DE2002200A1 - Method for breeding a L-asparaginase-rich bacterial cell mass - Google Patents

Description

Patent attorney Dipl. -Ing. F.We te km at ν, ZüU Z 2 UU

H-WEiCKMANN, graduate phys. Dr. K. Fincke . F. A. Weickmann, Dipl.-Chem. B. Huber

HZW 8 MUNICH 86, DEN

. ; PO Box 860 820

MÖHLSTRASSE 22, NUMBER 4S 3921/22

BOEHEIIiGER IiAMHEIM GMBH *, Mannheim

Method of growing a; L-asparaginase-rich bacteria

cell race ■.

The invention relates to a method for growing a ! -Asparaginase submit bacterial seed mass, in particular continuous breeding process.

! -Asparaginase, a hydrolytically active enzyme that L-asparagine splits into aspartic acid and ammonia in animal and plant tissues and body fluids ao. as in yerschisdenen microorganisms. Particularly noteworthy is the occurrence of an! -asparaginase in guinea pig serum, which have an inhibiting effect on various has transplantable mouse and rat lymphomas. It also shows a high level of activity against certain leukemic Cells in humans. The same is known for L-asparaginases from E. coil and some other microorganisms such as Serratia marcescens and Erwinia carotovora. Be followed everywhere efforts to develop best practices to meet the corresponding need for ti-asparaginase preparations reported with antileukemic efficacy.

A method is known from OS 19 04 330 in which a Microorganism of the genus Serratia in one to one for Growth of the microorganism sufficient value limited

ventilation is cultivated in an aqueous nutrient medium which contains at most 1 wt .- # of a carbon source. From OS 19 04 850 a method for cultivating L-asparaginase-rich E. coli is known, in which cultivation is carried out in a glycerol-containing nutrient medium which optionally contains casein or soybean meal degraded with pancreatic ferment, sodium chloride and dipotassium phosphate. Au3 US Patent 3,440,142 I3T a method is known, in which E. coli grown with vigorous aeration for 18 hours at 37 ⁰ C on a medium containing dextrose, yeast extract, peptone and potassium phosphates.

Common to all known methods is the disadvantage that the cultivation results in relatively low levels of asparaginase in the harvested Microorganism leads. Another disadvantage is that the process does not allow for continuous growth are suitable.

There is therefore a need for a growing method for Microorganisms, which the content of the desired! -Asparaginase increases. What is also desired is a process which can be carried out continuously.

These disadvantages are overcome by the invention.

In kinetic investigations of the breeding process in batch culture under certain conditions, a two-phase Growth of the microorganisms is observed, which is noticeable in a sharp kink in the linear curve power that is obtained when in a known manner the natural logarithms of the cell masses that have grown over applied over time. Surprisingly, it was found that, depending on the test conditions, after 3 to 5 hours initially rapid growth there is a significant slowdown in growth. Only step in the second phase considerable L-asparaginase narrowing while in the first Phase forming copious amounts of intracellular asparagine, the

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decreases sharply in phase 2.

The method according to the invention for growing an L-asparaginase series Bacterial cell mass, therefore, consists in first placing the bacteria in a precursor under strong ventilation of the citric acid cycle and amino acids, especially those of the aspartic acid family, containing sewing medium at .pH 7 to 8.5 lets grow until the critical point of the growth curve is reached and then allowed to mature in the medium depleted in oxygen and nutrients while maintaining the pH value and the cell mass gains.

Acetic acid is preferred as the precursor of the citric acid cycle or / and glutamic acid is used. It is important that in the first phase it is strongly ventilated, i.e. a lot of oxygen is supplied will. Sufficient intracellular adenosine phosphate as Energy donator are formed. ,

The addition of glucose surprisingly has a disadvantageous effect, and the addition of glycerol is also undesirable.

In the course of the cultivation an increase in the pH value can be observed, so that acids are preferred to keep the pH constant be admitted. The acetic acid used with preference acts as a self-consuming neutralizing agent. Lactic acid was also found to be suitable, but it does lead not to the enzyme quantities achievable with acetic acid *

After reaching the break point of the growth curve, the second phase of the method according to the invention begins, the so-called Starvation phase. During this phase of hunger, oxygen is depleted and at the same time the already emaciated breeding ground no longer added. However, the pH also becomes preferred in the second stage kept constant. .

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The amino acids of the so-called Aspartic Acid Farailie are expediently added in such an amount that an excessively high population density is not obtained in the cell broth. Getting too much Amino acids added, the growth broth becomes too thick and foams. The appropriate amino acid concentration can be easily determined determine that these amino acids have been used up in the area of the break point of the aforementioned growth curve.

In the figure of the accompanying drawing, the aforementioned growth curve shown. It gives the dry mass of bacteria in natural logarithms over time. You can see clearly that at about 5 hours the growth curve, which can be represented by a straight line, has a sharp kink and goes along with it a second growth curve intersects.

This physiological behavior of the cultures enables the cultivation of cell masses optimally containing L-asparaginase. According to the invention, this is possible in batch operation, but requires a corresponding effort in terms of monitoring. This leaves avoid each other while achieving optimal operation through continuous breeding.

A preferred embodiment of the invention is therefore that it is carried out continuously by a first as much fresh nutrient medium is supplied to the strongly ventilated fermenter that the bacterial density is constant and close to the value of the critical point of the growth curve (intersection of the two Straight line) and the cell broth formed is fed in the same amount to a second larger fermenter, whose Cell broth content is such that at constant volume the bacterial density is constant and above the value of the first fermenter is held. "

The continuous culture forms an open system that is in constant equilibrium. With a continuous culture. the environmental conditions and the system are kept constant

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can be easily automated with it. The static one Culture (Bateh culture) dominant time factor with start-up phase, exponential growth phase and stationary phase of growth the withering phase is followed by the continuous Culture turned off. The conditions of equilibrium can be calculated from the growth curve:

Change in bacterial density = growth - washout rate = 0 or in mathematical formulation r "■ '" ■

- μΧ * DX = 0

If the growth rate μ and the dilution rate D are equal to one another in this differential equation, the result is that the bacterial density X in the culture vessel or fermenter remains constant. The exponential multiplication of the cells is compensated for by aen negative exponentieilen process of leaching, μ (i / h) is the specific Waehstumsrate and D (1 / h), the dilution rate, flow rate F (L / h) divided by the working volume V (1) , ie D indicates the volume change per hour. For the case of the growth curve shown in the attached drawing, this results in:

I, D = μ = tg * = £ 1 * 1 =% I £ = 0.375

II. D = μ = tgÄ = | - | ^ = ^ 29 ₌ o, O58

The above equations for the two stages of the invention continuous process so that one in the Chosen execution form in the I. stage with great dilution rate D works and processes this accordingly in the second stage. . diminishes. According to the invention, this is done with the same flow rate F achieved by using appropriate working volumes V.

In the I. stage of the continuous process is preferred Turbidostatic work, i.e. keeping a certain bacterial density or turbidity constant at a dilution rate with maximum bacterial yield. The point of intersection of the two logarithads serves as the working point for the bacterial density Curves, corresponding to the kink in the figure of the attached drawing.

The bacterial culture thus obtained is now fed to the second fermenter, the working volume of which is so dimensioned that at same flow rate (l / h) the calculated low dilution rate is achieved. Matures in this second fermenter the culture producing the high activity of Ii-asparaginase. The process is carried out at constant pH values. the pH values are between 7 and 8.5, preferably between 7.5 and 7.8. The pH value is expedient in both process stages regulated separately. This pH regulation is preferably carried out by adding acetic acid.

The growth curve shown in the drawing applies to E. coli. If another microorganism is used, the angles ^ ¹ and f "change somewhat, as can easily be determined by experiments. The principle remains the same in each case and is based on the fact that a strong formation of! -Asparaginase always only occurs in the starvation phase , ie takes place in the second branch of the growth curve.

The method according to the invention allows a considerable increase the Ii-asparaginase content in the microorganisms. in the Batch processes were here values up to 1 * 5 .... iatfiraationale MnJie.iten_jBrj3 "jiig..Xro ^ e_iji, reached. As a unit here is defines the amount that releases 1 μΜοΙ ammonia from asparagine in one minute under the given üJestbedingungen. In relation to the volume, the achievable activities are 3 IU / ml. In contrast, the OS 19 04-330 maximum 0.66 IU / mg protein and for the procedure

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OS 19 04 850 stated a maximum of 1.62 IU / ml. According to the invention a yield increase of at least 100 # is achieved.

The following example explains a preferred embodiment of the method according to the invention.

example

A plant is used that contains two fermenters. The working volume of the created is 20 liters, that of the second 80 1. The fermenters are equipped with aeration device and stirrers equipped. The filling of the first fermenter is 1.0 l, that of the second 60 1. ^

750 liters of a nutrient medium are placed in a separate container the following composition is made and sterilized;

Acetic acid 0.5 #

Glutamic acid. 0.5 %
Yeast extract 0.1 #. . : ■ · .- ■ "'"'

Protein hydrolyzate (high content of methionine »threonine,. Alanine and lysine) 2.0 $> .

small amounts! phosphate

The substances are tap water in the specified amount dissolved, the pH was adjusted to 7.0 with KOH and the solution sterilized *

First, 60 l of the nutrient medium are added to the second fermenter a vaccination culture door of E * eoli vaccinated separately as SchütteBcultür-was used on the same nutrient medium in 15 hours. This culture is 10 hours without external influx grow through vigorous ventilation (600 1 LUft per hour) ^ calmly. The pH of the fermentation medium is now 7.2. Now 10 1 of the Rultu broth obtained are added to the. first ^ er'-

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menter introduced. The turbidity measurement is used to determine whether the critical point in the growth curve has already been reached. As soon as this is the case, the pumping of fresh nutrient solution is started. At the same time there is vigorous ventilation (600 liters per hour). With an average flow rate of 3 nutrient solutions per hour, 690 liters can be applied fully automatically in 230 hours. With the selected working volumes, the result for the first stage is a dilution rate D of 0.3 »in the second stage of 0.05. An output is obtained with an L-asparaginase activity of 2800 to 3000 TU / l and a specific activity (Iü / mg protein) between 1.3 and 1.5. The yield is 4 to 4.5 g of dry bacterial mass per culture solution is collected in a coolable storage container and the bacterial mass is separated from it on a continuous centrifuge. The pH value control is carried out separately in stages I and II, a total of about 10 g of 100% acetic acid being required per hour.

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Claims (10)

■ 2002280 P a t e η t an s ρ r ü c h e Method for growing an L-asparaginase rich Bacterial cell dimensions, characterized in that the bacteria are first placed in a precursor of the Citric acid cycle and amino acids, especially those .. of the aspartic acid family, containing nutrient medium at pH 7 up to 8.5 can grow until the critical point of the growth curve is reached and then in that of oxygen and nutrients The depleted medium is allowed to mature while maintaining the pH value and the cell mass gains. - / 2. The method according to claim 1, characterized in that work is carried out continuously by adding enough fresh nutrient medium to a first strongly ventilated fermenter that the bacterial density is kept constant and near the value of the critical point of the growth curve and formed Cell broth is fed in the same amount to a second larger fermenter, the Zellbrühg'ehalt is such that at constant volume the bacterial density is kept constant and above the value of the first fermenter. 3. The method according to claim 2, characterized in that the working volume of the second fermenter is 3 to 10 times, preferably 5 to 7 times, the first fermenter. 4. The method according to any one of claims 1 to 3, characterized in that acetic acid is used as the citric acid cycle precursor. 5. The method according to any one of the preceding claims, characterized in that yeast extract, a protein hydrolyzate rich in essential amino acids and phosphate is added to the medium. 10 984771512 6. The method according to claim 2 to 5 »characterized in that that E. coli is grown and a dilution rate of 0.3 and 0.05 is used in the first and second stages, respectively. 7. The method "according to any one of the preceding claims, characterized characterized in that a pH of 7.5 to 7.8 is maintained. 8._ The method according to any one of claims 2 to 7, characterized in that that the pH value is regulated separately in both stages. 9. The method according to any one of the preceding claims, characterized in that the pH - value by adding acetic acid is kept constant. 109847/1512