CS240247B1 - A method for selecting and accumulating phenoxymethylpenicillin hydrolyzing microorganisms to 8-aminopenicillanic acid and a strain of Cryptococcus sp. CCY 17-22-1 - Google Patents

Abstract

The solution relates to a method for selecting and accumulating microorganisms hydrolyzing phenoxymethylpenicillin to 6-aminopenicillin acid and is characterized in that cultures of microorganisms are inoculated into a selective medium, where the sole source of assimilable nitrogen, and/or both nitrogen and carbon are phenoxyacetamide, 3-phenylpropionamide, 4-hydroxyphenoxyacetamide, phenylacrylamide, and/or where the sole sources of assimilable carbon are salts of 3-phenylpropionic, phenylacrylic, phenoxyacetic and 4-hydroxyphenoxyacetic acids, optionally with amides of these acids, serving as the sole sources of assimilable nitrogen, or with an inorganic source of nitrogen, preferably with ammonium ions, wherein the grown cultures of microorganisms are passaged in these media until the growth rate does not increase, after which the grown culture is inoculated and preserved. The invention also relates to a strain of the microorganism Cryptococcus sp., CCY 17-22-1 producing an intracellular V-penicillin amidase that preferentially hydrolyzes phenoxymethylpenicillin to 6-aminopenicillanic acid.

Description

The invention relates to a method for the selection and accumulation of microorganisms hydrolyzing phenoxymethylpenicillin to 6-amlnopenicillanic acid and a strain of Cryptococcus sp. CCY 17-22-1.

It is known that 6-aminopenicillanic acid (hereinafter 6-APK) serves as an intermediate for the production of so-called semi-synthetic penicillins, e.g. ampicillin. 6-APK can be prepared from natural penicillins, ie benzylpenicillin (G-penicillin) or phenoxymethylpenicillin (V-penicillin), either chemically or by enzymatic hydrolysis. Chemical hydrolysis of natural penicillins is economically disadvantageous. In contrast, enzymatic hydrolysis has significantly better economic parameters, which is used to produce 6-APK by a number of foreign pharmaceutical companies.

In most cases in the world, as well as in Czechoslovakia, 6-APK is produced by the enzymatic hydrolysis of benzylpenicillin. For this purpose, high-producing strains of bacteria synthesizing a high amount of the enzyme G-penicillin amidase, for example strains of Escherichia coli according to US Pat. No. 162 274 and 188 631.

However, phenoxymethylpenicillin (V-penicillin) is rarely enzymatically hydrolyzed by bacterial strains. E.g. only a few strains of bacteria of the genera Erwinia, Achromobacte, Micrococcus, Streptomyces and Actinoplanes have been described which are capable of synthesizing a specific V-penicillin amidase hydrolyzing V-penicillin to form 6-APK. However, the production of this enzyme in said bacterial genera is very low, in some species the enzyme is synthesized extracellularly (Streptomyces), so these strains of bacteria are not of industrial importance for the production of 6-APK (Vandamme, EJ, Economic Microbiology, Microbial Enzymes and Bioconversions) , Rose, AH, ed., Vol. 5, Chapter 9, pp. 467-522, Acad. Press, New York, 1980].

The cited author, on the other hand, states that in some eukaryotic microorganisms, the incidence of V-penicillin amidase is more widespread, particularly in some yeasts (eg, the genus Torulopsis, Zygosaccharomyces, Debaryomyces, Torula, Rhodotorula), fungi (eg the genus Penicillium, Aspergillus, Fusarium) higher fungi (eg, the genera Pleurotus, Bovista). The best producers of V-penicillin amidase are some strains of higher fungi, especially Pleurotus ostreatus and Bovista plumbea.

Managing the cultivation of these organisms in large-capacity fermenters is very demanding due to the high cost of cultivation control, nutrient composition and sterility of the culture during long-term submersive cultivation (of the order of 150 to 200 hours).

To date, very few V-penicillin amidase producers have been characterized in baseline parameters. In most cases, information is reported for fungi and higher fungi, rarely for bacteria and only for one yeast, Rhodotorula glutinis var.

glutinis (Vandamme, E. J. and Voets, J. P., Zeit. Allg. Microblol. 13, 701-710, 1973). Using a highly concentrated suspension of intact cells of this yeast, an 80% theoretical conversion of 1% w / v aqueous solution of V-penicillin.u potassium salt to 6-APK (28 ° C, pH 6.5, 30 hours reaction time) was achieved ).

The process according to the invention is characterized in that it is possible to select suspect strains of natural and collecting microorganisms with V-penicillin amidase activity in liquid or solid chemically defined nutrient media, where phenoxyacetamide serves as the only source of assimilable nitrogen and / or both nitrogen and carbon. -phenylpropionamide, phenylacrylamide (cinnamic amide) or derivatives thereof, wherein the ability to grow enzymatically active cells with the desired activity is indicated by the increase in turbidity or colony formation or growth of strains in the presence of the 3-phenylpropionic, phenylacrylic and phenoxyacetic acid salts , which serve as the only sources of assimilable carbon in a chemically defined culture medium. Single selection and passage in nutrient media containing these compounds and their derivatives can be combined with sequential selection in any combination in terms of utilization, induction, and substrate specificity of the enzyme. So. for example, strains rapidly growing on utilizable synthesis inducers, etc. can be obtained.

A single and sequential selection, preceded by extensive screening of collection and natural isolates, resulted in the strain of Cryptococcus sp. CCY 17-22-1, which is also a subject of the invention and whose properties, in particular the high specific activity of V-penicillin amidase, intracellular localization of the enzyme and the absence of β-lactamase, make it possible to use it for the enzymatic preparation of 6-APK from V-penicillin. by means of intact cells separated from the culture liquid, immobilized cells, optionally this strain can serve as a source of enzyme for its subsequent isolation and its further use in soluble or immobilized form. The basic morphological and biochemical properties of the subject strain and those related to the basic characterization of the enzyme in intact cells are summarized in the following summary.

Properties of the strain Cryptococcus sp. CCY 17-22-1 '

Basic characteristics of the strain

The yeast, growing well on the wort agar, forms a creamy colonies, a polar bud, a refracted capsule. When grown in broths containing rapidly assimilable carbon sources (glucose), extra240247 forms a highly viscous polysaccharide cellular. Extracellular polysaccharide formation can be eliminated by the nutrient composition and C: N ratio. It grows well on corn extract, amino acid mixtures and yeast hydrolyzate. It also grows well on chemically defined mineral nutrient media with a suitable carbon source; it uses ammonium ions as a nitrogen source.

Growth on the compounds listed below as N-Penicillin strain properties

1. enzyme localization;

2. Presence of β-lactamase:

3. Induction of enzyme synthesis:

4. substrate specificity of the enzyme:

5. Catabolic repression of enzyme synthesis:

6. Optimum pH of enzyme activity:

7. Specific activity of intact cells *

8. Enzyme conversion products:

the only sources of assimilable carbon in liquid nutrient soils.

Glucose (+), sucrose (+), fructose (+), galactose (-), lactose (+), glycerol (-), DL-lactate (-), succinate (+), citrate (+), ethanol (+ ], phenylacetic acid (-), phenoxyacetic (-), 4-hydroxyphenoxyacetic (-), 3-phenylpropionic (+), phenylacrylic (+) salts.

intact cells in intact cells;

negative;

3-phenylpropionic acid (+++), phenylacrylic acid (+), 4-hydroxyphenoxyacetic acid (++), phenoxyacetic acid (+), phenylacetic acid (-); V-penicillin (+++), G-penicillin (-), ampicillin (-), glutaric acid monoamide (++), phenoxyacetamide (+), 4-hydroxyphenoxyacetamide (+), 3-phenylpropionamide. (-), phenylacrylic acid amide (+), phenylacetamide (-);

In the presence of glucose and sucrose, the enzyme is not synthesized even if any of its inducers are present;

8.0;

0.5 to 12.0, «mol 6-APK. mg ¹ dry cell mass. The specific activity achieved depends on the composition of the culture medium, the culture conditions and the physiological state of the culture; Cellulose thin layer chromatography and high pressure liquid chromatography showed that contact of intact cells with aqueous solutions of V-penicillin K-salt produced only 6-APK and phenoxyacetic acid.

* The unit of V-penicillin amidase enzyme activity is the amount of enzyme that catalyzes the hydrolysis of an aqueous solution of V-penicillin K-salt as a substrate to produce one micromole of 6-APK in one milliliter per hour under zero order enzyme reaction kinetics at pH 8 , 0 and 37 ° C. Dimension of activity: gmol 6-APK. ml ^-1 . h '¹; dimension of specific activity: μίησί 6-APK. mg ^-1 dry cell mass. To determine the concentration of 6-APK was used the procedure given in MS. No. 116,959.

According to the morphological, growth and biochemical behavior of the culture, the strain obtained was classified into the genus Cryptococcus in accordance with the taxonomic criteria set forth in: The Yeasts, A taxonomy study, second edition, J. Lodder, ed., North-Holland Publishing Company - Amsterdam - London, 1970; J.A. Barnett, R.W. Payne and D. Yarrow, Yeasts, Characterization and Identification, Cambridge, London, New York, New Rochelle, Melbourne, Sydney, 1983. Subject. Cryptococcus sp. le saved in MS. Czechoslovak Collection of Yeasts in Bratislava under No. CCY 17-22-1.

The principle of the method of single and sequential selection and accumulation of strains of microorganisms, in particular yeasts, hydrolyzing phenoxymethylpenicillin to 6-amlnopenicillanic acid according to the invention consists in inoculating the cultures of microorganisms into selective chemically defined nutrient media where the only source of assimilable nitrogen; nitrogen, and carbon are provided by phenoxyacetamide, 4-hydroxyphenoxyacetamide, 3-phenylpropionamide, phenylacrylamide, and / or wherein salts, 3-phenylpropionic acid, phenylacrylic, phenoxyacetic, hydrophenoxyacetic acids, or amides of these acids serve as the sole sources of assimilable Carbon. a single source of assimilable nitrogen, or an inorganic source of nitrogen, preferably ammonium ions, wherein the grown cultures of the microorganisms pass in these soils until growth rate is increased, whereupon the grown culture is seeded and conserved. vuje.

The present invention further provides a strain of Cryptococcus sp. CCY 17-22-1, which was obtained as described in the preceding paragraph, and which produces intracellular V-penicillin amidase which preferentially hydrolyzes phenoxymethylpenicillin to 1,6-aminopenicillanic acid.

The invention is further exemplified without being limited thereto.

EXAMPLE 1 Collection and 8 wild-type yeast strains which could be taxonomically classified into Saccharomyces, Debaryomyces, Candida, Cryptococcus, Rhodotorula, Torulopsis and Trichosporon according to the literature cited herein were inoculated with sloping agar loops into flasks containing 50 ml of liquid broth containing: 0.2% phenoxyacetamide, glucose 0.5 to 1.0 why. KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.2%, yeast extract 0.05%, MgSO ₄ 0.05%, phenoxyacetic acid Na-salt 0.05%; pH of the culture medium 6.5. The inoculated flask cultures were placed on a shaker and the submerged culture was carried out at 28 ° C.

Of the 30 strains tested, 19 cultures grew under the indicated conditions at various times. After centrifugation of the suspension, the cell sediment was analyzed to demonstrate the formation of 6-APK from V-penicillin according to the procedures described herein. However, V-penicillin amidase activity was not demonstrated in any of the grown cultures.

In addition, 11 non-growing strains were discarded and 19 growing strains in the broth, where phenoxyacetamide served as the sole nitrogen source, were inoculated into flasks containing 50 ml of non-selective liquid broth containing: casein hydrolyzate 0.5% , peptone 0.5%, yeast extract 0.1%, KH ₂ PO ₄ 0.3%, NH ₄ H ₂ PO ₄ 0.5%, MgSO ₄ 0.05%, phenoxyacetic acid sodium salt 0.05 ° /O; pH of the culture medium 6.5. The inoculated flask cultures were placed on a shaker and the submerged cultures were run at 28 ° C for 24 to 48 hours. Intact cell sediments after centrifugation were again analyzed for 6-APK formation from V-penicillin.

In 19 cultures grown under these conditions, 5 strains were analytically proven to produce 6-APK from V-penicillin. These were strains of Candida utilis, Rhodotorula sp., Rhodotorula pulcherina, Rhodotorula glutinis and Cryptococcus sp.

These 5 strains were evaluated in quantitative terms for the amount of V-penicillin amidase produced. The cultures were inoculated into flasks containing 50 ml of liquid non-selective broth containing: corn extract - precipitated, filtered 2 '% (wet mass), KH ₂ PO ₄ 0.1 ° / o, K _and HPO ₄ 0.2 ° / o , MgSO ₄ 0.05 θ / o, Phenoxyacetic acid Na-salt 0.05%; pH of the culture medium 6.9. Submerged cultivation was carried out at 28 degrees Celsius for 30 hours. After centrifugation of the cultures, the sediments were quantitatively analyzed for V-penicillin amidase content in the cells.

The specific activity of this enzyme was demonstrated in all strains, but the quantitative differences between the strains grown under these conditions were several orders of magnitude. The highest specific activity was obtained in the Cryptococcus sp. (0.28 μΐηοΐ 6-APK .mg ^-1 dry matter cells) and Rhodotorula glutinis (0.012 μΐηοΐ 6-APK. Mg ¹ dry matter cells). Therefore, the Cryptococcus sp. Strain was chosen as the starting material for further selection and accumulation of the above production strains, since its specific activity was more than orders of magnitude higher compared to the second isolate obtained.

Example 1 a d 2

Investigating the specificity of V-penicillin amidase induction during submersive cultivation of the Cryptococcus sp. in nutrient broths containing compounds chemically identical or chemically close to the side chains of natural and semi-synthetic penicillins, it has been found that the highest specific activity of the enzyme in cells is achieved in the presence of salts of 3-phenylpropionic, 4-hydroxyphenoxyacetic, cinnamic and phenoxyacetic acid. This is reported in the review of the Cryptococcus sp. in the description of the present invention. Some of these compounds are used as the sole sources of assimilable carbon, others are not. Thus, some of these substances act as assimilatable inducers, others as "seemingly" inducers. These findings have been utilized to select selective chemically defined nutrient media to select and accumulate fast-growing strains on said compounds as the only sources of assimilable carbon, especially since most of these are highly toxic and inhibit growth.

The strain Cryptococcus sp., Obtained as in Example 1, was inoculated from sloping wort agar into flasks containing 150 ml of liquid broth containing: corn extract - precipitated, filtered 2% (wet mass), KH ₂ PO ₄ 0.1 K ₂ HPO ₄ 0.2 ° / o, yeast extract 0.05%, thiamine 0.01% / o, MgSO ₄ 0.05%, K-salt of 3-phenylproponic acid 0.1%, nutrient pH soil 7,2. Submerged culture was carried out at 28 ° C for 24 hours.

The grown culture was used as an inoculum (5 vol% suspension) to inoculate flasks containing 150 ml of liquid broth containing: K-salt of 3-phenylpropionic acid 0.5%, KH ₂ PO ₄ 0.2%, NaCl 0.05 %, yeast extract 0.03 ° / o, (NH ₄ j ₂ SO ₄ 0.3 ° / o, NH ₄ H ₂ PO ₄ 0.2%, CaCl ₂ 0.002%, FeSO ₄ 0.0005 why. MgSO ₄ 0.01% pH of the broth 7.3.

Submerged culture was performed by alternating passage of the cultures at 28 ° C, each time by transferring 5 vol% of the grown culture from the first to the second, second to the third, and so on. The first subculture was grown in 120 hours to dry cell mass at approximately 1 mg. ml ¹ , the second subculture in 70 hours at the same dry matter, the third subculture in 50 hours, the fourth in 70 hours and the fifth subculture in 45 hours at 1.2 mg su240247 sine. ml ^-1 . In the sixth and subsequent subcultures, the growth rate and cell dry matter yield did not increase.

The accumulated population of faster-growing cells in the broth containing 3-phenylpropionic acid K-salt as the only assimilable carbon source was sown from the last subculture on wort agar petri dishes, inoculated with individual colonies to obtain a faster growing 3-phenylpropionic acid utilizing strain carbon. The specific activity of V-penlcilin amidase in this strain is about 4-6 µmol 6-APK. mg ¹ dry cell culture in submersive culture in flasks in liquid medium containing: corn extract - precipitated, filtered 0,5% (wet mass), K-salt, 3-phenylpropionic acid 0,5%, KH ₂ PO ₄ 0 1%, K 2 HPO 4 0.2%, yeast extract 0.05%, thiamine 0.01%, MgSO ₄ 0.01%; pH of broth 7.2. The yield of dry cell mass is about 1.5 to 1.8 mg. ml ^- . ¹ at 46 hours culture at 28 ° C.

Example 1 a d 3

When investigating the substrate specificity of V penicillin amidase in intact Cryptococcus sp. Cells, to compounds containing amidic CO-NH bond in the molecule, especially amides of acids chemically close to the side chains of natural and semi-synthetic penicillins, some have been found to be substrates, the rate of enzyme hydrolysis is shifted in the order of several orders of magnitude to favor the hydrolysis of V-penicillin as a substrate. Thus, these are all very poor substrates compared to V-penicillin. This is shown in the review of the Cryptococcus sp. in the description of the present invention. However, these facts have been utilized to select selective chemically defined nutrient media in order to accumulate the production strains using amides of said substances as the only sources of assimilable nitrogen.

The strain Cryptococcus sp. obtained as in Example 2 was inoculated from sloping wort agar into flasks containing 150 ml of liquid broth of the composition shown in Example 2, last paragraph. After a 46-hour submersible culture at 28 ° C, this culture was used as an inoculum (5 vol / o) to inoculate the selective liquid broth in flasks filled with 150 ml of soil containing: 4-hydroxyphenoxyacetamide 0.2% / o, K-salt 3-phenylprononic acids 0.5%, KH ₂ PO ₄ 0.1%, K ₂ HPO ₄ 0.2 why. yeast extract 0.03%, thiamine 0.01 why. MgSO ₄ 0.01%, pH of broth 7.2.

Submerged cultivation is carried out by alternating passage of the cultures at 28 ^° C in each case by transferring 5 vol.% Of the grown culture from the first to the second and subsequent subcultures until the growth rate has not changed.

A heterogeneous cell population obtained by sequential selection in a culture medium, wherein 4-hydroxyphenoxyacetamide served as the only source of assimilable nitrogen and 3-phenylpro- K-salt was the only source of assimilable carbon. pionic acid was plated on wort agar petri dishes, inoculated with individual colonies to obtain a strain with increased V-penicillin amidase content in the cells.

The specific activity of the enzyme in this strain is between 6 and 12 μπιοΙ 6-APK. . mg ^-1 dry cell mass in its submersible culture in flasks in liquid medium containing: K-salt of 3-phenylpropionic acid 0,5%, KH ₂ PO ₄ 0,2%, NH ₄ H ₂ PO ₄ 0,2%, (NH ₄ J ₉ SO ₄ 0.3%, yeast extract 0.05%, thiamine 0.01%, MgSO ₄ 0.01%, CaCl ₂ 0.002 why., FeSO ₄ 0.0005%; pH of nutrient medium 7.2. the yield of cell dry matter is about 1.3 mg. ml ^-1 for 48 hours at 28 ° C.

Claims (2)

SUBJECT Method for the selection and accumulation of microorganisms hydrolyzing phenox-ymethylpenilcilin to 6-aminopenicillanic acid, characterized in that the cultures of microorganisms are inoculated into selective chemically defined nutrient media, where phenoxyacetamide serves as the only source of assimilable nitrogen and / or nitrogen and carbon. , 4-hydroxyphenoxyacetamide, 3-phenylpropionaraid, phenylacrylamide, and / or wherein the only sources of assimilable carbon are the salts of 3-phenylpropionic acid, phenylacrylic acid, phenoxyacetic acid, 4-hydroxyphenoxyacetic acid, or amides thereof OF THE INVENTION as the sole assimilable nitrogen source or with an inorganic nitrogen source, preferably with ammonium ions, wherein the grown cultures of the microorganisms pass through the soils until the growth rate is increased, whereupon the grown culture is seeded and preserved. 2. The strain of Cryptococcus sp. CCY 17-22-1, producing intracellular V-penicillin amidase which preferably hydrolyzes phenoxymethypenicillin to 6-aminopenicillanic acid, obtained by the method of item 1.