 2 pcs, AKV, explosion-proof design, duplicator, brine cooling, sight glass for separation of two liquid phases, stirrer.

17. UVF-400 ultrafiltration unit, 1 pc, performance 1 m ³ . h ¹ , CP-20 ultrafiltration membranes. ¹

Description of production of 6-APK from V-penicillin stabilized yeast

The wet biocatalyst in the form of an aqueous suspension, or a dry biocatalyst pre-swollen in water for 24 hours, the preparation method of which was given in Example 1, is stored in containers 5, where it is cooled to 5 to 15 ° C. The suspension concentration is defined by dry matter.

1500 liters of demineralized water and an amount of slurry from reservoir 5 are pumped into reactor 5 to produce a final volume of a homogeneous slurry of 1920 liters containing 6-7% yeast dry matter. The suspension is stirred at 37 DEG C. while stirring, and the pH is adjusted with dilute ammonia water (1: 1) to a pH in the range of 7.9 to 8.0 to that of the reservoir T. The amount of dry matter of the stabilized biomass is selected according to the conversion test given in the description of the present invention. 130 kg of the potassium salt of V-penicillin are gradually added to the reactor during stirring. Enzyme hydrolysis lasts a maximum of 2 hours. The consumption of ammonia water and the increments of 6-APK concentrations are monitored continually. The end of 259996 hydrolysis is indicated by the cessation of consumption of the alkali solution and analytically. Upon completion of the hydrolysis, the reactor contents are immediately discharged into storage tank 3. The slurry flowing into tank 1 is stirred and cooled and immediately pumped to a plate cooler 5, where it is cooled to 5 ° C with brine. From the cooler it returns to the tank JL. From there, after cooling, it is pumped immediately to the sedimentation centrifuge 3.

The centrifuge 2 is connected and pre-tested and started in time for water. The liquid flow rate is set to 800 liters. h ^- ^. Partial centrifuge centrifugation in a volume of 5 to 10 liters at intervals of 5 to 8 minutes. After the centrifuge is switched to the cooled suspension, these parameters are controlled so that the flow rate is as high as possible at the highest value of the sediment and filtrate slightly opalescent but without cells (microscopic control). The supernatant is fed to the crystallization boiler 9. A dense sediment of cells with part of the reaction mixture is collected in chilled boilers 4.

In one of the crystallization tanks, the supernatant is cooled with stirring. After all of the supernatant has been pumped into the crystallization tank 9, the supernatant is clarified according to the procedure of Art. Certificate No. 218 182 as follows:

The pH of the solution is adjusted to 6.4 to 6.8 with a 40% v / v solution of sulfuric acid with stirring, then 10 liters of diluted Sedipur CL 930 solutions prepared according to the invention are added to the supernatant from the portable plastic container while stirring. below. The liquid is stirred for about 10 minutes and then left to stand. The system is allowed to flocculate for about 15 minutes. After this time, the flocculated cooling system is metered at a pressure of 0.15 to 0.20 MPa onto the pressure filter 11.

The clear, sparkling filtrate is collected in another crystallized chilled tank 9. After filtration, the entire apparatus is purged with compressed air and purged with about 50 liters of demineralized water. The Sedipur CL 930 stock solution is prepared as follows: dissolve 0.5 kg of Sedipur in 4.5 liters of distilled water and dissolve completely with stirring. The so-called Sedipur working solution is prepared from this stock solution. 250 to 400 ml of Sedipur stock solution is poured into a plastic container in which 10 liters of distilled water have been previously submitted. This working solution is mixed and used to clarify the supernatant liquid according to the following criteria. If the supernatant is slightly cloudy, 250 ml is used to prepare the working solution (259996 supernatant transmittance).

At a higher turbidity level, 350-400 ml of Sedipur stock solution was used in 10 liters of water (supernatant transmittance of 60% or less). The turbidity of the supernatant is measured at 600 nm. Sedipur stock solution (0.5 kg / 4.5 liters) must not be older than 1 week.

To about 1700 to 1800 liters of the clarified, cooled supernatant after filtration in a crystallization cauldron is added 500 liters of distilled butyl acetate with stirring and continuously acidified with a 40% v / v sulfuric acid solution from a measuring cup 10 to adjust the pH to 4.3. After the final pH adjustment, the suspension is allowed to stir for about 1 hour and then allowed to crystallize under cooling for 1 to 2 hours. Then the stirrer is turned on and the product is fed to the filter centrifuge 12. The mother liquors are fed into the container 15. The wet product on the filter centrifuge 12 is covered with about 10 liters of cold distilled water and suspended in a 60 liters of cooled acetone / ethanol (3). The suspension is returned to the filter centrifuge 12 and covered with about 5 liters of ice-cold acetone. All wash solutions must be pre-cooled to 5 ° C. The washed wet product is placed on trays covered with polyethylene foils and dried in the apparatus 14 for 24 hours. After drying, the dry product is homogenized and weighed.

The mother liquors with overlaps of organic solvents are collected in reservoir 15. The content of 6-APK in the aqueous phase is determined analytically. The 66-APK concentration in the mother liquors ranges from 2000 to 30 µg. ml ^- ^. The aqueous phase of the mother liquors is gravitationally separated through a sight glass and is disposed of. The organic phase is forced into the extraction boiler 16, and about 300 liters of demineralized water are added with stirring and the pH is adjusted to 40% w / v with a MaOH solution in the range of 7.8 to 8.0. After stirring for about 30 minutes, the two phases are separated by gravity, the aqueous phase is forced into the reservoir 15, and the organic phase is recovered by distillation. The aqueous phase is forced from the reservoir 15 back to the extraction boiler 16 and regenerated from phenoxyacetic acid as follows: the stirred aqueous phase is acidified to pH 2.0 with 40% w / v sulfuric acid solution and cooled vigorously (5 ° C). The stirrer is then stopped and the phenoxyacetic acid is allowed to crystallize for 2-3 hours. Thereafter, the wet product is passed to a filter centrifuge 12 with stirring, the centrifuged second product is washed with about 25 liters of cold demineralized water and dried on trays separately in the apparatus 14. This regenerates about 85% of phenoxyacetic acid.

-14elements. Mother liquors are disposed of.

The used biocatalyst in the form of a concentrated refrigerated suspension of stabilized yeast containing part of the reaction mixture from the previous cycle is pumped back to the reactor 6, where 1500 liters of demineralized water are submitted. After the suspension of the biocatalyst used has been pumped, the dry matter content is checked, the reactor volume is again adjusted to 1920 liters by the addition of demineralized water, the suspension is allowed to warm to 37 DEG C. with stirring and the next cycle of enzymatic hydrolysis of V-penicillin is started. The time delay between cycles should not exceed 4 hours. After prolonged storage of the used biocatalyst with part of the reaction mixture from the previous cycle, the growth of 6-APK degradation products starts to be adversely affected.

In order to improve the quality of the 6-APK, further purification of the supernatant after conversion and clarification prior to isolation by ultrafiltration may optionally be included as follows: a clear, sparkling, chilled supernatant after filtration on the apparatus 11; during ultrafiltration it recirculates through the second plate cooler JB so that its temperature does not rise above 5 ° C. The permeate obtained is pumped to another crystallization vessel 9 where it is cooled and stirred. About 1600 liters of permeate and about 200 liters of concentrate are obtained. Crystallization of the product from the permeate is carried out separately in the presence of organic solvents as described above. The ultrafiltration concentrate is treated separately as follows: the cooled concentrate is adjusted to 2.0-2.1 with a 40% sulfuric acid solution under vigorous stirring. After adjusting the pH, a sufficient amount of solid ammonium sulfate was added with stirring to form a solution with 60% saturation. The pH should be kept within the stated values during the addition of solid ammonium sulfate. After all the ammonium sulfate has been introduced, the solution is stirred for about 30 minutes and then left to stand for 30 minutes. The solution was vacuum filtered through a pad of diatomaceous earth with a filter cloth inserted. The filtered solution is transferred to a cooled and stirred vessel and the pH is adjusted to 4.3 with dilute NaOH with stirring.

After crystallization, the product is treated as described above, except that the wet product obtained is covered with cold distilled water on the filter centrifuge, suspended first with cold butyl acetate and separated on the filter centrifuge again, covered with distilled water and suspended in acetone / ethanol and filtered again. , covered with ice-cold acetone and dried separately. Organic solvents after permeate treatment and con259996

The concentrates are recovered as well as phenoxyacetic acid as described above.

The yield of 6-APK from cycle 1 is between 50 and 65% of theory. The yield of 6-APK in the second and subsequent cycles ranges from 82 to 85% of theory. In the mother liquors, the concentration of 6-APK ranges from 2.0 to 3.0 g of 6-APK. liter ” ¹ . The average value in mother liquors is 2.5 g of 6-APK. L ^'1, which is about 10%. The active ingredient content of the dry product is between 98 and 100%. The handling losses of the stabilized yeast biocatalyst during its semi-continuous cycling amount to a maximum of 1% dry matter / cycle loss and are compensated by the addition of a storage unused biocatalyst.

Claims (5)

A method for the enzymatic production of 6-aminopenicitin in phenoxymethylpenicillin stabilized yeast cells, characterized in that the enzymatic hydrolysis is carried out by a suspension of yeast comprising the enzyme V-penicillin amidase, in particular by a suspension of yeast Cryptococcus sp. CCY 17-22-1, previously chemically stabilized by the action of a bifunctional reagent such as dialdehydes of dicarboxylic acids, preferably glutaraldehyde, and previously washed solutions of inorganic salts of strong acids and / or bases, preferably ammonium sulfate, and using a solution of phenoxymethylpenicillin at a concentration 1.0 to 10.0% w / v, preferably 4 to 7% w / v, at pH 7.0 to 8.5, preferably 8.0, at a temperature of 10 to 45 ° C, preferably 37% C, in a stirred reactor and after complete hydrolysis and cooling of the reaction mixture to 0-5 ° C, the stabilized and washed yeast suspension is separated and used to hydrolyze a fresh batch of phenoxymethylpenicillin solution and recover from the supernatant or mother liquor after pH and temperature adjustment 6-aminopenicillanic acid, optionally regenerating phenoxyacetic acid, wherein enzymatic hydrolysis of phenoxymethylpenicillin and isolation of 6-aminopenicillanic acid, optionally regeneration of phenoxyacetic acid, at least twice. 2. The process according to claim 1, wherein the substrate is a solution of phenoxymethylpenicillin in the form of an ammonium, potassium or sodium salt. 3. The process of claim 1, wherein the substrate is a crude acid solution of phenoxymethylpenicillin obtained from the fermentation of the fermented broth after the cultivation, or intermediates obtained from the various phases of isolation and purification of phenoxymethylpenicillin. 4. Process according to claim 1, characterized in that the stabilization of the aqueous yeast suspension and the washing thereof are carried out with stirring at temperatures of 10 to 40 [deg.] C., in the glutaraldehyde concentration range of 0.5 to 10% v / v. / volume, at a cell concentration of 1 to 10% w / v dry matter, preferably 5 to 8% w / v dry matter, for 0.5 to 5 hours, preferably 3 hours, after which the stabilized yeast suspension is added Dilute with water, separate cells, resuspend in water and treat with stirring by introducing solid ammonium sulfate over a range of concentrations in an aqueous suspension of 0.1 to 5.0 mol. L ^"1, preferably 2.0 mol. ¹ liter, for 1 to 10 hours, preferably 2 hours, the suspension is diluted with water, the yeast is separated and washed at least twice with water. 5. The process according to claim 1, wherein the stabilized, washed and washed yeasts are stored in a refrigerated container at a temperature in the range of from 1 to 30 [deg.] C, preferably from 5 to 30 [deg.] C., prior to production of the 6-aminopenicillanic acid. 15 ° C, or the concentrated suspension is treated briefly with heavily subcooled acetone or ethanol under agitation in a ratio of 1 volume of cell suspension to 2-5 volumes of acetone or ethanol, after which the cells are separated by centrifugation or filtration, the wet cake of stabilized cells is homogenized and dried at 10 to 45 ° C, preferably 20 to 30 ° C, the dry mass of the stabilized cells is mechanically homogenized and stored in well sealed containers at temperatures of 1 to 40 ° C, preferably 15 to 20 ° C before use.