DE2366505C2 - Use of the strain Pseudomonas ATCC 21973 for the in situ production of an aminotransferase - Google Patents

Description

The present invention relates to the use of the strain Pseudomonas ATCC 21 973 for in situ production an aminotransferase, which is thermally stable and resistant to physicochemical conditions Conversion medium is insensitive and fumaric acid to L-aspartic acid at high speed able to convert, in which under aerobic conditions the microorganism is cultivated in a nutrient medium which contains a carbon source, a phosphorus source, a nitrogen source and oligo elements

The carbon source preferably consists of sugar beet molasses or sugar cane molasses.

The invention is summarized in the preceding claim

Aminotransferases are enzymes that transfer an amino group from one substrate (donor) to a second substrate (Acceptor) transferred. An aminotransferase is used, for example, to produce aspartic acid from fumaric acid. There are already numerous processes for the production or isolation of Aspartic acid is known from natural products, but only those processes that rely on a bio-conversion are known fall back, applicable on an industrial scale. However, all previously known methods are has the disadvantage that the reaction rates are slow, and their implementation is costly is, so that they were only carried out insofar as they led to the manufacture of products that are in the Pharmacy are used.

The use of aspartic acid as a starting compound for the synthesis of various peptides opens up very interesting sales opportunities for this amino acid. However, this requires for implementation on an industrial scale the availability of a very pure aspartic acid, which is under economic Conditions can be established.

The production of aminotransferase, which is responsible for the conversion of fumaric acid into L-aspartic acid is required, the most suitable microorganisms require a complex culture medium for their growth contains sources of organic nitrogen in addition to a source of assimilable carbon, usually Protein hydrolysates, which are starting materials with a non-constant composition that are relatively are expensive. Due to this fact, the known methods using these microorganisms can can be viewed as very complex and unsuitable for industrial production. A well-known one Method using a microorganism of this type and the disadvantages described above is described in FR-PS 13 94 722. The bioconversion of the fumaric acid takes place here Pseudomonas trifolii. This microorganism grows in a nutrient medium made up of glucose and peptone bred. The conversion which takes place at 37 ° C in a neutral medium is very slow.

A new microorganism of the family Pseudomonodaceae has now been found, which is not the disadvantages described above adhere more. This microorganism can use cheaper Means the bio-conversion of fumaric acid to L-aspartic acid at considerable rates effect, these speeds being significantly greater than those of the methods described above. The yields obtained are very high, with the product having a high degree of purity in easy to extract. Production and purification of L-aspartic acid are in the German patent 23 45 271.

This microorganism was found in France near Nesle in the Somme department (80) from liquid waste water a plant that produces sugar from sugar beet. It belongs to the genus Pseudomonas, must however, to be regarded as new because it differs from bacteria of this type by one or more reproducible ones and distinguishes stable properties according to the criteria set out in the 1967 edition of »BERGEY'S MANUAL« are described. These properties are as follows:

Rods: short, isolated or in pairs, little or no mobility

Gram discoloration: negative | »

Colonies: ψ

a) On agar: Round, whitish to pale chamois, shiny translucent at edges, diameter | $ 3 mm after 4 days of incubation at 30 ⁰ C pi

b) on gelatine: round, whitish to yellow, shiny Puncture in a gelatin base: no liquefaction f; \

King's B medium: Creation of a green fluorescent pigment which after 24 hours of incubation ^ '

is not diffusible at 30 ° C · ',?

Milk: not alkalized
Nitrate: no reduction to nitrite

b5 growth on glucose, lactose, sucrose, levulose, mannitol. Glycerine, without acidification

Tyrosine medium: no development
Phenol: not attacked
No generation of indole, hydrogen sulfide or gas

No lysine decarboxylase

Aerobia: good growth between 30 and 37 ° C

The isolation of the microorganism can take place under the following conditions and in compliance with the following The usual isolation and cleaning methods described are carried out.

0.1 ml samples of the drainage from a factory producing sugar from sugar beet are spread out in a Petri dish containing a solid agar-agar-med ^: um A of the following classic composition:

Beef extract 20 g / l Peptone 20 g / l Glucose 4 g / l NaCl 5 g / l Agar Agar 15 g / l

10

Incubation is established after 24 to 30 hours incubation is to the occurrence of numerous colonies at about 30 ⁰ C.

Each of these colonies is removed by spreading on the medium described above as well to be cleaned under the specified conditions.

A microscopic examination is sufficient to determine the morphological homogeneity of a bacteria to confirm each isolated and purified Pseudomonas colony.

The pure culture strains thus obtained are based on that described above Α-medium preserved in a test tube that has been cooled in an oblique position.

Finally, the strains are examined for their ability to produce L-aspartic acid, namely by growing in a substrate containing fumaric acid. The following comparison table shows the production capability of 3 Pseudomonas strains isolated during the same separation procedure ("Screening").

Cultivation time of the microorganism in hours 115 178 18th 115 178 24 115 178 14th 36 36 Content of fumaric acid for 36 Start time, g / l Extent of bio-conversion according to 5-day bio-conversion (in %) 0 0 1 1 1.2 1 Tribe: 4th 7th 0 5 6th 1 6th 7th PO 205 0 0 0 3 0 0 3 0 0 PO 7111 (invention) 1 0 0 PO 91 0

35 40

In contrast to the previously known other microorganisms that are used as aminotransferase producers have been, the strain of the invention can develop in a simple medium that for example essentially composed of the residues of the sugar extraction, d. H. from beet sugar or cane sugar molasses which have been provided with a mineral phosphorus addition, for example in the form of a phosphate or in the form of phosphoric acid, as well as an addition of nitrogen, for example in the form of urea and ammonia, some oligoelements also having been added.

The use of sugar beet molasses or sugar cane molasses as the carbon source is preferred.
H The following example shows how the use of molasses has a beneficial effect.

I example 1

^b! '' Two culture media are produced in parallel, which have the following compositions:

55

Artificial medium (medium 1)

60

Glucose 60 g / l Maize macerating liquid 40 g / l MgSO ₄ · _{7H 2} O 0.5 g / l Monopotassium phosphate 5 g / l Fumaric acid lg / 1 Molasses medium (medium 2) Molasses expressed as total sugar 60 g / l Monopotassium phosphate 5 g / l MgSO ₄ · 7 H ₂ O 0.5 g / l urea 8 g / l

After sterilizing the medium by heating to 121 ° C for a period of 20 minutes the pH is adjusted to 7 to 7.2. For this purpose, gaseous ammonia is used under sterile conditions.

Each medium is then inoculated with a salt suspension of the strain PO 7111. The breeding takes place in 6 liter containers containing 1 l of the medium, using a stirrer.

Bacterial development is determined by measuring the optical density after diluting it by 20 times determined The development of the aminotransferase activity is determined according to the method described in »Methods in enzymology «.

Breeding Medium 1 Activity, Medium2 activity duration, hours bacteria expressed in bacteria expressed in population/ intern. amino population/ intern. amino ml transferase ml transferase Units / 1 / hour Units / 1 / hour 0 IxIO ⁸ 18.8 IxIO ⁸ 26th 10 0.8 xi O ⁹ 18th 0.9 xl O ⁹ 24 15th 1x10 '" 19th 0.9x10 '" 32.6 20th 1.1x10 '" 18.8 1.1x10 '" 31.2 24 1.1x10 '" 1.0x10 '"

It can be noted that if the bacterial development is almost identical in the two media, the Aminotransferase activities are increased by 50 to 60% when the microorganism is in the medium 2 is bred.

In general it can be stated that the production of aminotransferase soft fumaric acid converts, the most suitable nutrient medium corresponds approximately to the following composition:

15 to 60 g / l of sugary molasses (expressed as total sugar), preferably 40 g / l, expressed as Total sugar.

1 to 4 g / l phosphoric acid or mineral phosphates (expressed as phosphoric acid), preferably 2 to 2.6 g / l.

0.1 to 1 g / l Mg (expressed as magnesium sulfate), preferably 0.4 to 0.6 g / l magnesium sulfate.

0.5 to 10 g / l fumaric acid, preferably 0.9 to 1.1 g / l.
the pH of the aqueous medium obtained in this way is adjusted to 7 to 8.5, preferably to a value in the vicinity of 7.

During the cultivation period, which can be between 9 and 15 hours and preferably between If the temperature fluctuates between 10 and 12 hours, the temperature must not exceed 38 ° C.

Likewise, a temperature below 25 ⁰ C prevents the microorganism from growing. An optimal bacterial population density is obtained at 3O ⁰ C.

Traces of oligoelements can be added to the culture medium in an amount of up to 10 ppm either in the form of salts, or one can proceed in the manner that is customary, non-mineralized Add water to adjust the volume of the medium to 1 liter.

The microorganisms used so far have the property that the enzyme they produce thermally unstable and very sensitive to physical / chemical influences of the bioconversion medium especially with respect to pH and temperature.

A systematic investigation of the strain PO 7111 has shown that this microorganism in a Culture medium on the basis of molasses an enzyme can enrich its activity within broader so process conditions for bioconversion of fumaric acid is effective. This is especially true with regard to the Temperature and pH.

Various studies have shown that breeding of the strain PO 7111 on molasses the Formation of a medium which contains an enzyme that is responsible for the amination of fumaric acid in L-aspartic acid is responsible, with its activity increasing with an increase in the transition temperature increases. The activity develops within a temperature range of 20 to 60 ° C. Simultaneously was found that an increase in the pH value maintained during bioconversion has a beneficial effect exerts on the rate of conversion within a pH range of 6 to 12.

In order to investigate the influence of these two parameters on the rate of bioconversion, the Strain PO 7111 grown on a molasses medium as described in Example 1 above. There were 101 of these media to perform each of the examples described below manufactured.

speed

Example 2
Influence of temperature on the bio-wall

2 kg of ammonium fumarate are added to 10 1 of the culture produced after the pH has reached 8 and the temperature has been set to the selected value. During the conversion, the pH

held at 8 by adding gaseous ammonia.

The activity is measured by dosing according to the method as described in "Methods in enzymology", Volume 13, after 1 hour of reaction with stirring and then every hour for a period of 4 hours. The value given in the table corresponds to the mean of five measurements made in this way.

Bioconversion temperature, ⁰ C 20th 30th 37 40 50 57 60 Aminotransferase activity in 0.01 0.05 0.07 0.08 0.12 0.14 0.14 mmol / l / hour

It is seen that the activity almost increases linearly with Bioumwandlungstemperatur between 20 and 50 ⁰ C and then more slowly between 50 and 60 ° C, it is not destroyed even at 60 ° C.

It is thus felt by the bioconversion between 30 and 6O ⁰ C, preferably between 50 and 60 ⁰ C and especially 55-57 ° C as the optimum temperature appears to be at 55 to 57 ° C.

Influence of pH on the rate of biodegradation

The same molasses media fractions as when carrying out Example 2 are used. the Fractions of 10 1 are placed in a vessel equipped with a stirrer and kept at 57 ° C. After 2 kg of diammonium fumarate has been added, the rate of bioconversion becomes in the same way as when carrying out experiment 2.

The values given in the following table represent the mean of five consecutive measurements Measurements (see experiment 2).

pH of the medium during the 7th 7.5 8th 8.5 9 9.5 10 Bioconversion Aminotransferase activity 0.07 0.11 0.22 0.30 0.32 0.34 0.34 in mmol / l / hour

It should be noted that above pH 8.75 the medium tends to release ammonia. To avoid loss of ammonia, the vessel is kept under slight pressure.

The results show that the rate is proportional to the pH between 7 and 8.75 and then slowly rises between 8.75 and 9.50 and then stabilizes above this value. Hence the Bioconversion is carried out at a pH between 8.5 and 10 and preferably between 9 and 9.5.

An investigation into the influence of the concentration of the substrate to be converted, i.e. H. of fumaric acid, has shown that the rate of conversion is best when the content of fumaric acid in the Medium is close to 10%, but the speed is not influenced by the content of it formed L-aspartic acid (within the saturation limits of the solution). Hence it is recommended that procedure by successive additions of the substrate to be carried out in such a way that during one The concentration of fumaric acid is kept in the vicinity of 10% for as long as possible. To however too large an amount of unconverted in the solution used to extract the amino acid To avoid starting product, the supply is cut off during the last hours of the process.

According to a preferred embodiment, the production of the aminotransferase is necessary for bio-conversion is required, carried out in such a way that the strain PO 7111 is incubated aerobically in a culture medium which beet molasses or cane molasses as the only carbon source as well as a limited one Amount of fumaric acid as a potentiating agent for the accumulation of aminotransferase by this microorganism contains

If the optimal content has been reached, then the cultivation is stopped, whereupon the substrate to Conversion to aspartic acid is used

The activity of the enzyme is not influenced by the presence of other microorganisms. It is not necessary to work in a sterile medium during the bioconversion of fumaric acid to L-aspartic acid .

It is therefore possible to use a substrate which has not been subjected to any special treatment. In particular, it is possible to use a pure crystallized fumaric acid such as is conventionally commercially available You can also use a fumarate.

The solid is added to the bioconversion medium without prior sterilization in such an amount that a concentration in the vicinity of 10% is maintained

This bioconversion phase takes place without aeration in order to avoid the formation of undesirable by-products, in particular oxidation products of the ethylene carboxylic acid. The attacked substance is then the diammonium fumarate, if the fumaric acid has been introduced into an ammoniacal molasses medium

The reaction is carried out under shielding from a neutral gas such as nitrogen

During the conversion, the temperature is preferably 55 to 57 ° C and the pH is 9 to 9.5 held by the addition of gaseous ammonia.

After the reaction has ended, that is, when the residual content of fumaric acid has fallen to such values that

the yield is optimal, the mass is subjected to a separation operation, using the usual and can use known separation methods, for example centrifugation, filtration, etc., to the Eliminate microorganisms and all other insoluble compounds.

In this way, a concentrated solution of aspartic acid is obtained, from which the amino acid in is removed in a known manner, for example by ^ solubilization by changing the pH up to to the isoelectric point.

The crystallized product obtained under these conditions is of a purity suitable for industrial use Purposes is sufficient; however, it may be advantageous to further purify until a product is obtained which conforms to pharmaceutical standards by simple recrystallization after decolorization.

Example 4

The optimal conditions for the cultivation of the strain PO 7111 are maintained on an industrial scale in the operation of a pilot plant, which ^{enables the production of 1 m 3 of} a culture which is rich in aminotransferase.

Making the culture

In a fermentation device equipped with a stirrer and a heating device, 1000 l made of a medium with the following composition:

molasses 40 g / l, expressed as total sugar phosphoric acid 2.3 g / l Magnesium sulfate 0.5 g / l Fumaric acid lg / 1 Ordinary water qspl

Before sterilization, the pH is adjusted to 7 using ammonia. The sterilization will carried out by heating to 123 ° C for a period of 30 minutes.

The medium produced in this way is mixed with 201 of the preculture of the Pseudomonas strain PO 7111 inoculates.

The cultivation is carried out in sterile medium at a controlled temperature with stirring as well as with aeration carried out.

The selected process conditions are as follows:

Temperature: 30 ⁰ C

Agitation: 100 rpm

Aeration speed: 0.8 volume / volume / minute

After 1 hour of cultivation, the bacterial population has reached 10 ° bacteria / ml, the pH is 8.95. the enzymatic activity is therefore maximal.

The strain PO 7111 was deposited under the number ATCC 21 973 with the "American Type Culture Collection", and under the number Ferm-P-No. 2269 at the Japanese Agency of Industrial Science and Technology, Research Institute "now" Fermentation Research Institute ".

so comparison experiment

The effectiveness of the ATCC 21 973 strain compared to Pseudomonas trifolii ATCC 14 537, ATCC 12 287 and B-26-9-PY-5 according to FR-PS 13 94 722 shows the following:

Example of
FR-PS 13 94 722

Amount of fumaric acid used

Obtained aspartic acid
lot

Bulge

ATCC 14537

ATCC 14537

ATCC 14537

ATCC 14537

ATCC 12287

B26-9-PY-5

invention
ATCC 21973

75 g / l

50 g / l

100 g / l

120 g / l

75 g / l

75 g / l

100 ») g / l

86 g / l

57.3 g / l
100.25 g / l
134.88 g / l

48 g / l

83 g / l
211 g / l

100 mol%
100 mol%
87.5 mol%
98 mol%
57 mol%
98.8 mol%
95 mol%

*) During the bioconversion, the concentration is always kept at 100 g / l (10%) by adding fumaric acid.

If you compare Examples 1, 2, 5 and 6 of FR-PS 13 94 722 with the invention, you can see that in practically the same length of bioconversion and with a yield of practically 100% according to FR-PS 13 94 722 much more dilute solutions can be obtained.

In Examples 3 and 4, where more concentrated solutions are used according to FR-PS 13 94 722, are required a conversion time that is more than twice as long in order to achieve comparable yields.

Example 4 of the present invention shows that the remaining fumaric acid is 0.7% that is 3.6%, based on the fumaric acid used, which is good with the value 4% according to Table 1 of FR-PS 13 94 722 is comparable.

After all, it is more important for industrial production to use relatively highly concentrated solutions uses and such can be obtained in a relatively short time than that you work on 100% yield and therefore much longer times or more dilute solutions are required. According to the invention, you can always use it work a 10% solution and get a yield of 95 mol% in a relatively short time 20% solution of quite pure aspartic acid.

Claims (1)

Claim: Use of the strain Pseudomonas ATCC 21 973 for the in situ production of an aminotransferase which thermally stable and insensitive to physico-chemical conditions of a conversion medium and is able to convert fumaric acid to L-aspartic acid at high speed by aerobic cultivation of this microorganism in a nutrient medium that contains a source of carbon, a source of nitrogen, contains a source of phosphorus and oligoelements.