CZ282791B6 - Method of submersible cultivation of cryptococcus sp. ccy 17-22-1 with a high content of intracellular v-penicillin amidase - Google Patents

Abstract

A method of submersible feeding cultivation of Cryptococcus sp. CCY 17-22-1 yeast with a high content of intracellular V-penicillin amidase in the presence of phenylpropionic acid as a metabolising inductor, characterized by the fact that phenylpropionic acid at a concentration of 0.1 to 0.3% by weight in combination with p-hydroxyphenoxyacetic acid is added to the cultivation medium, and that phenylpropionic acid at a total concentration of 0.1 to 0.3% by weight, ethanol at a total concentration of 0.8 to 1.5% by weight, and ammonium sulphate at a total concentration of 0.06 to 0.2% by weight according to the volume of the cultivation medium are used for feeding the cultivation. Individual doses of phenylpropionic acid range from 0.005 to 0.1% by weight, batches of ethanol range from 0.02 to 0.2% by weight according to the volume of the cultivation medium and the concentration of ammonia ions in the cultivation medium range from 0.001 to 0.04% by weight, and the concentration of dissolved oxygen in the cultivation medium during the feeding phase is controlled within a range of 3 to 15% of the coefficient of the solubility of gasses, and with the pH controlled at 7.6 to 7.8.

Description

Technical field

The present invention relates to a method for the submersive feed culture of yeast Cryptococcus sp. CCY 17-22-1 with a high content of intracellular V-penicillin amidase.

BACKGROUND OF THE INVENTION

6-Aminopenicillanic acid (6-APK), which is an important intermediate for the preparation of semisynthetic derivatives of natural penicillins, can be prepared by enzymatic hydrolysis of benzylpenicillin (G-penicillin) or phenoxymethylpenicillin (V-penicillin). Enzymes that catalyze the selective hydrolysis of the CO-NH bond between the side chain and the backbone of both natural penicillins (penicillin amidase EC 3.5.1.11) are substrate specific according to the type of side chain of the parent penicillin. Although the vast majority of 6-APKs are currently produced from G-penicillin, alternative V-penicillin-based technologies have a number of advantages, notably due to the higher stability of V-penicillin in aqueous solutions depending on pH and temperature, with which it is associated considerably lower presence of degradation products (mainly phenoxymethylpenicillanic acid) and hence better 6-APK quality and higher isolation yield. NOVO offers a biocatalyst of fungal origin with a specific activity of 130 U / g for the production of 6-APK from V-penicillin. BIOCHEMIE KUNDEL produces a similar biocatalyst with a specific activity of 183 U / g, the starting microorganism being Pleurotus sp. We have developed technology for the preparation of 6-APK from V-penicillin, which included a high-production non-pathogenic strain of Cryptococcus sp. CCY 17-1-22 (Czech author's certificate no. 240 247, hygienic-epidemiological expertise on the non-pathogenicity of the strain), its submersive supplementary cultivation (Czech author's certificate no. 250 245) and its immobilization (Czech author's certificate no. 259) 996 and Czech author's certificate No. 259 997). The specific activity of biocatalysts prepared by this technology varied between 60 - 100 U / g.

In MS. No. 240,247 describes a method for selecting microorganisms, especially yeast, and a mutant strain of Cryptococcus sp. CCY 17-22-1 with high intracellular V-penicillin amidase activity. The V-penicillin amidase contained in these yeasts has the advantage of being able to hydrolyze not only V-penicillin but also its p-hydroxy derivative, which is present in some commercially-produced V-penicillins. In MS. No. 250,495 discloses a method for submersive feed culture of this strain in fermentation tanks. The cultivation method used significant amounts of phenylpropionic acid, a metabolizable inducer of this enzyme, for the feed (and hence to achieve a high specific activity of V-penicillin amidase) as the main carbon source. Biomass dry weight of 5.5 - 6.5 g / l, V-penicillin amidase activity of 1000 - 1300 U / l fermentation medium and specific activity of 150 - 210 U / g dry cell weight were achieved by the method described, consuming 1% phenylpropionic acid per fermentation liquid volume (w / v). Due to the high cost of phenylpropionic acid, this cultivation method and hence the whole technology of preparing 6-APK from V-penicillin was a very expensive matter.

This disadvantage is overcome by the method of submersive cultivation of the Cryptococcus sp. CCY 17-22-1 according to the invention, which at the same time allows to achieve substantially higher specific activities of V-penicillin inamidase.

- 1 GB 282791 B6

SUMMARY OF THE INVENTION

The subject of the invention is a method of submersible cultivation of yeast Cryptococcus sp. CCY 17-22-1 with a high content of intracellular V-penicillin amidase in the presence of phenylpropionic acid 5 as a metabolisable inducer, comprising adding to the culture medium phenylpropionic acid at a concentration of 0.1 to 0.3% by weight. in combination with p-hydroxyphenoxyacetic acid, preferably at a concentration of 0.01% to 0.3% by weight, and that for the baby food, phenylpropionic acid is used in a total concentration of 0.1 to 0.3% by weight, ethanol in a total concentration 0.8 to 1.5 wt. and ammonium sulfate in a total concentration of 0.06 to 0.2 wt. % by volume of the culture medium, the single doses of phenylpropionic acid being in the range of 0.005 to 0.1 wt. % ethanol in the range of 0.02 to 0.2 wt. per volume of culture medium and the concentration of ammonium ions in the culture medium is in the range of 0.001 to 0.04 wt. The dissolved oxygen concentration in the culture medium is maintained between 3 and 15% of the gas solubility coefficient (saturation) during the feed phase and the pH is maintained between 7.6 and 8. Sufficient biomass during cultivation is provided by the following main sources of carbon and nitrogen: corn extract, ethanol, sodium citrate and ammonium sulfate. The total amount of phenylpropionic acid, which is a metabolizable inducer, is significantly lower in this cultivation method than in the cultivation method according to U.S. Pat. No. 250,245 and the specific activity of V-penicillin amidase at the end of the culture is higher. Ethanol 20 in combination with inducers and an inorganic nitrogen source increases the production of V-penicillin amidase.

P-hydroxyphenoxyacetic acid is a non-metabolizable inducer and is therefore 0.02% by weight. concentrations are sufficient, and therefore, after the other media components have been added, the supernatant from previous cultures containing unchanged p-hydroxyphenoxyacetic acid can be used for further cultivations.

DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate the nature of the invention without limiting the claims.

The unit of V-penicillin amidase activity (U) in the examples is defined as the amount of enzyme catalysing the formation of 1 µm (micromole) of 6-APK from V-penicillin potassium salt at an initial substrate concentration of 2% (w / v) per minute at 37 ° C; pH 8.0 in the range of zero order reaction kinetics.

(U = 1 pmol 6-APK / min.)

Specific activity is then expressed as U / gram cell dry weight.

Example 1

Submerged cultivation was carried out in a 10 L Chemap fermenter with a working volume of 10 L. The cultivation parameters were as follows: temperature 28 ° C, stirring 400 rpm. (approx. 1.57 m / s), aeration 45 2.5 l / min, overpressure 10 kPa. The pH was maintained during the feed phase of the culture

7.7 with a 20% sulfuric acid solution, the dissolved oxygen (pO2) concentration in the fermentation broth was maintained within 5-10% saturation (oxygen electrode).

A 48-hour culture of Cryptococcus sp. CCY 17-22-1 50 grown in flasks on a reciprocating shaker at 28 ° C on a liquid medium of the following composition: 0.3% phenylpropionic acid (potassium salt), 0.02% p-hydroxyphenoxyacetic acid (potassium salt), 1% corn extract, 0.25% sodium citrate, 0.25% acid potassium phosphate, 0.1% yeast autolysate, and 0.05% magnesium sulfate heptahydrate (w / v), pH 7.5. For vaccination

A 2% inoculum (v / v) was used in the fermentor. The composition of the fermentor medium corresponded to that of the inoculum, except that 0.3 ml SB 2020 Structure (Schill and Schleicher GmbH, Hamburg, Germany) was added as antifoam. The patch phase of the culture lasted 35 hours and at the end of the process a dry matter of 2.5 g / l, a V-penicillinamidase activity of 421 U / l and a specific activity of 170 U / g dry cell cells were achieved.

Thereafter, simultaneous dosing of the following baby foods was started: ammonium sulfate, ethanol and phenylpropionic acid according to the analysis results. Because high concentrations of ammonium ions suppress enzyme synthesis, ammonium sulfate, which served as a nitrogen source, was dosed so that the concentration of ammonium ions in the fermentation medium was about 0.004%. Ethanol and phenylpropionic acid, which served as carbon sources, were dosed so that the ethanol concentration in the medium did not exceed 0.05% and the phenylpropionic acid concentration did not exceed 0.02% (w / v). A total of 0.1% phenylpropionic acid per volume of fermentation medium, 0.8% ethanol per volume of fermentation medium, and 0.08% ammonium sulfate per volume of fermentation liquid was consumed for feed. At the end of the meal culture (at the 69th hour of cultivation), the dry food consumption reached 4.1 g / l, the V-penicillinamidase activity of 1510 U / l and the specific activity of 368 U / g.

The culture supernatant, which still contained non-metabolizable p-hydroxyphenoxyacetic acid, was used for repeated culture after replenishment of the other media components, the amount of induction remaining unchanged.

Example 2

Submerged power culture was performed in a 751 Bioengeneering fermenter with a working volume of 301. A 48-hour culture of Cryptococcus sp. CCY 17-22-1 grown on flasks on a reciprocating shaker at 28 ° C on liquid medium of the following composition: 1% corn extract; 0.25% KH ₂ PO _4; 0.1% yeast autolysate, 0.3% phenylpropionic acid (K-salt), 0.02% p-hydroxyphenoxyacetic acid, 0.2% ethanol, 0.002% CaCl ₂ .6H ₂ O, and 0.05% Mg50 ₄ · 7H ₂ O, pH before sterilization 7.5, at 2% per fermenter.

The fermenter medium differed from the inoculum medium only in that 1 ml of SB 2020 Structure (Schill and Schleicher GmbH, Hamburg, Germany) was added as antifoam. Culture parameters: temperature 28 ° C, stirring 250 rpm. (approx. 1.57 m / s), aeration 8 l / min, overpressure 10 kPa, pH control 7.7 (20% H ₂ 50 ₄ ), pO2 control within 10-15% saturation.

At the end of the batch phase (at 36 h culture) a dry matter of 3.2 g / l was achieved, the activity of V-penicillin amidase was 453 U / l and a specific activity of 141 U / g dry matter.

During the fed batch phase, the baby foods were dosed according to the analysis results so that the actual concentrations of the individual baby foods did not exceed the values given in Example 1. In the first 8 hours of the fed batch culture phase, only ethanol and ammonium sulfate solution were dosed. - phenylpropionic acid-ethanol salts (1 + 1) and ammonium sulfate solution, then only ethanol and ammonium sulfate solution for a further 10 hours. At 74 hours, the culture was stopped. A dry weight of 6.8 g / l, a V-penicillinamidase activity of 2339 U / l and a specific activity of 346 U / g dry matter were achieved. A total of 0.167% ammonium sulfate, 0.989% ethanol, and 0.253% phenylpropionic acid per volume of fermentation medium were consumed during the fed batch culture.

Example 3

Submerged culture was carried out in a 75 L Bioengeneering fermenter with a working volume of 30 L. The composition of the inoculum medium, the fermenter medium, the inoculum size and the culture parameters were the same as in Example 2.

At the end of the batch phase of culture (at 32 nd cult, hour), a dry weight of 2.8 g / l, a V-penicillin amidase activity of 450 U / l and a specific activity of 162 U / g dry matter were achieved.

During the first 9 hours of fed batch culture, ethanol and ammonium sulphate were fed as feed and 3.8 g / l dry matter, 786 U / l V-penicillin amidase activity and 200 U / g dry matter specific activity were achieved. An additional 37 hours of culture was dosed with a 1 + 10 mixture of a solution of phenylpropionic acid potassium salt and ethanol together with an ammonium sulfate solution. At the end of the culture (78 hours), a dry matter of 6.1 g / l was achieved, the activity of V-penicillin amidase was 1931 U / l and a specific activity of 309 U / g dry matter. With this culture arrangement, the consumption of phenylpropionic acid for baby foods required to achieve said activity was only 0.1% per volume of fermentation liquid. Ethanol was consumed 1% and ammonium sulfate 0.65% per volume of fermentation medium.

Example 4

The cultivation was carried out as described in Example 3, except that at 78 hours the cultivation was not complete but the ammonium sulfate solution was added together with the carbon source solution, which was prepared containing ethanol and phenylpropionic acid in a ratio of 3. +1. During the next 9 hours of supplementary culture (up to 87 h), the activity of V-penicillin amidase increased to 2328 U / l and the specific activity to 380 U / g dry matter with almost unchanged dry matter (6.2 g / l). In this case, the consumption of phenylpropionic acid for baby foods from 0:00 hour was 0.065%, the consumption of ethanol 0.195% and the consumption of ammonium sulfate 0.0135% per volume of fermentation medium (w / v). Thus, the total feed consumption for the fed batch culture phase was 0.165% phenylpropionic acid, 1.195% ethanol and 0.6635% ammonium sulfate per volume of fermentation liquid.

Industrial applicability

The submersible feed culture method of the invention can be used to prepare an enzymatically active biomass from which a biocatalyst capable of hydrolyzing V-penicillin to 6-APK can be prepared as part of the technology for producing 6-aminopenicillanic acid from V-penicillin.

PATENT CLAIMS

Claims (2)

1. A method of submersive feed culture of yeast Cryptococcus sp. CCY 17-22-1 with a high content of intracellular V-penicillin amidase in the presence of phenylpropionic acid as a metabolizable inducer, characterized in that phenylpropionic acid is added to the culture medium in a concentration of 0.1 to 0.3% by weight. in combination with p-hydroxyphenoxyacetic acid, and that for the baby food, phenylpropionic acid is used in a total concentration of 0.1 to 0.3% by weight. and ammonium sulfate in a total concentration of 0.06 to 0.2 wt. per volume of culture medium, the single doses of phenylpropionic acid being in the range of 0.005 to 0.1 wt. benefits % Ethanol in the range of 0.02 to 0.2 wt. per volume of culture medium and the concentration of ammonium ions in the culture medium is in the range of 0.001 to 0.04 wt. and the dissolved oxygen concentration in the culture medium is maintained within the range of 3 to 15% of the gas solubility coefficient during the feed phase and the pH is maintained at 7.6 to 8. Method according to claim 1, characterized in that, after the addition of the other components of the medium, the supernatant from the previous cultivations containing unmetabolizable p-hydroxyphenoxyacetic acid in an unchanged amount is used for further cultivations.