CS201338B1 - Method of producing penicilin v - Google Patents

Description

The invention relates to a process for the production of penicillin V by culturing a production microorganism Penicilium ehrysogenum in a liquid culture medium containing a source of assimilable carbon which is dosed during cultivation and a source of assimilable nitrogen, a mineral nutrient salt and a phenoxyacetic acid precursor under submerged conditions.

The yield of fermentation and isolation in the biosynthetic production of penicillin V is determined by the ratio of the desired product, i.e. penicillin V, to the undesired by-product, i.e. the penicillin phydroxy derivative, in the content of precursor residue in the fermentation broth. The more fermentation broth at the end of the fermentation of penicillin V and less of the p-hydroxy derivative of penicillin V and the unused precursor, the more favorable the economy of penicillin V biosynthetic production including isolation.

The formation of an undesired p-hydroxy derivative of penicillin V by transforming penicillin V into the production microorganism during fermentation was avoided by carrying out model experiments in which the substance of penicillin V was added at different concentrations and at different times of fermentation to the fermentation broth containing no precursor. During the fermentation at 25 ° C for 10 days, the composition of the penicillins was monitored and the penicillin content was found to be constant and that its p-hydroxy derivative was not formed.

It is apparent from the results of these experiments that the undesired p-hydroxy = penicillin V derivative is formed during fermentation so that the penicillin V precursor, i.e. phenoxyacetic acid, is first transformed into the p-hydroxy derivative of penicillin V, from which it is only a further biochemical reaction. a p-hydroxy derivative of penicillin V is formed

During the overall fermentation process analysis, the content of penicillin V and its p-hydroxy derivative, phenoxyacetic acid and p-hydroxy phenoxyacetic acid was monitored in detail during the whole fermentation, when either the entire amount of precursor was present in the medium as early as 0 hour of fermentation or precursor dosed in different amounts at different stages of fermentation. Under these conditions, the formation of the penicillin V p-hydroxy derivative can be inferred from the autolysis of the culture or the deficiency of the substrate suitable for oxidation after the finished dosing of sucrose. In fact, in a standard procedure, saciharose is not added until the end of the fermentation; Therefore, further experiments were carried out in which autolysis was intentionally promoted by premature stopping of sucrose dosing and vice versa, by increasing the dosing time of sucrose, an oxidizable substrate was provided for the culture.

The results obtained indicate that the content of penicillin V p-hydroxy derivative in the fermentation broth can be controlled by dosing sucrose. In fact, it has been discovered that the longer the sucrose is dosed during fermentation, the less p-hydroxy derivative of penicillin V is produced in the fermentation broth.

Based on these findings, a method for producing penicillin V has been developed by culturing a production microorganism Penicillium chrysogenum in a liquid culture medium containing a source of assimilable carbon which is dosed during cultivation and a source of assimilable nitrogen, mineral nutrient salt and phenoxyacetic acid as precursor under submerged conditions. in which the assimilable carbon source is dosed into the fermentation broth until the end of the growth phase of the microorganism, in the next production phase the assimilable carbon source is reduced to the amount needed to maintain the culture, after which the p-hydroxyderivative content of penicillin In the soil, dosing resumes the assimilable carbon source as in the growth phase, with the last dose being added 12 to 24 hours before the end of the production phase.

By means of this simple measure, the fermentation process of penicillin V was effectively improved and the content of the undesired by-product, i.e., the p-hydroxy derivative, and the content of the unused precursor were reduced to an acceptable level. This also simplifies the isolation of penicillin V and improves its quality.

The following examples illustrate the process according to the invention but do not limit it in any way. Example 1

Laboratory scale fermentation was carried out with the production microorganism Penicillium chrysogenum in brewing flasks on a rotary shaker. The inoculum was prepared by standard procedure on broth containing maize extract, sucrose and nutrient salts, and broth containing soybean flour, precursor, chalk and a sulfur source were used in the production phase. Sucrose was used as a carbon source and was continuously dosed until the 4th or 8th day of fermentation. The dependence of penicillin V phydroxy derivative on sucrose dosage is shown in Table 1.

Table 1

Days fer- mentace Sucrose dosed into 4th day of fermentation 8th day of fermentation p-hydroxy- derivative mg / ml prekur- zor mg / ml p-hydroxy- derivative mg / ml prekur- zor mg / ml 7. 1.1 2.9 1.0 3.0 8. 1,2 2.1 0.9 2,0 9. 1.3 1,8 0.7 1.4 10. 1.6 1.7 0.6 1,2 11. 1.9 1.5 0.5 0.6

If the dosing time of sucrose is prolonged, a favorable ratio of penicillin V to its p-hydroxy derivative and the remainder of the precursor is achieved. The content of p-hydroxy derivative of penicillin V is not higher than 0.5 mg / ml, the content of phenoxyacetic acid does not exceed 0.6 mg / ml. Example 2

Fermentation in 20 liter fermentation tanks was performed with the production microorganism Penicillium chrysogenum. The inoculum was prepared on soil containing corn extract, sucrose and nutrient salts or soy flour, sucrose and salts. The fermentation broth contained soy flour, precursor, chalk and a source of sulfur. Sucrose was continuously dosed either up to 100 hours or until the end of fermentation, i.e. up to 160 hours. The dose dependence of sucrose p-hydroxy derivative for penicillin V is shown in Table 2.

Table 2

Days of fermentation Sucrose dosed into 100. hod. 160. hod. p-hydroxy- derivative mg / ml prekur- zor mg / ml p-hydroxy- derivative mg / ml prekur- zor mg / ml 25 0.1 6.3 0.1 5.8 53 0.2 5.8 0.2 5.0 75 0.3 4.3 0.2 4.3 100 ALIGN! 0.6 3.3 0.5 3.1 125 0.7 2.5 0.6 2.2 150 1,2 2.5 0.6 1,8 164 1,2 2.4 0.6 0.9

When dosing sucrose throughout the fermentation, the content of penicillin p-hydroxy derivative V is 0.6 mg / ml and the unused precursor content is less than 1 mg / ml after the fermentation is complete.

SUBJECT OF THE INVENTION

Claims (1)

A method of producing penicillin in culturing a production microorganism of Penicillium chrysogenum in a liquid culture medium containing an assimilable carbon source which is dosed during cultivation and a source of assimilable nitrogen, mineral nutrient salt and phenoxyacetic acid as a precursor under submerged conditions, characterized in that the source is assimilable The amount of assimilable carbon source is reduced to the amount needed to maintain the culture in the subsequent production phase, after which the assimilation of the source of assimilable carbon source as in the growth phase is restored after starting increasing the content of penicillin V p-hydroxy derivative in the soil. The last batch is added 12 to 24 hours before the end of the production phase.