DE2753422A1 - METHOD FOR PRODUCING L-SERINE BY MICROBIOLOGICAL WAYS - Google Patents

Description

u.z. : M 4-51

Case: 206-4

KYOWA HAiOCO KOGYO CO. , LTD.

Tokyo, Japan

"Process for the production of L-serine in a microbiological way"

The invention relates to a process for the production of L-serine from glycine in a microbiological way.

There is a great need for the amino acid L-serine for medical purposes.

There are a number of methods used to make L-serine known. For example, it can be made through protein hydrolysis produce.

A process for the production of L-serine from glycine on a microbiological basis Routes using a microorganism belonging to the genus Nocardia is disclosed in Japanese Patent Publication 9391/76 known. However, the L-serine yields are only low achieved.

On page 80 of the executive summary of the Congress

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of Fermentation Technology, Japan 1975, it is known that increased yields of L-serine can be obtained by using a mutant of the species Corynebacterium glycinophilum, which is able to convert glycine to L-serine and which one decreased Has the ability to decompose L-serine.

Further methods for the production of L-serine are also available microbiological known, in which strains of the genera Arthrobacter, Corynebacterium, Brevibacterium, Escherichia, Micrococcus, Pichia or Candida in a nutrient medium or in cultured in a nutrient medium containing glycine.

However, there is still a need for processes for the production of L-serine which can be carried out on an industrial scale run in high yields.

When investigating with the aim of a process for the production of Developing L-serine from glycine has been invented found that mutants of the genus Nocardia, which are able to form L-serine from glycine and which have no or have a reduced ability to decompose L-serine when grown in a nutrient medium containing glycine accumulate considerable amounts of L-serine in the fermentation liquor.

It was also found that mutants of the genus Nocardia, which are capable of producing L-serine and which are against at least one antagonist of glycine, serine, methionine, glutamine, histidine, leucine, isoleucine, valine, purine, pyrimidine and / or are resistant to folic acid, when grown in a nutrient medium containing glycine, also considerable amounts of L-serine form in the fermentation liquid.

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The inventive method can be improved Achieve large-scale yields of L-serine.

According to the invention, increased yields of L-serine are achieved, by placing glycine in an aqueous medium containing microorganism cells of the genus Nocardia necessary for conversion from glycine to L-serine are able to convert to L-serine.

The mutant used according to the invention has no or one reduced ability to decompose L-serine and / or resistance to at least one glycine antagonist, Serine, methionine, glutamine, histidine, leucine, isoleucine, valine, purine, pyrimidine or poly acid.

The conversion of glycine to L-serine occurs during cultivation the mutant carried out in a nutrient medium containing glycine (method I).

The reaction can also be carried out by culturing the mutant in a nutrient medium to produce cells of microorganisms to obtain, and the microorganism cells in an aqueous medium containing glycine (method II).

The conversion of glycine to L-serine can lead to increased yields of L-serine can be achieved by adding an additive to the medium. Examples of such additives are Hydrocarbons, alcohols, ketones, ethers, esters, polyalcohols and derivatives of polyalcohols.

Furthermore, when the mutant is grown in a nutrient medium with a glycine content, increased yields can be achieved L-Serine in the fermentation liquid can be obtained by adding the medium

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mixed with phosphate, so that a medium with a high phosphate ion concentration is obtained.

In method II, the productivity of L-serine increase if immobilized cells are used as microorganism cells.

Any mutant strains of the genus Nocardia, which are able to convert glycine to L-serine and which have no or one reduced susceptibility to decomposition of L-serine and / or resistance to at least one metabolic antagonist of glycine, serine, methionine, glutamine, histidine, leucine, isoleucine, valine, purine, pyrimidine or folic acid, be used.

Such strains can be obtained by using a microorganism strain of the genus Nocardia, which is capable of converting glycine to L-serine, the ability to decompose of L-serine in whole or in part and / or resistance to at least one of the aforementioned Imparts metabolic antagonists.

Furthermore, such strains can also be obtained by using a microorganism strain of the genus Nocardia, the no or reduced ability to decompose L-serine and / or resistance to at least one of the above "has metabolic antagonists that confers the ability to convert glycine to L-serine.

In addition, strains can be used in the method according to the invention which have properties contributing to the formation of L-serine other than those mentioned above.

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Below are examples of metabolic antagonists used for Induction of mutants indicated.

Table I.

Glycine Glycinhydroxaat, Arainomethylsulfonsäure,

Chloromethylglycine, glycidic acid, N-methylglycine;

Serine hydroxamate, D-serine, homoserine;

Methionine ethionine, trifluoromethionine, a-methyl methionine Methionine hydroxarnate, selenomethionine;

Glutamine methionine sulfone, methionine sulfoximine, azaserine, Alanosine, duazomycin;

Histidine thiazolalanine, triazolalanine, aminomethyltriazole, oc-methylhistidine, histidine hydroxamate;

Leucine corleucine, trifluorleucine, azaleucine, leucine hydroxamate;

Isoleucine Thiaisoleucine, O-MethyIthreonine, Isoleucine Hydroxamate, D-isoleucine, norleucine;

Norvaline, valine hydroxamate, D-valine, norleucine;

6-mercaptopurine, 8-azaguanine, 2-fluoroadenine;

Pyriinidine 5-I ¹ IuOrUrBCiI, 2-thiouracil, 5-fluorocitosine, 6-azauracil;

Folic acid amethopterin, aminopterin, trimethoprim, pyrimethamine.

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For the production of mutants which can be used according to the invention standard procedures can be used, for example Irradiation with UV light, X-rays or Co 60, or treatment with mutagenic compounds such as N-methyl-N'-nitro-N-nitrosoguanidine.

A simple method of selecting strains with none or a decreased ability to decompose L-serine from the colonies obtained after the mutation treatment given below:

From the colonies obtained by the mutation, one colony is selected and placed in an L-serine as the only carbon source, only source of nitrogen or only source of other nutrients. Strains mentioned in the foregoing Medium show no growth or growth that is reduced compared to the starting strain, have none or a reduced growth Ability to decompose L-serine.

Nocardia butanica KY 7985 is an example of a mutant with a reduced ability to decompose L-serine. This mutant was created by mutating the L-serine-forming agent Parent strain Nocardia butanica ATCC 21197 was obtained in the manner described below.

The strains listed in Table II are examples of Mutants with a resistance to metabolic antagonists. These strains were mutated by Nocardia butanica KY 7985 is obtained in the manner described below.

The microbiological properties of the species Nocardia butanica are in Japanese patent publication 48673/72 described.

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The mutants mentioned above became upon fermentation Research Institute, Agency of Industrial Science and Technology, Chiba-ken, Japan. The strain KY 7985 has the depository number FERM-P 3782. The deposit numbers of the remaining strains are given in Table II.

These mutants were also found at the Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois, deposited. The KY 7985 strain was given the deposit number NRRL 11189. The deposit numbers of the remaining strains are also given in Table II.

FERM-P-
No. Tabel II antagonist Internal
No. 3764 NRRL-
No. Trimethopurine (0.35) 20IMP-14
KY 7983 3765 11187 6-mercaptopurine (0.5) 20MP-24
KY 7984 3766 11188 Serine hydroxamate (1) 21SX-2
KY 7986 3767 11190 Glycine hydroxamate (1) 21GX-1
KY 7987 3768 11191 Glycine hydroxamate (1),
Ethionine (5) i
Norleucine (1) IE- 36
KY 7988 3769 IIO59 Glycine hydroxamate (1),
6-azauracil (0.5) IAU-3
KY 7989 3770 11192 Glycine hydroxamate (1),
Ethionine (5) »
Methyl sulfone (5) 36MSF-2
KY 7990 3771 11193 Glycine hydroxamate (1),
Ethionine (5)>
Triazole alanine (1) 36TRA-1
KY 7991 11194 The numbers in brackets indicate the Annotation:

Isolation of the mutant medium used in the concentration (mg / ml) of the metabolic antagonist present.

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KY 7985, NRRL 11189 strain was obtained in the following manner. Microorganism cells of the starting strain Nocardia butanica ATCC 21197 are suspended in a concentration of about 10 cells per ml in 0.1 M tris-maleate buffer with a pH of 6.0. The suspension is mixed with 0.5 mg / ml of N-methyl-N'-nitro-N-nitrosoguanidine. The mixture is allowed to stand at room temperature for 30 minutes. The suspension obtained is then placed on an agar plate of a nutrient medium with a content of 0.5 g / dl glucose, 0.5 g / dl yeast extract, 1 g / dl peptone, 1 g / dl meat extract, 0.5 g / dl NaCl and Spread out 2 g / dl agar (pH 7.2) and ^{incubate at 50 °} C. for 2 days. The cells of the colonies formed are placed on an agar plate of a minimal medium with a content of 0.5 g / dl glucose, 0.2 g / dl (NH ₄ ^ SO ₄ , 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ .7H ₂ O, 0.01 g / dl NaCl, 0.001 g / dl FeSO ₄ ^ H ₂ O, 0.001 g / dl MnSO ₄ .nH ₂ 0 , 0.001 g / dl CaCl ₃ .2H ₅ O, 100 µg / liter biotin, 1 mg / liter thiamine hydrochloride and 2 g / dl agar (pH 7.2) and further on an agar plate with a medium that contains the has the same composition as the above minimal medium, with the exception that instead of (NH ₄ ^ SO ₄ 0.2 g / dl L-serine is present, streaked out.

From the strains that did not grow on the latter medium show, but growing on the agar plate with the first-mentioned medium, i.e. the minimal medium, strains that lead to the formation of L-serine in good yields are able to be selected. This includes the strain Nocardia butanica KY 7985, NRRL 11189

The mutants listed in Table II are used from Nocardia butanica KY 7985, NERL 11189, obtained as a starting strain in the following manner. Microorganism cells of the starting strain are in a concentration of about 10 cells per ml in 0.1 M tris-maleate buffer with a pH of 6.0

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suspended. The suspension is made with N-methyl-N'-nitro-N-nitrosoguanidine added at a concentration of 0.5 mg / ml. The suspension is left to stand for 30 minutes at room temperature.

The suspension is then diluted. The dilution obtained is spread onto the agar plate with a medium whose composition corresponds to the minimal medium given above with the exception that the antagonist is present in the concentration given in Table II and ^{incubated at 30 °} C. for 2 to 10 days.

In this way, the mutants listed in Table II are isolated from the colonies growing on the medium. The mutants differ from the parent strain in that that they are resistant to the metabolic antagonists.

The property of whether the mutant has a reduced ability to decompose L-serine compared to the parent strain, can be determined by the following procedure.

A selected strain is grown in a nutrient medium that does not contain either glycine or L-serine to confirm that the strain does not produce L-serine. Then the trunk grown in a nutrient medium containing L-serine and no glycine to determine the ability to decompose L-serine. If the amount of L-serine remaining in the fermentation liquid is greater than in the original strain after cultivation is complete, If the strain has no or a reduced ability to decompose L-serine.

In other methods, cells from the logarithmic phase, cells from the stationary phase, cell extracts or

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disrupted cells in an aqueous medium with a L-serine content incubated for a sufficiently long time. Afterward the amount of L-serine in the mixture is determined and compared with the amount of L-serine present at the beginning of the incubation.

The experiments described below show that Nocardia butanica KY 7985 has a reduced ability to decompose of L-serine.

Attempt 1

The strain Nocardia butanica KY 7985, NRRL 11189, is grown on an agar slant culture of pH 7.2 with a content of 0.5 g / dl glucose, 0.5 g / dl yeast extract, 2 g / dl peptone, 0, 5 g / dl NaCl and 2 g / dl agar ^{cultured at 30 °} C. for 2 days.

The cultivation culture obtained is placed with an eyelet on 7 nil of a cultivation medium with a pH of 7 »2 with a content of 4 g / dl glucose, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO _4, 0.05 g / dl MgSO _4. 7H ₂ O, 0.3 g / dl urea, 50 ig / dl biotin, 0.5 g / dl yeast extract and 2 g / dl peptone in a large test tube (20 χ 190 mm) inoculated. The cultivation is carried out with shaking at 30 ^° C. for 24 hours.

0.25 ml of the culture culture obtained are per 5 nil one L-serine containing medium of the following composition, which is in a large test tube, inoculated:

glucose 5 g / dl (NH ₄ ) ₂ S0 ₄ 0.2 g / dl urea 0.2 g / dl KH ₂ PO ₄ 0.15 g / dl K ₂ HPO ₄ 0.05 g / dl MgSO ₄ .7H ₂ O 0.05 g / dl NaCl 0.01 g / dl

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FeSO.,. 7H _o 0 0.001 g / dl MnSO ₄ .ηΗ ₂ 0 0.001 g / dl Biotin 50 µg / liter Yeast extract 0.1 g / dl L-serine 0.5 g / dl CaCO ₃ 3 g / dl (PH value 7.2)

The cultivation is carried out for 3 days at 30 ^° C. with shaking.

The quantitative determination of L-serine is done by paper chromatography using Toyo filter paper No. 50 using a mixture of n-butanol, acetone, water and diethylamine (10: 10: 5 * 2 parts by volume) carried out as the mobile phase.

The decreased amount of L-serine is 1.0 mg / ml. To the Control, the procedure described above is repeated using Nocardia butanica ATCC 21197. the Amount of L-serine is 4.6 mg / ml.

Attempt 2

The cultivation of Nocardia butanica KY 7985, NREiL 11189, is carried out according to experiment 1. 1 ml of the culture culture obtained is added to 20 ml of a fermentation medium of the following Composition in a 300 ml conical flask is inoculated:

glucose 5 g / dl (NH ₄ ) pSO ^ 0.2 g / dl Glycine 0.5 g / dl L-serine 1 g / dl

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urea 0.2 g / dl Yeast extract 0.5 6 / dl Biotin 50 µg / liter KH ₂ PO ₄ 0.15 g / dl K ₂ HPO ₄ 0.05 g / dl MgSO ₄ .7H ₂ O 0.05 g / dl NaCl 0.01 g / dl FeSO ₄ .7H ₂ O 0.001 g / dl MnSO ₄ .nH ₂ 0 0.001 g / dl CaCl ₂ ^ H ₂ O 0.001 g / dl (PH value 7.2)

The cultivation is carried out for 40 hours at 3O ⁰ C with shaking.

50 ml of the culture liquid obtained in this fermentation are centrifuged to obtain the microorganism cells. The microorganism cells are washed with isotonic sodium chloride solution and then in 0.1 M pyrophosphate buffer of pH 9.0 with a content of 5 x 10 "^ m

-Ji. —LL

Pyridoxalphosphoric acid, 5 x 10 m J2DTA and 10 m mercaptoethanol suspended.

The suspension obtained is used to break up the cells 30 Treated minutes at 20 KHz with an ultrasonic grinder. 0.5 nil of the supernatant solution (protein concentration 6.0 mg / ml) and 0.5 ml of the aforementioned 0.1 M pyrophosphate buffer of pH 9.0 with a content of 1.4 g / dl L-serine are combined. The mixture is at 14 hours Left to stand at 37 ° C.

According to experiment 1, the reduced amount of L-serine is paper chromatographed determined to be 1.6 mg / ml. At a control

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experiment using Nocardia butanica ATCC 21197, the amount of L-serine is 4.2 mg / ml.

According to the invention, when breeding a mutant in one Nutrient medium containing glycine for the formation of L-serine In the fermentation liquid the productivity of L-Serine can be further increased by using the culture medium either at the beginning during cultivation or during the growth phase of the cells with phosphate in a concentration of 0.037 M phosphate ions (P07 ~) offset.

The influence of high phosphate ion concentrations in the culture medium on the productivity of L-serine when using Nocardia butanica KY 7988 is being investigated. The results are shown in Experiment 3.

Attempt 3

Nocardia butanica KY 7988, NRRL 11059, is added to 5 ml of a fermentation medium with a content of 5 g / dl glucose, 1 g / dl (NH ₄ ) ₂ SO ^, 2.5 g / dl glycine, 0.05 g / dl K ₂ HPO ^ (corresponding to 0.003 m phosphate ions), 0.15 g / dl KH ₂ PO ₄ (corresponding to 0.011 m phosphate ions), 0.05 g / dl MgSO ₄ .7H ₂ O, 0.001 g / dl FeSO ₄ .7H ₂ O, 0.001 g / dl MnSO ₄ .nH ₂ 0.1 g / dl peptone, 3 g / dl CaCO, and 0 to 2 g / dl Mg ₅ (PO ₄ ) ^. 8H ₂ O (pH 7.2) inoculated into a large test tube. The cultivation is carried out for 5 days at 28 ^° C. with shaking. The yields of L-serine are given in Table III.

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Table III Yield of L-serine
(mp; / inl) Addition to
Mg ₃ (PO ₄ ) ^ SH ₂ O
(κ / dl weight / volume) Total concentration
of phosphate ions
(m) 1.1 0 0.014 2.0 0.1 0.019 ² J ⁹ 0.2 0.024 h ² 0.3 0.029 3.5 0.4 0.034 3.9 0.5 0.039 4.5 0.6 0.044 5.0 0.7 0.043 5.2 0.8 0.053 5.5 0.9 0.058 6.3 1.0 0.063 7.3 0.089 7.0 2.0 0.112

From Table III it can be seen that the yields of L-serine increase with increasing phosphate ion concentration (maximum 6-fold).

The above procedure is repeated, except that 0 to 2 g / dl MgSO ^ .7H ₂ O instead of Mg ₅ (PO ^) ₃ . 8H ₂ O can be used. It turns out that increasing amounts of MgSO ^ .7H ₂ O do not contribute to the good yields of L-serine.

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Examples of phosphate compounds that can be used in the process according to the invention are: NaH ₂ PO ₄ , Na ₂ HPO ₄ , Na ₃ PO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ , K ₅ PO ₄ , NH ₄ ^ ₂ PO ₄ , (NH ₄ ) ₂ HP0 ₄ , (NH ₄ ) ₃ P0 ₄ , Μ ₆ (Η ₂ Ρ0 ₄ ) ₂ , MgHPO ₄ , Mg ₃ (P0 ₄ ) ₂ , Ca (H ₂ P0 ₄ ) ₂ , CaHPO ₄ , Ca ₃ (P0 ₄ ) ₂ , Mn (H ₂ P0 ₄ ) ₂ , MnHPO ₄ , Mn ₃ (P0 ₄ ) ₂ , ZnHPO ₄ , Zn ₃ (PO ₄ ) ₂ , FeHPO ₄ , Fe ₃ (P0 ₄ ) ₂ , Co ₃ (P0 ₄ ) ₂ , K (NH ₄ ) HPO ₄ and Na (I ^) ₂ PO ₄ and hydrates of these salts.

Orthophosphates are generally used as phosphates. Meta-, pyro- and polyphosphates can of course also be used be used.

Materials with a content of phosphates are also suitable, such as phosphate ores, industrial chemicals such as phosphate ion-adsorbing ion exchange resins and phosphate ions adsorbing activated carbon.

These phosphates are used either alone or in a mixture of two or more phosphates.

The phosphate concentration in the medium, which promotes the conversion of glycine to L-serine, is generally more than 0.057 mol / liter and preferably 0.05 to 0.4 mol / liter.

In the conversion of glycine to L-serine according to the invention the productivity of L-serine can be increased further by adding the aqueous medium with at least one additive from the Group hydrocarbons, alcohols, ketones, ethers, .esters, Polyalcohols and derivatives of polyalcohols are added.

If the mutant is grown in a glycine-containing medium to accumulate L-serine in the fermentation liquor, the Productivity of L-serine by adding the additives to the medium

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at any time during the cultivation of the microorganisms increase. In this case, the additive is generally added when the microorganism grows is finished. Furthermore, the addition to the glycine can be added to the medium or it can be added after the addition of the additive glycine can be added.

Examples of additives which can be used according to the invention are listed in Table IV.

Hydrocarbons:

Alcohols:

Ketones:

Table IV

Ligroin, petroleum spirit, petroleum ether, cyclohexane, hexane, Kerosene, hexadecane;

Methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol, n-amyl alcohol, Cyclohexanol, furfuryl alcohol;

Acetone, methyl ethyl ketone, diethyl ketone, ethyl ether, isopropyl ether, n-butyl ether, 1,2-propylene oxide, 1,4-dioxane, furan, Furfural, tetrahydrofuran;

Ethyl formate, methyl acetate, ethyl acetate, citric acid tributyl ester, dioctyl phthalate, Ethyl propionate, ethyl benzoate;

Polyalcohols

(polyvalent alcohols): ethylene glycol, propylene glycol,

1,3-butanediol, glycerin;

Ester:

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Derivatives of

Polyalcohols: diethylene glycol, triethylene glycol,

Ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, Ethylene glycol monomethyl ether acetate, ethylene glycol monobutyl ether, Glycerine triacetate, glycerine ether.

These additives are generally used in concentrations of 0.01 to 5 percent (volume / volume).

Any synthetic or natural media which contain appropriate carbon sources, nitrogen sources, inorganic constituents and trace amounts of nutrients which are required for the growth of the specific mutant can be used as fermentation media for culturing the mutant in the process according to the invention.

The nutrient medium can contain any carbon and nitrogen sources as long as these can be used by the microorganism. Examples of carbon sources are carbohydrates such as glucose, fructose, sucrose, maltose and mannose, sugar alcohols such as sorbitol and mannitol, glycerin, starch, starch hydrolyzate liquids and molasses. Furthermore , various organic acids such as pyruvic acid, lactic acid, acetic acid, fumaric acid and gluconic acid, lower alcohols such as methanol and ethanol, glycols such as ethylene glycol , and hydrocarbons such as ethane, propane, butane, n-paraffin and kerosene can be used . Examples of appropriate nitrogen sources are: ammonia, various inorganic and organic ammonium salts, such as ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium nitrate and ammonium acetate, urea and other nitrogenous materials

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alien and nitrogen-containing organic products such as peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, fish meal or its hydrolyzate and chrysali hydrolyzate.

Examples of inorganic components are potassium dihydrogen phosphate, Dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, Iron (II) sulfate, manganese sulfate and calcium carbonate.

If further nutrients are required for the mutants to grow these must of course be contained in the nutrient medium. However, it is not required that these are added separately to the medium, provided that they are added together with the above-mentioned components of the nutrient media are fed. Certain natural products therefore adequately supply the special growth factors.

The cultivation is carried out under aerobic conditions, for example with shaking or with aeration and agitation. The growth temperatures are generally 20 to 40 ° C. Preferably the pH of the medium is adjusted during cultivation kept near the neutral point in order to achieve high yields. Compliance with these temperature and pH conditions however, it is not absolutely necessary for the practical implementation of the process according to the invention. The cultivation time is generally 1 to 7 days.

After cultivation is complete, the fermentation liquid is centrifuged and filtered to obtain the microorganism cells or subjected to appropriate treatment. The microorganism cells are used directly or initially with suitable devices, for example an ultrasonic

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- »- η ^2753-22

Crusher, broken open.

If the cultivation of the mutant results in the formation of L-serine in the fermentation liquid is carried out in process I in the glycine-containing nutrient medium, the same fermentation medium, as indicated above for the recovery of the microorganism cells can be used with the Exception that the medium contains glycine. The cultivation conditions can be the same as those given above correspond.

In this case, glycine can be added to the nutrient medium at a concentration of 0.1 to 5 percent (weight / volume) either at the beginning fermentation or during the growth phase of the cells.

If the implementation is carried out according to procedure II, the cells in the phosphate buffer solution of pH 7 »0 with a content of 0.5 to 20 percent glycine in one concentration from 5 to 200 mg / ml, based on the dry matter, suspended. The reaction then takes 5 to 30 hours performed at room temperature to L-serine in aqueous Enrich medium.

If in the process according to the invention as microorganism cells immobilized cells are used, which by conventional methods, for example by entrapment, such as fiinschliessen in gel or microcapsules, by adsorption, or by covalent bonding, L-serine can advantageously be Manufacture on an industrial scale.

When immobilizing the cells by enclosing them in gel a suspension of microorganism cells with a mono-

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kidney compound, such as acrylic acid amide, Ν, Ν'-methylene-bis-acrylic acid amide, Acrylic acid or methacrylic acid amide, a polymerization initiator, such as ammonium persulfate or potassium persulfate, and a polymerization accelerator such as Ν, Ν, Ν ', Ν'-tetramethylethylenediamine, offset. A suspension polymerization is then carried out at 0 ° to 40 ° C. for 1 hour. When immobilized by enclosing in microcapsules, a substance is used that leads to the formation of a semipermeable Membrane, like ethyl cellulose or polystyrene, in a water-immiscible organic solvent, whose boiling point is lower than water, such as benzene or cyclohexane, dissolved. Microorganism cells then become in the solution suspended to initially a water-in-oil emulsion to build. Then the emulsion becomes a a protective colloid, such as gelatin or polyvinyl alcohol, containing suspension added with stirring, with a second Emulsion is created. The second emulsion becomes the solvent removed, thereby forming microcapsules.

The contact of glycine with the immobilized cells obtained in this way can be intermittent or continuous, for example in the column or fluidized bed process.

When carried out in batches, the immobilized cells are suspended in a concentration of 5 to 15 g / dl in a glycine solution. The suspension is reacted for 5 to 30 hours with stirring at 20 to 40 ^° C. in order to convert glycine into L-serine. After the completion of the reaction, the resulting mixture is filtered or centrifuged, whereby a filtrate or a supernatant containing L-serine is obtained. The glycine concentration in the glycine solution is preferably

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5 to 50 g / liter, based on the total volume of glycine solution and immobilized cells. The immobilized cells are separated from the reaction mixture and can be used again be used.

When using columns, 5 to 50 g / liter of glycine solution are required passed through a column packed with immobilized cells, whereby L-serine is formed in the solution.

In the fluidized bed process, the immobilized cells are placed in the reactor using air and a phosphate buffer with a content of 5 to 50 g / liter glycine and 0.5 to 5 g / dl of an inorganic compound such as magnesium sulfate, Manganese sulfate and phosphate, kept as a fluidized bed, whereby L-serine is formed in the solution. the Fluidized bed reaction is carried out at a pH of 5 to 9 and Temperatures of 20 to 40 ° C carried out. After finished The microorganism cells and the precipitate are converted from the mixture or the precipitate by conventional methods Removed fermentation liquid. Subsequently, L-serine is obtained from the solution obtained by conventional methods, for example by treatment with an ion exchange resin.

The examples illustrate the invention.

Example 1 Fermentation

The strain Nocardia butanica KY 7985, FERM-P 3782, NRRL 11189 is used. An eyelet of a seed culture, the day-2 by culturing at 30 ⁰ C on an agar plate containing 0.5 g / dl of glucose, 0.5 g / dl yeast extract, 2 g / dl peptone, 0.5 g / dl NaCl and 2 g / dl agar (pH 7.2) has been obtained,

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is based on 7 ml of a culture medium with a content of 4 g / dl glucose, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ . 7H ₂ O, 0.3 g / dl urea, 50 μg / liter biotin, 0.5 g / dl yeast extract and 2 g / dl peptone (pH 7.2), which is in a large test tube (20 χ 190 mm) is inoculated. The cultivation is carried out with shaking at 30 ^{° C. for 24 hours.} 1 ml of the culture culture obtained is added to 20 ml of a fermentation medium with a content of 3 g / dl glucose, 1 g / dl (NH ₄ ) ₂ S0 ^, 0.2 g / dl yeast extract, 1 g / dl glycine, 0.001 g / dl FeSO ₄ .7H ₂ .0, 0.001 g / dl MnSO ₄ .nH ₂ 0, 0.05 g / dl MgSO ₄ .7H ₂ O, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ and 3 g / dl CaCO (pH 7.0), which is in a 300 ml Erlenmeyer flask, inoculated. The cultivation is carried out for 4 days at 30 ^° C. with shaking. L-serine is obtained in a yield of 3.5 mg / ml,

cleaning

After cultivation is complete, 1 liter of fermentation liquid is centrifuged to remove microorganism cells and precipitate. The obtained supernatant is placed on a column packed with 400 ml of a strongly acidic cation exchange resin (H ⁺ form) to adsorb L-serine thereon. After washing the synthetic resin with 1.5 liters of water, it is eluted with 0.5 N aqueous ammonia. The fractions containing L-serine are collected and concentrated to a volume of 15 ml. 0.1 M citrate buffer with a pH of 3.41 is then prepared by dissolving 21.01 g of citric acid in 200 ml of 1 η sodium hydroxide solution and adding 110 ml of 1 η hydrochloric acid and 5 ml of thioglycol and diluting the resulting solution with water to 1 Liter fills up. After adjusting the pH of the above concentrate to 3.41 with citric acid, the solution obtained is loaded onto a 3.2 χ 85 cm column packed ^{with strongly acidic cation exchange resin in the Na + form, the}

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has been equilibrated with the citrate buffer, given. The L-serine is adsorbed on this column. The elution is carried out with the above citrate buffer of pH 3 »4-1. During elution, the column is maintained at a temperature of 37.5 ⁰ C. The fractions containing L-serine were collected and passed through a column packed with 385 nil strongly acidic cation exchange resin in the H ^{+ form.} The synthetic resin is then washed with water and eluted with 0.5 η aqueous ammonia. The fractions containing L-serine are collected and concentrated to 100 ml under reduced pressure. The concentrate obtained is decolorized with activated charcoal and concentrated to 20 ml. The solution obtained is cooled to 5 ° C. and 70% (volume / volume) ethanol, cooled to 5 ° C., is slowly added, L-serine crystallizing. The crude crystals obtained are recrystallized from alcohol. 1.9 g of crystalline L-serine are obtained.

As a control, the same fermentation process is repeated, with the modification that Kocardia butanica ATCC 21197 is used. L-serine is obtained in a yield of 2.0 mg / ml.

Example 2

The distantation process of Example 1 is repeated, with the modification that a medium containing 8 g / dl glucose and 2.5 g / dl glycine is used as the fermentation medium instead of 3 g / dl glucose and 1 g / dl glycine and that 50 hours after the inoculation 1 % (volume / volume) propylene glycol is added. Cultivation is carried out for 73 hours. L-serine is formed in an amount of 9.5 mg / ml.

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- η

Example 3

The L-serine-producing mutants Nocardia butanica IE-36, KY 7988, NBEL 11059 and Nocardia butanica KY 7985, NREL 11189 are used. The mutants are grown on 7 ml of a culture medium with a pH of 7 »2 with a content of 4 g / dl glucose, 0.3 g / dl urea, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ .7H ₂ O, 0.5 g / dl yeast extract, 2 g / dl peptone and 50% / liter biotin, which is contained in a 50 ml large test tube (20 χ 190 mm) is inoculated. The cultivation is carried out ^{at 30 ° C. for 24 hours.} 2 ml of the culture culture obtained are added to 20 ml of a separation medium with a content of 5 g / dl glucose, 0.5 g / dl NH ₄ Cl, 2 g / dl glycine, 0.15 g / dl KH ₂ PO ₄ , 0, 05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ .TH ₂ O, 0.01 g / dl NaCl, 1 mg / dl FeSO ₄ .7H ₅ O, 1 mg / dl MnSO ₄ . nH ₂ 0.1 g / dl peptone, 2 g / dl Mg ₅ (PO ₄ ) ^. 8H ₃ O (pH 8.2), which is in a 300 ml Erlenmeyer flask. The cultivation is carried out ^{at 30 °} C. for 5 days with shaking. L-serine is formed in a yield of 8.6 mg / ml or 6.5 mg / ml. The amount of residual glycine is 1.0 mg / ml and 6.0 mg / ml, respectively.

For control purposes, the procedure described above is repeated, with the modification that Nocardia butanica ATCC 21197 is used. 4.8 mg / ml L-serine are obtained.

After the cultivation of Nocardia butanica NRBL 11059 becomes purified in a manner similar to Example 1. You get 5 »1 g crystalline L-serine.

Example 4

The L-serine-forming compounds shown in Table V are used Mutants used. For comparison, Nocardia butanica is used

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ATCC 21197 used.

0.25 ml of a seed culture, which has been obtained by culturing the microorganisms shown in Table V genrics Example 3, are on 5 nil one. fermentation medium with a pH of 6.1 with a content of 5 g / dl glucose, 1 g / dl (NH ₄ ) pSO ₄ , 2 g / dl. Glycine, 0.15 g / dl KH ₂ PO ₄ , 0.05 ß / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ .7H ₂ O, 0.01 g / dl HaCl ₁ 1 mg / dl FeSO ₄ .7H ₃ O, 1 mg / dl MnSO ₄ .nlipO, 1 g / dl peptone and 3 g / dl CaCO _2. , Which is in a large test tube, inoculated. The cultivation is carried out for 4 days with shaking at 30 ⁰ C. The yields of L-serine are given in Table V.

Table V Microorganisms Yield of L-Serine (mg; / ml )

Nocardia butanica KY 7983, NRRL 11187 4.1

¹¹ "KY 7984, NRRL 11188 4.1

"" KY 7986, IiRRL 11190 2.9

¹¹ "KY 7987, NRRL 11191 2.8

"KY 7985, NRRL 11189 2.0

"" ATCC 21197 0.5

Example 5

The microorganisms shown in Table VI are used. The procedure of Example 3 is repeated, with the modification that 0.25 ^ffl l of a seed culture obtained according to Example 3 is inoculated onto 5 ml of the fermentation medium from Example 3. The yields of L-serine are given in Table VI.

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Table VI Microorganism Yield of L-Serine (mg / ml)

Nocardia butanica KY 7989, NRRL 11192 7.0

^{11 n} KY 7990, NRRL 11193 9.5

η ti _KY 799 ^ ₁ NRRL 11194 9.0

¹¹ KY 7985, NRRL 11189 6.0

¹¹ "ATCC 2II97 4.5

Example 6

Nocardia butanica IS-56, KY 7988, FERM-P 3768, NRRL IIO59 is used. A loop obtained by culturing for 2 days at 30 ^C C in a yeast broth slant culture with a content of 0.5 g / dl yeast extract, 1 g / dl peptone, 1 g / dl meat extract, 0.5 g / dl NaCl and 2 g / dl agar (pH 7.2) is grown on 7 ml of a culture medium with a content of 4 g / dl glucose, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dL MgSO ₄ . 7H ₂ O, 0.5 g / dl yeast extract and 2 g / dl peptone (pH 7.2), which is in a 50 ml large test tube (20 χ 190 mm), inoculated. The cultivation is carried out ^{at 30 ° C. for 24 hours.} 1 ml of the culture culture obtained is inoculated into 20 ml of a fermentation medium of the following composition, which is in a 250 ml Erlenmeyer flask:

Glucose 5 g / dl (NH ₄ ) ₂ SO ₄ 1 g / dl glycine 2.5 g / dl

KH ₂ PO ₄ 0.15 g / dl (0.0Ii η phosphate ions)

K ₀ HPO. 0.05 g / dl (0.003 m phosphate * ions)

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Mg ₃ (P0 _{4) 2} · 8H ₂ 0 3 g / dl (0.147 m phosphate ions) MgSO ₄ .7H ₂ O 0.05 g / dl Fe SO, .. 7HpO 0.001 g / dl MnSO ₄ .nH ₂ 0 0.001 g / dl Peptone 1 g / dl CaCO ₅ 3 g / dl (PH value 7.0)

The cultivation is carried out ^{at 30 °} C. for 5 days with shaking. 8.4 mg / ml L-serine are formed.

For comparison, the above procedure is repeated with the modification that a fermentation medium without Mg ;, (PO ₄ ) ₂ · 8H ₂ O, which is 0.014 m in phosphate ions, is used. 2.5 mg / ml L-serine are formed. After the completion of the cultivation, 1 liter of the fermentation liquor obtained above is purified in a manner similar to that in Example 1. 4.0 g of crystalline L-serine are obtained.

Example7

Nocardia butanica KY 7988, NRRL 11059 is used. The seed culture is carried out according to Example 6. 0.25 ml of the The culture culture obtained are transferred to 5 ml of fermentation media the compositions given below (medium I and II), which are in 50 ml test tubes, inoculated.

Medium I.

glucose 5 g / dl (NH ₄ ) ₂ S0 ₄ 0.4 g / dl KH ₂ PO ₄ 0.15 g / dl K ₂ HPO ₄ 0.05 g / dl

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MgSO ₄ .7H ₂ O 0.05 g / dl PeSO ₄ .7H ₂ O 0.001 g / dl MnSO ₄ . XiH ₂ O 0.001 g / dl Glycine 2 g / dl Peptone 1 g / dl CaCO ₃ 3 g / dl PH value 7.0

This medium is 0.014 m in phosphate ions.

Medium II

This medium has the same composition as medium I, with the additional amounts of phosphate given in Table VII available.

The cultivation is carried out according to Example 6. The yields of L-serine are given in Table VII .

(2) Table VII Yield of L-serine
(mg / ml) Phosphate addition
(%, W / v) (2) Total concentration
of phosphate ions 7.0 Hg ₅ (PO ₄ VSH ₂ O (2) 0.112 7.5 MgHPO ₄ .3H ₂ O (2)
(2) 0.129 2.5 Ca ₃ (PO ₄ ) ₂ (2) 0.143 1.8
2.0 Mg ₂ (P ₂ O ₇ ). 3H ₂ O
Zn - ^ (PO,) ". 4H _o 0 (3) 0.159
0.102 6.0 K ₃ P0 ₄ .nH ₂ 0 0.089 * 1.0 + Mg ₂ SO ₄ .7H ₂ O
(none) 0.014

• K ₃ PO ₄ .nH ₂ 0 calculated as trihydrate

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Example 8

Nocardia butanica KY 7990, KRRL 11193 is added to 30 ml of a growing medium with a content of 4 g / dl glucose, 0.15 g / dl KH ₂ PO ^, 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl M ₅ SO ₄ .7H ₂ O, 0.5 g / dl yeast extract and 2 g / dl peptone (pH 7.2), which is in a 3OO ml Erlenmeyer flask, inoculated. The cultivation is carried out with shaking at 30 ^{° C. for 24 hours.} 1 ml of the culture culture obtained is added to 30 ml of a fermentation medium with a content of 8 g / dl glucose, 1 g / dl (NH ^) ₂ SO ₄ , 1 g / dl peptone, 3 g / dl Mg ₃ (PO ₄ ) _{2 #} 8H ₂ O, 0.15 g / dL KH ₃ PO ₄ , 0.05 g / dL K ₂ HPO ₄ , 0.05 g / dL MgSO ₄ .7H ₂ O, 0.001 g / dL FeSO ₄ .7H ₃ O and 0.001 g / dl MnSO ₄ .4H ₂ O (pH 7.2), which is in 15 300 ml Iirlenmeyer flasks equipped with baffle plates. The cultivation is carried out with shaking at 30 ° C for 24 hours. Then 2 ml of a glycine solution with a content of 2 g / dl are added to the medium. The cultivation is carried out for another 16 hours. The fermentation liquids obtained are combined. The mixture is centrifuged to collect the microorganism cells. The cells obtained are suspended in 0.2 M phosphate buffer with a content of 2.5 g / dl glycine. A suspension is obtained with a content of 60 mg / ml, based on the dry weight of the cells. The suspension is then poured into a 300 ml Erlenmeyer flask. The reaction is carried out ^{at 30 °} C. for 20 hours with shaking. L-serine is formed in a yield of 12 mg / ml.

Example 9

Nocardia butanica KY 7988, NRRL 11059 is used. The seed culture is carried out according to Example 6. 2 ml of the culture culture obtained are added to 20 ml of a fermentation medium with a content of 5 g / dl glucose, 1 g / dl (NH ₄ ) ₂ S0 ₄ ,

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2.5 g / dl glycine, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ .7H ₂ O, 0.001 g / dl FeSO ₄ . 7H ₅ O, 0.001 g / dl MnSO ₄ .nH ₂ 0, 1 g / dl peptone and 3 g / dl Mg ₅ (PO ₄ ) ₂ .8H ₂ O (pH 7.0), which is in a 300 ml Erlenmeyer flask is inoculated. The cultivation is carried out with shaking at 30 ° C. for 48 hours. The glucose concentration after cultivation is less than 1 g / dl. At this point in time, the growth of the microorganism has ended, as can be seen from the following explanations. 0.5 ml of 6 η hydrochloric acid are added to 0.5 ml of the fermentation liquid in order to dissolve any insoluble constituents present in the fermentation liquid, with the exception of the microorganism cells. The optical density of the suspension obtained after adding 24 ml of water is then measured at a wavelength of 660 μm using a spectrophotometer. The result is a value of 0.27. This value corresponds to the value measured in the same way 2 hours earlier.

The additives indicated in Table VIII are then added in the indicated concentrations. Breeding will 72 hours continued. The yields of L-serine are given in Table VIII.

When the cultivation is complete, 1 liter of the fermentation liquor obtained using acetone as an additive in the cultivation becomes cleaned. The cleaning is carried out according to Example 1. 6.2 g of crystalline L-serine are obtained.

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Table VIII 3 Yield of L-serine
(mg / ml) concentration
Addition (%, Vol./Vol.) 3 13.0 Ligroin 1 10.4- Cyclohexane 1 10.8 Methanol 0.5 9.0 Ethanol 1 11.9 acetone o, 5 9.5 Methyl ethyl ketone 1 10.5 Ethyl acetate 0.3 10.0 Methyl acetate 2 10.0 Citric acid tributyl ester 1 9.5 1,4-dioxane 0.5 9.8 Tetrahydrofuran 0.5 10.0 Propylene glycol 0.13 9.5 1,3-butanediol 0.13 9.0 Ethylene glycol monoethyl ether - 9.0 Ethylene glycol monomethyl
ether acetate 8.2 no addition

Example 10

The seed culture is carried out according to Example 8. 100 ml of the seed culture are inoculated onto 3 liters of the fermentation medium according to Example 8, which additionally contains 0.01 g / dl CaClp.2HpO and is located in a 5 liter fermenter. The cultivation is carried out for 48 hours under aeration and Begegung at 30 ⁰ C. The fermentation liquid is then centrifuged. The microorganism cells obtained are washed twice with 0.1 M phosphate buffer of pH 7.0 and in

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Stored frozen in phosphate buffer. 5 g ethyl cellulose (50 to 100 Cp) are in a solvent mixture of 72.5 g of benzene and 25.5 g of η-hexane with a Dissolved content of sorbitan monolaurate as a dispersant.

Furthermore, 5 g of frozen microorganism cells (dry weight) are suspended in 50 ml of 0.1 M phosphate buffer with a pH of 7.0 and containing 100 mg of Mg (H ₃ PO ^) ₂ and 100 mg of Mg, (P0 ^) _2. The suspension obtained is added to the ethyl cellulose solution. The mixture is stirred thoroughly to form a first emulsion. The emulsion is then added to 500 ml of a 1 percent (weight / volume) polyethylene glycol solution (average molecular weight 6000) at pH 8 to 9 and cooled ^{to 10 to 15 ° C. with ice.} The mixture is stirred to form a second emulsion. This emulsion is continuously fed in a rate of 10 to I5 ml / min to cooled with ice water, n-hexane (5 to 10 ⁰ C) to crystallize, the benzene to remove and ethylcellulose. After 1 hour, immobilized cells enclosed in ethyl cellulose are obtained. The particle diameter is 5 to 100 µ.

The activity of the immobilized cells is about 50 to 60 percent of the activity of the cells before immobilization. The amount of enclosed cells is 500 to 350 mg cells / ml microcapsules. 100 ml of the immobilized cells are kept in a 1 liter reaction vessel (90 mm 150 mm φ ) using air and 0.1 M phosphate buffer solution with a pH of 6.1 in a fluidized bed. The phosphate buffer solution contains 0.2 g / ml glycine, 5 g / ml Mg (H ₂ PO ^) ₂ and 5 g / ml Mg, (PO ^) ₂ . This solution is fed to the reactor with a residence time of 15 to 20 hours. It becomes an overflow solution continuously for 50 hours

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with an L-serine content of 10 to 15 mg / ml.

Example 11

500 ml of cyclohexane as a dispersion medium and 4 g of sorbitan monolaurate as a dispersant are mixed and stirred at 10 to 15 ° C. while cooling with ice and under nitrogen as an inert gas. The mixture is then mixed with 19.5 g of acrylamide and 0.5 6 Ν, Ν'-bisacrylamide, which is dissolved or suspended in 100 ml of 0.1 M phosphate buffer solution with a pH of 6.1, and with 10 g of frozen cells ( based on the dry components), which have been prepared according to Example 10, added. The mixture obtained is admixed with 10 ml of 2.5 percent (weight / volume) N, N, N ¹ , N'-tetramethylethylenediamine as a polymerization stabilizer and 125 ml of 2.5 percent (weight / volume) ammonium persulfate as a polymerization initiator. The polymerization reaction is carried out for 1 hour, whereby immobilized cells are formed, which are enclosed in gel and have a diameter of 500 to 400 μ .

The activity of the immobilized cells is about 50 to 40 percent of the cells before immobilization. The amount of cells enclosed in the gel is 60 to 70 mg cells / ml gel.

Then 100 ml of gel and 800 ml of a glycine solution with a content of 25 mg / ml are poured into a 1 liter reactor. The fieaktion is carried out batchwise for 25 hours at 50 ⁰ C. L-serine forms in a concentration of 12.5 mg / ml in the reaction mixture. The gel is then recovered from the reaction mixture. The above procedure is repeated 2 times using the recovered gel. The results are shown in Table IX.

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Table IX

Conversion number of the concentration of the concentration of the degree Uses Glycine (mg / ml) L-Serine (mg / ml) (%)

1 25th 12.5 50.0 2 25th 11.8 47.2 3 25th 10.5 42.0

1 liter of the first reaction mixture is filtered to remove the immobilized cells. The pH of the supernatant piltrate is adjusted to 2.0 with hydrochloric acid. Subsequently, the FiItrat is a with a strongly acidic cation exchange resin (Diaion SK-1B) packed Given column. Elution is carried out with 2 η aqueous ammonia solution carried out. The eluate is made under reduced pressure evaporated. 8.5 g of L-serine are obtained.

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Claims (1)

VOSSIUS · VOSSIUS HIlTL ^t ALSO PATENT Attorneys 7753Δ22 SIEBERTSTRASSE 4 BOOO MÖNCHEN 86 PHONE: (O89) 474O7B CABLE: BENZOLPATENT MÖNCHEN TELEX S-S9 453VOPAT D u.z .: M 451 November 30, 1977 Case: 206-4 KYOWA HAKKO KOGYO CO., LTD. Tokyo, Japan "Process for the production of L-serine in a microbiological way" Priority: November 50, 1976, Japan, No. 143 748/1976 December 8, 1976, Japan, No. 147 389/1976 December 8, 1976, Japan, No. 147 390/1976 December 20, 1976, Japan, No. 153 024/1976 April 15, 1977, Japan, No. 42 556/1977 Claims Process for the production of L-Serine on a microbiological basis Ways, characterized in that one glycine in an aqueous medium in the presence of microorganism cells a mutant of the genus Nocardia, which is able to convert glycine to L-serine and the no or a reduced ability to decompose L-serine and / or a resistance to at least one Metabolic antagonists of glycine, serine, methionine, glutamine, histidine, leucine, isoleucine, valine, purine, pyrimidine and / or has folic acid, converts it to L-serine, L-serine enriched in the medium and isolated therefrom. 809822/0998 2. The method according to claim 1, characterized in that using a mutant belonging to the species Nocardia butanica. 3. The method according to claim 1, characterized in that that Nocardia butanica NERL 11059, Nocardia butanica NRRL 11187, Nocardia butanica NRRL 11188, Nocardia butanica NRRL 11189, Nocardia butanica NRRL 11190, Nocardia butanica NRRL 11191, Nocardia butanica NRRL 11192, Nocardia butanica NRRL 11193 or Nocardia butanica NRRL 11194- used. 4 ·. Method according to claim 1, characterized in that that the conversion of glycine to L-serine with cultivation of the mutant in a glycine containing Carries out nutrient medium. 5. The method according to claim 4, characterized in that that the nutrient medium contains at least 0.037 mol / liter of phosphate ions. 6. The method according to claim 4, characterized in that that the nutrient medium is 0.05 to 0.1 mol / liter Contains phosphate ions. 7. The method according to claim 4, characterized in that one carries out the breeding 1 to 7 days at temperatures of 20 to 4O ⁰ C. 8. The method according to claim 1, characterized in that that the reaction is carried out in an aqueous medium, the glycine and by culturing the mutant contains obtained microorganism cells. 809822/0998 r / 53422 9. The method according to claim 8, characterized in that that glycine is used at a concentration of 0.5 to 20 percent. 10. The method according to claim 1, characterized in that that the conversion of glycine to L-serine in the presence of at least one additive from the Group hydrocarbons, alcohols, ketones, JSster, ethers, Performs polyalcohols and derivatives of polyhydric alcohols. 11. The method according to claim 10, characterized in that the additive in one concentration from 0.01 to 5 percent by volume, based on the volume of the medium, is used. 12. The method according to claim 8, characterized in that the microorganism cells are immobilized Cells used. 809822/0998