SK280600B6 - Submersion feeding cultivation method of yeast cryptococcus sp. ccy 17-22-1 with high content of intracellular v-penicillin amidase - Google Patents

Abstract

Disclosed is a submersion feeding cultivation method of yeast Cryptococcus sp. CCY 17-22-1 with high content of intracellular V-penicillin amidase, which method can be characterised in that a cultivation medium is contacted with phenylpropionic acid having a concentration of 0.1 - 0.3 wt.% together with p-hydroxyphenoxyacetic acid, and in that feeding cultivation contains phenylpropionic acid having the total concentration of 0.1 - 0.3 wt.%, ethanol having the total concentration of 0.8 - 1.5 wt.%, and ammonium sulphate having the total concentration of 0.06 - 0.2 wt.% per one volume of a cultivation medium, whilst individual dosing units of phenylpropionic acid are ranging from 0.005 to 0.1 wt.%, dosing units of ethanol are ranging from 0.02 to 0.2 wt.%, and a concentration of ammonium ions being present in the cultivation medium ranges between 0.001 to 0.04 wt.%, a concentration of in the said cultivation medium dissolved oxygen is kept, during the feeding phase, in a range between 5 and 15 % saturation, and a pH value is kept at 7.6 - 7.8.

Description

Technical field

The invention relates to a method for submerged incremental culture of yeast Cryptococcus sp. CC Y 17-22-1 with high intracellular V-penicillin amidase content.

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 to the type of side chain of the parent penicillin. Although the vast majority of G-penicillin 6-APKs are currently being produced, alternative V-penicillin-based technologies have a number of advantages, in particular in increasing the stability of V-penicillin in aqueous solutions as a function of pH and temperature, associated considerably lower presence of degradation products (especially phenoxymethylpenicillanic acid) and hence improved 6-APK quality and higher recovery yield. For the production of 6-APK from V-penicillin, NOVO offers a biocatalyst of fungic origin with a specific activity of 130 U / g, BIOCHEMIE KUNDEL produces a similar biocatalyst with a specific activity of 183 U / g, the original 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 (MSC No. 240 247, hygienic-epidemiological expertise on the non-pathogenicity of the strain), its submerged feed culture (MSC No. 250 245) and its immobilization (MSC No. 259) 996 and Czechoslovak author's certificate No. 259 997). The specific activity of the biocatalysts prepared by this technology ranged between 60-100 U / g.

In MS. author's certificate no. 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 found in some commercially-produced V-penicillins. In MS. author's certificate no. 250,495 describes a method of submerged feed culture of this strain in fermentation tanks. The cultivation method utilized significant amounts of phenylpropionic acid, a metabolizable inducer of this enzyme, as a major source of carbon for food (and hence to achieve high specific activity of V-penicillin amidase). The method described yielded a biomass dry weight of 5.5-6.5 g / l, a V-penicillin amidase activity of 1000-1300 U / l fermentation medium and a specific activity of 150-210 U / l dry weight of cells, consuming 1% phenylpropionic acid per volume fermentation liquid (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 submerged cultivation of the Cryptococcus SP: Ccy 17-22-1 strain according to the invention, which at the same time allows to achieve substantially higher specific activities of V-penicillin amidase.

SUMMARY OF THE INVENTION

The subject of the invention is a method of submerged culture of yeast Cryptococcus sp. CCY 17-22-1 with a high content of intracellular V-penicillin amidase, comprising adding to the culture medium phenylpropionic acid at a concentration of 0.1 to 0.3% by weight. together with p-hydroxyphenoxyacetic acid, preferably at a concentration of 0.01-0.3% by weight, and that for the baby food, phenylpropionic acid at a total concentration of 0.1-0.3% by weight, ethanol at a total concentration of 0.8 % to 1.5 wt. and ammonium sulfate with a total concentration of 0.06 to 0.2 wt. per volume of culture medium, wherein the individual doses of phenylpropionic acid are in the range of 0.005 to 0.1% by weight, the doses of ethanol in the range of 0.02 to 0.2% by weight. 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% saturation during the feed phase and the pH is maintained between 7.6 and 7.8. Additional 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 of US. of the author's certificate no. 250,245 and the specific activity of V-penicillin amidase is higher at the end of the culture. Ethanol in combination with inducers and an inorganic nitrogen source increases V-penicillin amidase production. P-Hydroxyphenoxyacetic acid is a non-metabolizable inducer, so a 0.02% concentration is sufficient, and therefore, after the other media components have been added for further cultivation, a supernatant from previous cultures containing unchanged p-hydroxyphenoxyacetic acid can be used.

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 m (micromole) of 6-APK from V-penicillin potassium salt at an initial substrate concentration of 2% (w / v) per minute at 37 C and pH 8.0 over a range of zero reaction kinetics.

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

The 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 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 grown in cells on reciprocal shaker at 28 ° C on 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% potassium phosphate acid, 0.1% yeast autolysate, and 0.05% magnesium sulfate heptahydrate (w / v), pH 7.5. A 2% inoculum (v / v) was used to inoculate the fermenter. The composition of the fermentor medium corresponded to that of the inoculum medium 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 reached a dry matter of 2.5 g / L, a V-penicillin amidase activity of 421 U / L and a specific activity of 170 U / g dry cell cells.

Thereafter, the following feeds were started: ammonium sulfate, ethanol and phenylpropionic acid according to the results of the analyzes. Because high concentrations of ammonium ions suppress enzyme synthesis, the ammonium sulfate, which served as a nitrogen source, was dosed so that the concentration of ammonium ions in the fermentation broth 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). The feedstuffs consumed 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. At the end of the feed culture (at the 69th hour of culture), this feed consumption achieved a dry matter of 4.1 g / l, a V-penicillin amidase activity of 1510 U / l and a 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, with the amount of induction remaining unchanged.

Example 2

Submerged feed culture was performed in a 75 L Bioengeneering fermentor with a working volume of 30 L. A yeast culture of Cryptococcus sp. Yeast was used as the inoculum for the fermenter. CC Y 17-22-1 grown in cells on reciprocal shaker at 28 ° C on liquid medium of the following composition: 1% corn extract, 0.25% KH2PO4, 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% MgSO ₄ .7H ₂ O, pH before sterilization 7.5, at 2% on fermenter.

The fermenter medium differed from the inoculum medium only by adding 1 ml of SB 2020 Structure (Schill and Scheicher GmbH, Hamburg, Germany) as antifoam. Culture parameters: temperature 28 C, stirring 250 rpm. (approx. 1.75 m / s), aeration 8 l / min, overpressure 10 kPa, pH control 7.7 (20% H ₂ SO ₄ ), pO2 control in the range of 10-15% saturation.

At the end of the batch phase (at 36 hr of 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 feeds were dosed according to the analysis results so that the actual concentrations of the individual feeds did not exceed the values given in Example 1. In the first 8 hours of the fed batch cultivation 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 o'clock cultivation was complete. Dry matter was reached

6.8 g / l, V-penicillin amidase activity of 2339 U / l and specific activity of 346 U / g dry matter. During the fed batch cultivation phase, 0.167% ammonium sulfate, 0.989% ethanol and 0.253% phenylpropionic acid per volume of fermentation medium were consumed.

Example 3

Submerged culture was performed in a 75 L Bioengeneering fermentor with a working volume of 301.

The composition of the medium for the inoculum, the medium for the fermenter, the size of the inoculum and the cultivation parameters were the same as shown in Example 2.

At the end of batch culture (at 32 rd culture time), a dry matter 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 cultivation, ethanol and ammonium sulphate were fed as feed and a dry matter was achieved

3.8 g / l, V-penicillin amidase activity 786 U / l and specific activity 200 U / g dry matter. An additional 37 hours of culture was dosed with 1:10 phenylpropionic acid potassium salt and ethanol together with 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 was only required to achieve this activity of only 0.1% per volume of fermentation liquid. 1% ethanol and 0.65% ammonium sulfate were consumed 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: first During the next 9 hours of supplementary culture (up to 87 hours of culture), the V-penicillin amidase activity increased to 2328 U / l and the specific activity to 380 U / g dry matter with almost unchanged dry matter (6.2 g / l). The feed consumption of phenylpropionic acid from 78 hours was 0.065% in this case, 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 usability

The submerged 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.

Claims (2)

PATENT CLAIMS A method of submerged incremental culture of yeast Cryptococcus sp. CCY 17-22- / with a high content of intracellular V-penicillin amidase, characterized in that phenylpropionic acid at a concentration of 0.1 to 0.3% by weight is added to the culture medium. together with p-hydroxyphenoxy-acetic acid and that for the baby food phenylpropionic acid with a total concentration of 0.1 to 0.3% by weight, ethanol with a total concentration of 0.8 to 1.5 wt. and ammonium sulfate with a total concentration of 0.06 to 0.2 wt. per volume of culture medium, the individual doses of phenylpropionic acid being in the range of 0.005 to 0.1% by weight, the doses of ethanol in the range of 0.02 to 0.2% by weight. the volume of culture medium and the concentration of ammonium ions in the culture medium are in the range of 0.001 to 0.04% by weight, the dissolved oxygen concentration in the culture medium is maintained in the range of 5 to 15% saturation during the feed phase and the pH is maintained at 7, 6 to 7.8. Method according to claim 1, characterized in that, after the addition of the other components of the medium, the supernatant of the previous cultivations containing unchanged p-hydroxyphenoxyacetic acid in an unchanged amount is used for further cultivation. End of document