CS262502B1 - Process for the submersion cultivation of bacteria type pseudomonas producing hydrolase 7 beta-/4-carboxybutanamido /cephalosporic acid - Google Patents

Abstract

The solution concerns the submersible method single cultivation of bacteria producing intracellular hydrolase 7-beta ~ (4- -carboxybutanamido) cephalosporanic acid for the preparation of 7-amino compounds, in particular 7-aminocephalosporanic acids, which is an important intermediate for production semisynthetic cephalosporins. Way Submerged cultivation is that production strains, especially from the genus Pseudomonas they grow in liquid in the first stage of growth medium containing organic a source of assimilable nitrogen and in the other the growth phase is cultivated in liquid nutrient soil containing nitrogenous complex substrates, preferably protein hydrolysates, supplemented with another organic nitrogen source for example, with pre-precipitated corn extract, at a culture temperature of 18 to 35 ° C, aeration 0.2 to 1.0 air volume per volume of culture medium.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention hereinafter referred to as 7-ACK).

By the method of submersive single cultivation of production cells of bacteria of the genus Pseudomonas according to the present process, after separation of the cells from the fermentation liquid, a biomass with high activity of 7-beta- (4-carboxybutanamido) cephalosporic acid hydrolase (hereinafter glutaryl-7-ACK) is obtained. which is then useful for the single enzyme conversion of glutaryl-7-ACK to 7-ACK, which serves as a pharmaceutically important intermediate for the production of semisynthetic cephalosporins, particularly cefazoline and cephalotin.

Several production microorganisms capable of synthesizing said specific hydrolase are known, such as Pseudomonas ovalis, Comamonas (Japanese Patent Nos. 75,101,584, 7859095, Nos. 8,185,298 and 7,894,093), Bacillus, Arthrobacter, Alcaligenes ( Japanese Patent Nos. 77,128,293 and 7,886,094). In MS. No. 239 293 and No. 251 111, the strain Pseudomonas sp. CCM 3,635 and CCM 3,987.

Glutaryl-7-ACK, respectively. derivatives thereof, can be obtained from cephalosporins C or their derivatives either chemically (Japanese Patent Nos. 7,777,078 and 78,112,891), or by enzymatic action of native or immobilized and permeabilized cells with D-amino acid oxidase activity (British U.S. Patent Nos. 1,272,769, Japanese Patent Nos. 7,744,695, 7,788,694, 77,125,696, and 8,023,966).

For submersive cultivation of glutaryl-7-ACK hydrolase strains, suitable culture conditions and suitable substrates are required for the preparation of nutrient broths to ensure the production of enzyme active biomass. It is known that nitrogenous complex substrates such as peptone, yeast and beef extract and casein or dried fish extract (Bonito extract) supplemented with glutamic acid salts are used as nutrient broths for the production of cells with said intracellular hydrolase (Japanese patent specification). No. 75,101,584 and Agric. Biol. Chem. 452 225 (1981). However, the use of conventional complex substrates (peptone, yeast and bovine extract) results in low enzyme production and, in addition, a long culture time is required to achieve maximum hydrolase activity in the cells (up to 8 days). Other substrates are less readily available and require the addition of glutamate.

The present method of culturing bacterial cells with glutary-7-ACK hydrolase activity from -. It avoids these disadvantages since the starting materials for the preparation of nitrogenous complex substrates are advantageous in terms of cost and availability, do not require the addition of glutamic acid salts and when used as nutrient constituents, the maximum enzyme activity required is achieved in a relatively short cultivation time of 27 to 48 hours.

The nitrogenous complex substrates may preferably be acidic, alkaline or proteolytic digestion of the resulting protein hydrolyzates, starting with raw materials of microbial animal and / or vegetable origin, such as keratin, soya flour, peanut flour, gluten or malt sprouts. For the preparation of the nutrient broths, the hydrolyzates may be supplemented with another suitable complex substrate, such as yeast or corn extract.

The present method, submerged cultivation of bacteria consists in that in the first phase of the production microorganism is cultured in liquid medium containing assimilable organic source of nitrogen in an amount of 0.5 to 1.5 g / liter of broth ^-2, which also serves as the carbon source , such as peptone, corn or yeast extract.

The method further comprises culturing in a second phase of growth in a nutrient medium containing protein hydrolysates supplemented with another complex nitrogen source such as yeast and corn extract containing an assimilable nitrogen of 0.5 to 2.0 g.l ^{2 of} nutrient soil.

Prior to the addition of acidic protein hydrolysates to the nutrient broths, the hydrolysates may be neutralized by the addition of suitable alkali such as sodium hydroxide. The corn extract can advantageously be used in pre-precipitated form, i.e. after adjusting the pH of the aqueous corn extract solution in the pH range of 7.0 to 8.0, by boiling and separating the resulting precipitates, filtration or centrifugation. The use of pre-precipitated corn extract eliminates the formation of precipitates in the sterilization of nutrient media that contaminate biomass with the desired hydrolase activity separated from the nutrient media after cultivation.

The method further comprises culturing at a temperature of 18 to 35 ° C and aerating 0.2 to 1.0 air volumes per volume of culture medium. For the inoculation of the nutrient medium in the second growth phase, it is advantageous to use cultures characterized by its transition to a stationary growth phase.

The cultivation in the second phase is terminated after the maximum specific activity of said enzyme is reached, whereupon the cells are separated by centrifugation to give an enzyme-active biomass which can be used as a single source for the production of the mobilized cells according to the cf. No. 231 458 and solidify the resulting particles in accordance with the procedure of Art. No. 251 111 or may be used as a source for the isolation of the enzyme and, if appropriate, its subsequent immobilization.

Production microorganisms from the genus Pseudomonas, preferably Pseudomonas sp. CCM 3,635 and Pseudomonas sp. CCM 3 987. These strains synthesize glutaryl-7-ACK hydrolase in the absence of synthesis inducer in the broth and do not synthesize beta-lactamases.

The amount of this specific hydrolase was quantitatively monitored by measuring the initial rate of hydrolysis of glutaryl-7-ACK in 0.1 M phosphate buffer at pH 7.0 and 37 ° C in the region of zero-order enzyme kinetics. The reaction products were determined spectrophotometrically using prdimethylaminobenzaldehyde (Balasingham et al., Biochim. Biophys. Acta 276, 250, 1972). One unit (U) of glutaryl-7-ACK hydrolase enzyme activity is that amount of enzyme which corresponds to the formation of one micranol (1 µmol) of 7-ACK per minute under the above reaction conditions. Specific activity is defined as Ug ¹ dry cell mass. 7-ACK was monitored by chromatography, using a thin layer of cellulose in a n-propanol-water (7: 3) system with ninhydrin detection (R ^ 0.5) with a semiquantitative evaluation on a Vitatron TLD 100 densitometer. optical density of culture (OD) at 600 nm in 0.5 cm glass cells after centrifugation of cells, decanting the supernatant and suspending the cell sediment in distilled water. The cell dry matter was determined gravimetrically.

The invention is illustrated by the following examples.

Example 1

100 ml of nutrient broth were added to 500 ml boiling flasks: 1% peptone, 0.5% beef extract, 0.1% yeast extract, 0.5 * sodium chloride (pH 7, .2, NH 3-nitrogen content,

g.litre ^{-1 of} broth). The flasks were sealed with cellulose cotton plugs and autoclaved at 120 ° C and 1.2 atm for 20 minutes. They were then inoculated with a culture of Pseudomonas sp. CCM3635 sloped agar loop and cultured on a rotary shaker (240.min ¹ , eccentric 25 mm) at 28 ° C for 48 hours. Then, 5 ml of the grown culture was used to inoculate 100 ml of nutrient medium in 500 ml boiling flasks containing: 5% protein hydrolyzates (commercial product Vitana), 0.1% corn extract, NH ₂ -nitrogen content 1.5 g.litre ¹ nutrient soil, pH 7.2, sterilization at 120 ° C and 1.2 atm for 20 minutes. Cultivation was carried out under the same conditions as the growth of the first generation inoculum for 45 hours.

In one liter of culture liquid, glutaryl-7-ACK 80 U hydrolase production was achieved with an increase corresponding to 3.2 g dry matter. The specific activity was 25 µg ^-1 dry cell mass.

262502 '4

Example 2 g of corn extract (60 4 dry matter) was suspended in demineralized water, pH adjusted to 7.6 with sodium hydroxide solution, made up to one liter with water and heated at 100 ° C for 15 minutes. Thereafter, the resulting precipitates were removed by filtration. 100 ml of nutrient broth with the following composition were added to 500 ml boiling flasks: 1 4 peptone, 25 ml of pre-precipitated corn extract solution, soil pH 7.2, NH ₂ nitrogen content 0.8 g.l of nutrient broth. The flasks were closed with cellulose cotton plugs and sterilized at 120 ° C and 1.2 atm for 20 minutes. After cooling, the soil was aseptically inoculated with a culture of Pseudomonas sp. CCM 3 987 sloped agar loop. Submerged cultivation was performed on a rotary shaker 240.min ¹ , eccentric 25 mm at 29 ° C for 24 hours. Then, 5 ml of culture was used to inoculate 100 ml of broth in 500 ml flasks of the following composition:

4 protein hydrolyzate (commercial product Vitana), 50 ml pre-precipitated corn extract solution, NH3-nitrogen content 1.5 g.l. ¹ nutrient medium, pH soil 7.4, sterilization at 120 DEG C., 1.2 atm for 20 minutes.

After 35 hours of growth, the production of glutaryl-7-ACK 250 U hydrolase was achieved in one liter of culture liquid with a cell growth of 3.8 g dry matter. The specific activity was 65 ^Ug-1 of dry cell mass.

Example 3

To a 2 liter flask was added 150 g of malt sprouts, 450 ml of distilled water, and after stirring, 60 ml of 964 sulfuric acid was added gradually. After shaking, the mixture was heated at 120 ° C and 1.2 atm in an autoclave for 4 hours. After cooling, 350 ml of distilled water was added to the mixture and filtered. The filter material was washed with 250 mL of warm distilled water (60 ° C) and the filtrates were combined.

An inoculum of Pseudomonas sp. CCM 3,635 under the same conditions as in Example 2. 5 ml of grown culture was used to inoculate 100 ml of broth as follows: 20 ml of prepared malt germ hydrolyzate, 50 ml of pre-precipitated corn extract prepared as described in Example 2, the soil was supplemented with demineralized water, soil pH 7.2, sterilization at 120 ° C for 1.2 atm for 20 minutes. The flasks were cultured on a rotary shaker under the conditions described in Example 2 for 48 hours.

In one liter of culture liquid, glutaryl-7-ACK 75U hydrolase production and cell growth of 3 g dry matter were achieved. Specific activity was 25ug ^one dry cell mass.

Example 4

An inoculum of Pseudomonas sp. CCM 3 987 in 500 ml flasks containing 100 ml of broth of the same composition and the culture was grown in the same manner as described in Example 2. 200 ml (2 flasks) of the prepared first vegetative generation culture were used to aseptically inoculate the inoculation tank as follows:

In a 150-liter stainless steel tank, a pre-precipitated corn extract solution was prepared by dissolving 4.4 kg of the wet mass of the extract in 100 liters of demineralized water, adjusting the pH of the solution to pH 7.6 with 504 sodium hydroxide solution, water up to 110 liters. The solution was sterilized for 30 minutes at 120 ° C and 1.2 atm, then cooled and transferred to a Sharples separator, and after separation of the sediment, the clear supernatant was transferred to a storage vessel.

In a 1501 liter stainless steel fermentation tank that was pre-boiled and rinsed, equipped with a duplicator, one stainless steel turbine six-blade stirrer 30 cm in diameter, an aerating rim and four stops, 100 liters of nutrient broth were prepared as follows: 14 peptone, 25 liters of pre-precipitated corn extract filtrate ml of silicone antifoam SAG 471 (Belgium). The soil was made up to a total volume of 20 liters with drinking water and the pH of the broth was adjusted to 7.2 with 40% sodium hydroxide with stirring. The soil in the tank was sterilized for 40 minutes at 120 ° C and 1.2 atm. Then, the soil was cooled to 28 ° C and maintained at this temperature by thermoregulation. 200 ml of the first vegetative generation culture was also aseptically added to sterile fertile soil in the seed tank and cultivated at 28 ° C, aeration of 0.5 volume of air per volume of culture medium> at 300.min ¹ mixing frequency. During culture, growth of the culture was monitored by measuring the optical lensity culture. At the transition to the stationary phase of culture growth, at 8pm,

The inoculum grown in the seed tank was used to inoculate the production tank.

150 liters of nutrient medium were prepared in a 25 liter stainless steel tank, which was equipped analogously to the inoculation tank, except that two six-blade stirrers were placed on a common mixer axis and equipped with only one stop:

% acid protein hydrolyzate (commercial product Vitana), 75 liters pre-precipitated corn extract solution pumped from the reserve tank, 25 ml silicone antifoam SAG 471. The total tank volume was made up to 150 liters with drinking water and the pH of the broth adjusted with 40% sodium hydroxide solution to value 7.4. The soil in the tank was sterilized for 40 minutes at a temperature of 120 ° C and a pressure of 1.2 atm.

After cooling the soil, inoculation of sterile culture broth was performed by releasing 7.5 liters of culture grown in the seed tank through a line immediately steamed before inoculation (5% inoculum). Cultivation was carried out for 30 hours at 25 ° C under the same air and agitation parameters as the seed tank. During the cultivation, samples were taken at 4-hour intervals, after 18 hours of cultivation at 2-hour intervals to determine enzyme activity (washing the cell sediment with 0.1 M phosphate buffer pH 7.0), as described in the description of the invention, and washing the sediment with water).

In one liter of culture liquid, glutaryl-7-ACK 190 U hydrolase production was achieved with a cell growth of 3.8 g dry matter. After 30 hours of submersive culture, the entire volume of the fermentation liquid was discharged and the cells separated on a Sharples centrifuge. The sediment was further washed with 50 liters of demineralized water.

From 150 liters of fermentation liquid, 2.5 kg of wet cell paste with a dry matter content of 22.5% and a specific activity of 50 µg ^-1 dry cell mass, i.e. 562 g of dry matter cell with a total glutaryl-7-ACK hydrolase activity of 28,100 U, were obtained.

g of the washed cell paste was mixed with 90 ml of a 1% glutaryl-7-ACK solution (active ingredient content 95.5%), obtained by mixing 9 g of glutaryl-7-ACK with 50 ml of 0.1 M phosphate buffer pH 7.0, dosing with 1.1 M sodium hydroxide solution while stirring and continuously adjusting the pH to 7.0, and adding the same buffer to a total volume of 90 ml. The mixture was incubated with stirring at 37 ° C for 2 hours. After this time the cells were separated from the reaction mixture by centrifugation and the concentration of 7-ACK in the supernatant corresponding to 80% of the theory of conversion.

Example 5

In a 1 m ³ stainless steel tank, a pre-precipitated corn extract solution was prepared by dissolving 18 kg of corn extract in 420 liters of demineralized water, adjusting the pH of the solution to pH 7.6 with 40% sodium hydroxide solution, and total volume of 450 liters and further processed in the same manner as in Example 4.

A culture of the first vegetative generation inoculum of Pseudomonas sp. CCM 3 987 and the second vegetative generation in the seed tank under the conditions and method set forth in Example 4.

In a 1 m ³ stainless steel tank equipped with a duplicator, two agitators with six 35 cm blades, placed on a common axis at a distance of 120 cm and four stops.

800 liters of nutrient broth were prepared as follows: 5% acidic protein hydrolyzate (commercial product Vitana), 400 liters of pre-precipitated corn extract solution pumped from the reserve tank and 135 ml of SAG 471 silicone antifoam. The volume was made up to 800 liters with potable water. The pH correction and the sterilization of the broth were carried out as described in Example 4.

After cooling the soil, the sterile broth in the production tank was inoculated by pressure-relieving with a pre-steamed pipe of 40 liters of the second vegetative generation culture from the beginning of the stationary phase of growth, ie at 20 hours of culture (5% inoculum). Submerged cultivation and monitoring were performed in the same manner as in Example 4. The cultivation lasted 30 hours.

In one liter of culture liquid, glutaryl-7-ACK hydrolase production of 160 U was achieved at an increase of 3.7 g dry cell mass. After separating the cells in the same manner as in Example 4, 12.18 kg of wet cell paste with a dry matter content of 23% and a specific activity of 43.2 µg of dry cell mass were obtained from 800 liters of fermentation liquid, ie 2,801 g of dry cell content with a total activity of 121,003. AT.

Hydrolysis of the glutaryl-7-ACK solution was performed in the same manner as in Example 4. The conversion of glutaryl-7-ACK to 7-ACK was 85% of theory in 2 hours.

Example 6

An acid hydrolyzate of peanut flour was prepared as follows. To 17.5 liters of drinking water were added 2.5 liters of technical sulfuric acid (96%) and 3.75 kg of peanut flour. The suspension was hydrolyzed for 8 hours with stirring at 120 ° C and 1.2 atm. After this time, the hydrolyzed suspension was neutralized with a 40% sodium hydroxide solution to pH 7.0 and made up to a volume of 50 liters with potable water. In the same manner as in Example 4, a pre-precipitated corn extract solution was prepared.

A culture of the first vegetative generation of Pseudomonas sp. CCM 3 987 and the second vegetative generation inoculum of the same strain in a seed tank of the same equipment, with the same soil composition and embodiment as in Example 4.

A 150 liter stainless steel production tank, equipped as in Example 4, was prepared with 150 liters of broth of the same composition as in Example 4, but instead of the protein hydrolyzate, 37.5 liters of peanut flour acid hydrolyzate was added. Submerged culture was carried out for 48 hours under otherwise the same conditions as described in Example 4.

In one liter of culture liquid, glutaryl-7-ACK 80 U hydrolase production was achieved with a cell growth of 3.5 g dry cell mass. The yield of wet cell paste of 150 liters of the culture liquid after the separation was 2.25 kg dry matter content of 23% and a specific activity of 22.8 ^Ug-1 of dry cell mass, i.e. 517.5 g dry cell weight of the total activity of 11,799 U conversion of the solution of glutaryl The -7-ACK to 7-ACK under the reaction conditions of Example 4 was run for 4 hours, resulting in 78% of theory.

Example 7 kg of malt sprouts were suspended in 90 liters of demineralized water and, after stirring, 12 liters of sulfuric acid (96%) were gradually added. The mixture was heated with stirring at 120 ° C and 1.2 atm for 4 hours. After cooling, 70 liters of water were added and the contents were drained and filtered. The material was washed on the filter with 50 liters of warm drinking water (60 ° C) and the filtrates were combined and neutralized with a 40% sodium hydroxide solution to 7.0. In the same manner as in Example 4, a pre-precipitated corn extract solution was prepared.

The first and second vegetative inoculum of Pseudomonas sp. CCM 3 987 in the same manner as in Example 4. The method and procedure of submersible cultivation including the fermentation apparatus used was also the same as in Example 4. However, the cultivation was carried

48 48 hours in broth in which the acid hydrolyzate of malt sprouts was used in place of the protein hydrolyzate Vitana at a concentration of 20%, ie 30 liters per 150 liters of broth.

The production of glutaryl-7-ACK 150 U hydrolase was achieved in 1 liter of cutter liquid with a cell growth of 3 g dry matter. The yield of wet cell paste after separation from 150 liters of culture liquid was 1.87 kg with a dry matter content of 23% and a specific activity of 50 µg of ⁴ dry cell mass, i.e. 430 g dry matter cell with a total activity of 21,500 U.

The enzymatic conversion of glutaryl-7-ACK to 7-ACK under the reaction conditions of Example 4 was 80% of theory.

Claims (2)

A method of submersible single cultivation of 7-beta- (4-carboxybutanamido) -cephalosporanoic acid hydrolase producing bacteria of the genus Pseudomonas, characterized in that in a first growth phase the producing microorganism is cultured in a liquid nutrient medium containing an organic assimilable nitrogen source of 0 5 1.5 ⁴ g.litr soil and in the second phase growth is cultured in liquid medium containing nitrogenous complex substrates, preferably of microbial protein hydrolysates, animal and / or vegetal origin, supplemented with additional organic nitrogen source, preferably a solution of corn preshrinking resulting extract by boiling at pH 7.0 to 8.0 and subsequent separation of the precipitate, when the quantity of assimilable nitrogen of 0.5 to 2.0 g.litr ^- ^ broth at a cultivation temperature of 18-35 ° C, air flow 0, 2 to 1.0 volume of air per volume of nutrient medium; fits in the transition of culture to the stationary phase of growth. 2. Method according to claim 1, characterized in that bacterial strains of the genus Pseudomonas, preferably Pseudomonas sp. CCM 3,635 or Pseudomonas sp. CCM 3,987.