DE1642718A1 - Process for the production of amino acids from hydrocarbons - Google Patents

Description

KYOWA HAKKO KOGYO Co., Ltd., Tokyo, Japan

Process for the production of amino acids from coal

materials

The process for the preparation of amino acids, such as. B. L-glutamic acid, L-lysine, L-ornithine, L-valine and L-homoserine by fermentation from hydrocarbons as the main source of carbon involves the cultivation of a for Assimilation of hydrocarbons capable microorganism in a mixture with a for the production of amino acids capable microorganism under aerobic conditions in an aqueous nutrient medium which contains at least one Contains hydrocarbons. High yields of amino acids can be obtained if a mixed culture from, for example, Micrococcus glutamicus with a hydrocarbon assimilating microorganism such as Arthrobacter paraffineus or Brevibacterium ketoglutamicum.

Documents (Art. 7 i 1 Paragraph 2 No. I Clause 3 of the Xnrferunjjsges. Ν. 4.9.18 * 7)

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The invention relates to a method for producing Amino acids, in particular for the production of amino acids by fermentation and relates in particular to a process for the production of various amino acids by fermentation from hydrocarbons as starting material.

Processes for fermentative production have recently been used various useful substances from hydrocarbons using the so-called hydrocarbon assimilating microorganisms that grow in hydrocarbons, which serve as the main source of carbon and can reproduce in it. However, the type of fermentation product that can be obtained thereafter is limited until now. In addition, in most cases, insufficient product yield was obtained.

The invention relates to a process for the preparation of amino acids, which are used on a large scale in many fields of application Find use using hydrocarbons as a starting material, the hydrocarbons are easily accessible at low cost. Therefore, the method according to the invention can be used on an industrial scale Manufacturing are used.

One object of the invention is an improved method for the production of amino acids, which the after-

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parts and shortcomings of previous procedures eliminated.

The invention further provides a process for the production of amino acids by fermentation which can be carried out in an efficient and can be done easily. Another object of the invention is a method of manufacture various amino acids through fermentation, which is progressively carried out on an industrial scale under Ver f

using readily available raw materials and under Achieving high product yield can be carried out.

According to the invention it has been found that many useful substances, in particular amino acids in high yields of a hydrocarbon can be prepared as the main carbon source of a culture medium using at least by hydrocarbon assimilating microorganisms together with microorganisms having no or g low hydrocarbon Assimilierfähigkeit, to be cultivated. By cultivating a microorganism capable of assimilating hydrocarbons and a microorganism capable of producing amino acids together, it is possible to produce amino acids in the culture liquid in high yields.

It can be a wide range of hydrocarbon assimilating agents

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Microorganisms can be used in the method of the invention because of the direct ability to produce amino acids does not depend on it. As usable according to the invention Hydrocarbon assimilating microorganisms can therefore be used in a wider range than in the Production of amino acids by a single assimilating microorganism. This fact. Is one of the most essential Features of the invention. Another particular advantage of the invention is the fact that several. Amino acids can be generated with sufficient application of hydrocarbons, their application so far in the case of a fermentative process ^ in the one only assimilating microorganism was used, was considered difficult or unusable. Also can unsealed raw materials also easily in the present invention Process are used.

Although the scope of the invention is not intended to be limited by any theoretical explanations assumed that the fermentation mechanism of the present procedure is believed to be based on the following steps. The hydrocarbon is oxidized and transformed into metabolic intermediates, for example organic acids in the Tricarboxylic acid cycle, broken down by the hydrocarbon assimilating microorganisms, and then the

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Intermediate products converted into valuable amino acids by the action of the mixed microorganisms. It was also observed that even when a capable for producing an amino acid hydrocarbon assimilating microorganism is used directly, the amino acid in the selection of a suitable amino acid producing microorganism and cocultivation can thus be produced in a considerably higher yield than in the case of a simple KuI- ä door. This fact is possibly due to an effective progression of the conversion, which is brought about by a mutual supplementation of the metabolic intermediate, which is often present in insufficient quantities.

Assimilating microorganisms belonging to the genera Pseudomonas , Mycobacterium , Achromobacter and the like can be used in the method of the invention in addition to the above-mentioned hydrocarbon assimilating microorganisms, ie, Corynebacterium , Arthrobacter , Brevibacterium , Micrococcus and the like. Examples of the results of fermentation carried out using combinations of these hydrocarbon assimilating microorganisms with various kinds of amino acid producing microorganisms are shown in Table I.

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TABLE I.

Hydrocarbon

assimilating

Microorganism

Amino acid producing microorganism

Amount of amino acid formed

Arthrobacter
paraffineus
(ATCC 15591)

Micrococcus glutamicus (ATCC 13032) L-glutamic acid 35 mg / ml

Arthrobacter
paraffineus
(ATCC 15591)

Micrococcus glutamicus (ATCC 13058) L-glutamic acid 32 mg / ml

Arthrobacter
paraffineus
(ATCC 15591)

Micrococcus glutamicus (ATCC 13287) L-lysine
2.0 mg / ml

Arthrobacter
paraffineus
(ATCC 15591)

Micrococcus glutamicus (ATCC 13232) L-ornithine 1.8 mg / ml

Brevib acterium
ketoglutamicum
(ATCC 15588)

Micrococcus glutamicus (ATCC 14039) L-valine
1.5 mg / ml

Brevibacterium
ketoglutamicum
(ATCC 15588)

Miaecoccus glutamicus (ATCC 14296) L-homoserine 2.0 mg / ml

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The experiments were carried out as follows. The hydrocarbon assimilating microorganism was put into 20 ml of a Culture medium prepared in a 250 ml flask containing 0.25 Weight? Yeast extract, 0.5 wt. Meat extract, 0.5 wt. Peptone and 0.25 wt. Sodium chloride and had a pH of 7.2, pre-grown. The pre-cultivation took place for 24 hours with aerobic shaking.

Similarly, the amino acid producing microorganism in 20 ml of a 2 % glucose, 1 % peptone, 0.5? Meat extract and 0.25? Culture medium containing sodium chloride with a pH of 7.2.

200 ml of a fermentation medium containing 10 % hydrocarbons (a mixture of n-paraffins with 9 to 18 carbon atoms), 0.1? KH ₂ PO ₁₁ , 0.1? Na ₂ HPO ^. 12H ₂ O, 0.05? MgSO ₄ ^ H ₃ O, 0.002? MnSO ₄ .4HgO, 0.01? FeSO ₁₁ ^ H ₂ O, 0.001? ZnSO ₄ ^ H ₃ O, 1? (NH ₄ ) gSO ^, 0.1 corn steep liquor, 1.0? NZ- * Amin (trade name for a range of casein hydrolysates) and 2.5 / 1 biotin. The two types of pre-cultured for 24 hours, microorganisms were inoculated at the same time in a fermentation medium and cultured for 4 days at 30 ⁰ C under aerobic shaking. The pH was _{adjusted to about 7 S} 2 with urea solution during the cultivation.

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In the examples shown in Table I, two types of inoculum were mixed in equal volumes and inoculated at the same time. It should be noted, however, that the most favorable mixing ratio and the most favorable period of time in to which one or both microorganisms are added to the fermentation medium, can be selected in each case and that the mixing ratio and time are not limited to any particular specific limits.

Both a synthetic and a natural nutrient medium are required for carrying out the fermentation method according to the invention suitable as long as it has the essential nutrients for the growth of the particular strains used. Such Nutrients are known and these include substances such as B. a carbon source, a nitrogen source, inorganic Compounds and the like which are used in suitable quantities by the microorganisms used «As a nitrogen source various types of inorganic or organic salts or compounds, such as. B. urea or Ammonia or ammonium salts, such as. B. ammonium sulfate, ammonium chloride, Ammonium nitrate and the like, or natural nitrogenous substances, such as. B. Corn steep liquor, Peptone, meat extract, yeast extract, casein hydrolyzate, fish meal and the like can be used. Mixtures of two or

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several of these substances can be used. Among the inorganic compounds added to the culture medium include magnesium sulfate, potassium dihydrogen phosphate, Potassium monohydrogen phosphate, sodium chloride, iron sulfate as well as other salts of magnesium, iron, manganese, zinc, calcium and the like. Mixtures of such inorganic compounds can be used. Likewise, nutrients that are essential for the growth of the specially used " Strains are added to the medium, e.g. B. vitamins such as biotin, thiamine, pantothenic acid, nicotinic acid and the like

Hydrocarbons are used as the main source of carbon in the process of the invention. Usable hydrocarbons include straight-chain and branched-chain paraffins (alkanes) with 9 to 18 carbon atoms, ζ. Β. η-Νοηβη, n-decane, n-dodecane, n-hexadecane, n-octadecane, iso- | decane and the like, cycloparaffins, e.g. B. Cyclohexane and Cyclooctane, straight-chain and branched-chain olefins such as, for example, pentene-2, hexene-1, octene-1, octene-2, and the like. Cycloolefins, such as. B. Cyclohexene, aromatic hydrocarbons, such as Benzene, o-xylene, p-xylene and the like and mixtures thereof, as well as mixed hydrocarbons such as e.g. Kerosene, light oils, heavy oils, paraffin oils and the like.

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Small amounts of other carbon sources, such as B. glucose, Fructose, mannose, galactose, sucrose, starch, starch hydrolysates, waste molasses and the like can be used in the fermentation medium along with the hydrocarbons.

The fermentation of the mixed cultures is carried out under aerobic conditions, e.g. B. aerobic shaking of the culture or with stirring a submerged culture, carried out at a temperature of about 20 to 40 ⁰ C and a pH of about 4.0 to 9.0. After completion of the fermentation, the amino acids obtained are separated from the fermentation liquid by conventional means such as ion exchange resin treatment, extraction with solvents, precipitation with metal salts, chromatography or the like.

The following examples serve to illustrate the invention without limiting it. Unless otherwise stated, The percentages relate to the weight per liter of water.

example 1

20 ml of a culture medium containing 0.25% yeast extract, 0.5 % meat extract, 0.5% peptone and 0.25 % sodium chloride,

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which is contained in a 250 ml flask is inoculated with Arthrobacter paraffineus ATCC 15591. The medium has a pH of 7.2.

20 ml of a culture medium containing 2% glucose, 0.5 % meat extract , 1 % peptone and 0.25 % sodium chloride, prepared in a further 250 ml flask, is mixed with Miero

coccus glutamicus 541 ATCC 13058 inoculated. The nutrient medium ™

also has a pH of 7.2.

Both microorganisms are cultured for 24 hours under aerobic shaking at 30 ⁰ C. The cultures obtained are then inoculated into 200 ml of a fermentation medium which is contained in a 2 l flask and has the following composition:

10 % hydrocarbons (a mixture of η-Pa- _Λ

refines with 9-18 carbon \ 0.1 % KH ₂ PO ₁₁ carbon atoms)

0.1 %

0.05 ί

0.002 %

0.01 %

0.002 % ₁ Y ₂

1 % (NH ₁ Y) ₂ SO ₁ Y

0.1 % corn steep liquor.

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- 12 The medium has a pH value of 7.2.

The fermentation is then carried out while cultivating the mixture. Microorganisms at 30 ° C with aerobic shaking during Carried out 4 days. The drop in pH is achieved by adding EH ^ OH to the medium during cultivation set pretty much exactly to 7.2.- When the fermentation is complete, 35 mg / ml L-glutamic acid have been found in the Culture fluid formed.

The fermentation liquid is used to adjust the pH to 9.0 Calcium hydroxide is added and then the medium is heated. After the microorganism cells have been filtered off, the filtrate is concentrated. By adjusting the pH to 3.2 with hydrochloric acid and cooling the concentrated filtrate, the calcium salt of L-glutamic acid crystallizes out. After filtering off of the crystals obtained, 5.2 g of the calcium salt of L-glutamic acid are obtained.

Example 2

20 ml of a nutrient medium prepared in a 250 ml flask and containing 0.25 % yeast extract, 0.5 % meat extract, 0.5 % peptone and 0.25 % sodium chloride, and one

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Has a pH of 7> 2 are inoculated with Brevibacterium ketoglutamicum ATCC 15588. 20 ml of a medium prepared in another 250 ml flask containing 2 % glucose,

1 % peptone, 0.5 % meat extract and 0.25 % sodium chloride and has a pH value of 7 »2 are inoculated with Micrococcus glutamicus 901 ATCC 13287.

Both microorganisms are cultured for 24 hours at 30 ⁰ C under aerobic shaking. 200 ml one in one

2 1-flask containing fermentation medium, which follow the components:

10 % hydrocarbons (a mixture of n-Pa-

refined with 9-18 coals

0.1 ί KH ₂ PO ₁₁ substance atoms)

0.1 % Na ₂ HPO ₄ .12H ₂ O

0.05 %

0.002 %

0.Ol %

0.002 % ₁ Y ₂

1 % (NH ₄ I ₂ SO ₁₁

Ο, ΐί corn steep liquor 1 % NZ amine

10/1 biotin

are inoculated with the cultures obtained. The medium has a pH of 7.2.

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The fermentation is then carried out with the mixture of microorganisms by 4-day cultivation at 30 ° C under aerobic Shaking done. The drop in pH of the medium is caused by the addition of ammonium hydroxide to the medium adjusted to about 7> 2 during the cultivation. After completion After fermentation, 2.0 mg / ml L-lysine was formed in the culture medium.

After the microorganism cells are filtered off by centrifugal separation, the filtrate is treated with hydrogen chloride acidified. The L-lysine is purified and purified by an ordinary method using ion exchange resins severed. 0.31 g of L-lysine hydrochloride is obtained.

Example 3

20 ml of a nutrient medium prepared in a 250 ml flask, which contains 0.25 % yeast extract, 0.5 % meat extract, 0.5% peptone and 0.25 % sodium chloride and has a pH of 7.2, are added Pseudomonas aeruginosa ATCC 15246. The cultivation is carried out for 24 hours at 30 ° C. with aerobic shaking. The culture obtained is then poured into 200 ml of a fermentation medium contained in a 2 l flask and having the following composition:

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10 % kerosene

0.1 ί KH ₂ PO ₁₁

0.1 % Na ₂ HPO ₁ J. 12H ₂ O

0.05 % MgSO ₁₁ ^ H ₂ O

0.002 % MnSO ₁ , .4H ₂ O

0.01 % FeSO ₄ .7H ₂ O

0.002 % ZnSO..7H ₉ O

1 % (NH ₄ ) ₂ SO ₄ 0.1 % corn steep liquor

inoculated. The medium has a pH of 7 * 2.

The cultivation is performed for 4 days at 30 ⁰ C under aerobic shaking. After 24 hours, a culture of Micrococcus glutamicus 541 ATCC 13058 is added to the fermentation medium. The latter culture is obtained by adding 20 ml of a medium contained in another 250 ml flask j which contains 2 % glucose, 1 % peptone, 0.5 % meat extract, 0.25 % sodium chloride and has a pH of 7, 2, inoculated with Micrococcus glutamicus 541 ATCC 13058 and cultivated for 24 hours with aerobic shaking at 30 ° C. During fermentation of the mixed cultures, the pH of the medium is lowered by adding NH ₄ OH to the medium

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set about 7.2. After the fermentation has ended, there is 12 mg / ml L-glutamic acid in the fermentation liquid educated.

After filtering off the microorganism cells by centrifugal separation the filtrate is concentrated. By adjusting the pH to 3.2 with hydrochloric acid and cooling the filtrate, 2.1 g of crude crystals of L-glutamic acid are obtained. Obtained by recrystallization one 1.5 g of L-glutamic acid.

It can be seen that the principle of the present invention resides in the production of amino acids by fermentation using hydrocarbons as the main carbon source, with a hydrocarbon assimilating A microorganism and an amino acid producing microorganism are cultured together in admixture. The invention is therefore not restricted to any specific types of microorganisms, but to all of them Applicable to microorganisms falling within these two outlined categories. Microorganisms that have the ability possessing to assimilate hydrocarbons are known, and some of these microorganisms have been referred to above as Examples listed. In the same way, microorganisms are

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Men having the ability to produce amino acids by fermentation are known and these are in the process of the invention to use in a simple manner.

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Claims (1)

Patent application P 16 42 718.2-41 KYOWA HAKKO KOGYO CO. Our reference: K 789 Munich, the Claims 1. A process for the fermentative production of amino acids from hydrocarbons as the main carbon source, characterized in that a hydrocarbon assimilating microorganism and an amino acid producing microorganism are cultivated under aerobic conditions in an aqueous nutrient medium containing at least one hydrocarbon, the amino acids are collected in the culture liquid obtained and from the culture liquid wins. 2. The method according to claim 1, characterized in that a microorganism belonging to the genus Pseudomonas , Mycobacterium , Achromobacter , Corynebacterium , Arthrobacter , Brevibacterium or Micrococcus is used as the hydrocarbon assimilating microorganism. 3. The method according to claim 1, characterized in that there is used as the amino acid-producing microorganism Micrococcus glutamicus . Documents ^ it. The method of claim 3 »characterized in that the ₃ amino acid from L-glutamic acid * L-lysine, L-ornithine, L-valine and / or L-homoserine is. 5. The method of claim 1 to 4, characterized in that _a representative as the main carbon source used is a hydrocarbon η-paraffin having 9 to 18 carbon atoms. . | 6. The method according to claim 1 to S » characterized in that inoculation cultures of both microorganisms at the same time be added to the nutrient medium. 7. The method according to claim 1 to 5 » characterized in that inoculum cultures of the microorganisms used are added to the nutrient medium at different times. 8. A process for the fermentative production of an amino acid from hydrocarbons as the main source of carbon, characterized in that a mixture of (1) Micrococcus glutamicus and (2) a microorganism capable of assimilating hydrocarbons belonging to the genus Pseudomonas , Mycobaeterium, Achromobacter , Corynebacterium , Arthrobacter , Brevibacterium or Micrococcus - 20 - 2098U / 0238 belongs, under aerobic conditions in an aqueous nutrient medium containing at least one hydrocarbon cultivated, the amino acid in the obtained culture liquid collects and wins therefrom. 9. The method according to claim 8, characterized in that the amino acid consists of L-glutamic acid, L-lysine, I> ornithine, II-valine or L-homoserine. 10. The method according to claim 8, characterized in that an η-paraffin with 9 to 18 carbon atoms is used as the main carbon source representing hydrocarbon. 11. The method according to claim 8, characterized in that the cultivation is carried out at a temperature of about 20 to 40 ⁰ C and at a pH of about 4.0 to 9.0. 2098U / 0 238