DE2753422B2 - Process for the production of L-serine by microbiological means - Google Patents

Description

A number of processes are known for the production of L-serine, for example it can be passed through Making protein hydrolysis.

A process for the microbiological preparation of L-serine from glycine using a Microorganism belonging to the genus Nocardia is known from Japanese patent publication 9 391/76 However, only low L-serine yields are achieved here

On page 80 of the executive summary of the Congress of Fermentation Technology, Japan 19/5, It is known that increased levels of L-serine are obtained using a capable mutant of the species Corynebacterium glycinophilum. To convert glycine to L-serine and which has a reduced ability to utilize L-serine.

Further processes for the production of L-serine by microbiological means are also known from those strains of the genera Arthrobacter, Coryne bactei'ium, Brrvibacterium, Escherichia, Micrococcus, Pichia or Candida are grown in a nutrient medium or in a nutrient medium containing glycine will.

In DE-OS 23 58 496 is a process for the production of L-serine using Mutants of the species Corynebacterium glycinophilum described that have a need for at least one Have amino acid. In this process, the Yield of L-serine 1.14 to 1.47 times higher than when using the starting strain for the mutants. The starting point is in US-PS 36 23 952 described. Apparently it is C glycinophilum ATCC 21 341.

In investigations with the Zui to develop a process for the production of L-serine from glycine in a microbiological way, it was found according to the invention that mutants of the species Nocardia butanica, which are able to form L-serine from glycine and which no or a reduced ability to utilize of L-serine when grown in a glycine containing nutrient medium considerable amounts of

Enrich L-serine in the fermentation liquid. It was also found that mutants of the species

μ Nocardia butanica, which is responsible for the formation of L-serine in the Are able to withstand at least one antagonist of glycine, serine, methionine, glutamine, histidine, leucine, Resistant to isoleucine, valine, purine, pyrimidine and / or folic acid, if grown in a glycine

h ', the nutrient medium also contains considerable amounts Form L-Serine in the fermentation liquid.

The invention is therefore based on the object of an improved process for the production of L-serine from

To create glycine in a microbiological way. These The object is achieved according to the characterizing part of claim I. Pie claims 2 to U relate to preferred ones Embodiments of the method.

The yield of L-serine using the mutants used according to the invention is 135 to 8.2 times higher than when using the Starting strain for the mutants, namely Nocardia butanica ATCC 21 197.

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

The reaction can also be carried out by growing the mutant in a nutrient medium, to obtain microorganism cells, and the microorganism cells in an aqueous. Glycine-containing medium gives (Method II).

When converting glycine to L-serine, increased yields of L-serine can be achieved by the medium is mixed with an additive. Examples of such additives are hydrocarbons, alcohols, ketones, ethers, esters, polyalcohols and derivatives of Polyalcohols.

Furthermore, when cultivating the mutant in a nutrient medium containing glycine achieve increased yields of L-serine in the fermentation liquid by adding the medium mk phosphate, so that a medium having a high concentration of phosphate ions is obtained

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

In the method according to the invention, any mutant strains of the species Nocirdia butanica can be used which are able to convert G, ¹ a to L-serine and which have no or a reduced ability to utilize L-serine and / or a 'resistance to at least a metabolic antagonist of glycine, serine, methionine, glutamine, histidine. Leucine, isoleucine, valine, purine, pyrimidine or folic acid can be used.

Such strains can be obtained by using a microorganism strain of the species Nocardia butanica, which is able to convert glycine into L-serine, the ability to utilize L-serine fully or partially takes and / or resistance to at least one of the above gives metabolic antagonists mentioned.

Furthermore, such strains can also be obtained by adding a microorganism strain of the species Nocardia butanica, which has no or a reduced ability to utilize L-serine and / or a Has resistance to at least one of the above metabolic antagonists, the ability to Conversion of glycine into L-serine confers.

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

Examples of metabolic antagonists for the induction of t lutants are given below,

Table I. Glycine glycine hydroxamate, Ainomethylsulfonsäurc, Chloromethylglycine.glycidic acid c. N-methylglycine; Serine serine hydroxamate, D-serine. Homoserine; Methionine ethionine, trifluoromethione,

"-Metbylmethionine.Methioninehydrxamat,

Seienomethiontn; Glutamine Metbioninsulfon, Methioninsulfoximin,

Azaserine, alanosine, duazomycin; Histidine thiazole alanine, triazole lanine,

AminomethyltriazoL, ac-methylhistidine, Histidine hydroxamate;

Leucine norleucine, trifluorleucine, Azaleucine, leucine hydroxamate; Isoleucine thiaisoleucine, O-methylthreonine, Isoleucine hydroxamate, D-I & oleucine,

Norleucine; Valine norvaline, valine hydroxamate,

D-valine, norleucine; Purine 6-mercaptopurine, 8-azaguanine,

2-fluoroadenine;

Pyrimidine 5-fluorouracil2-thiouracil, 5-fluorocitosine, 6-azauracil;

Folic acid amethopterin, aminopterin. Trimethoprim, pyrimethamine. For the production of usable according to the invention

Mutants can be used standard procedures, for example irradiation with UV light, X-rays or Co 60, or treatment with mutagens Compounds such as N-methyl-N'-nitro-N-nitrosoguanidine.

jo A simple method for selecting strains with no or only reduced ability to decompose L-serine from the colonies obtained after the mutation treatment is given below:

j5 From the colonies obtained by the mutation a colony is selected and placed in an L-serine as the sole carbon source, sole nitrogen source or bred sole source of other nutrients. Strains which have no or an im Show, show no growth or a reduced growth compared to the parent strain Ability to decompose L-serine.

Nocardia butanica KY 7,985 is an example of a mutant with a reduced ability to decompose tion of L-serine. This mutant was created by mutation of the parent L-serine producing strain Nocardia butanica ATCC 21 197 to that described below

Way received. The strains listed in Table II are examples

for mutants with a resistance to metabolic antagonists. These strains were mutated by Nocardia butanica KY 7,985 was obtained in the manner described below. The microbiological properties of the species Nocardia butanica are described in Japanese patent publication 48 673/72.

The aforementioned mutants have been obtained from the Fermentation Research Institute, Agency of Industrial Science and Technology, Chiba-ken, Japan.

μ The strain KY 7 985 has the depository number FERM-P 3 782, 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

hi Street, Peoria. Illinois, deposited. The strain KY 7 985 has been assigned accession numbers NRRL 11 189. The accession numbers of the remaining strains are also given in Table II.

Table U 27 53 422 6th 5 Internal number, 20IMP-14 KY 7983 antagonist 20MP-24 KY 7984 FERM-P no. NRRL no. Trimethopurine (0.35) 21SX-2KY7986 3764 11187 6-mercaptopurine (0.5) 21GX-1 KY 7987 3765 11188 Serine hydroxamate (1) IE-36 KY 7988 3766 11190 Glycine hydroxate (1) 3767 11191 Glycine hydroxamate (1), 3768 11059 Ethionine (5), IAU-3 KY 7989 Norleucine (1) Glycine hydroxamate (1), 36MSF-2 KY 7990 3769 11192 6-azauracil (0.5) Glycine hydroxamate (1), 3770 11193 Ethionine (5), 36TRA-1 KY 7991 Methyl sulfone (5) Glycine hydroxamate (1), 3771 11194 Ethionine (5, Triazole alanine (I)

Annotation:

The numbers given in brackets indicate those used in for the isolation of the mutant Concentration (mg / ml) of the metabolic antagonist present in the medium.

The strain KY 7 8985, NRRLII 189 was obtained in the following manner. Microorganism cells of the starting strain Nocardia butanica ATCC 21 197 are suspended in a concentration of about 10 ⁸ cells per ml in 0.1 M tris-maleate buffer with a pH of 6.0. The suspension is made up of 0.5 mg / ml N-methyl ! -N'-nitro-N-nitrosoguanidine added. The mixture is left 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, 0.1 g / dl yeast extract, 1 g / dl peptone, 1 g / dl meat extract, 0.5 g / dl NaCl and 2 g / dl agar (pH 72) and incubated for 2 days at 30 ° C. The cells of the colonies formed are placed on an agar plate of a minimal medium with a content of 04 g / dl glucose, 0.2 g / dl (NH ₄ I ₂ SO ₄ , 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ · 7 H ₂ O, 0.01 g / dl NaCl, 0.001 g / dl FeSO ₄ · 7 H ₂ O, 0.001 g / dl MnSO ₄ · π H ₂ O, 0.001 g / dl CaCl ₂ · 2 H ₂ O, 100 μg / liter biotin, 1 mg / liter thiamine bvdroxhlorid and 2 g / dl agar (pH 7.2) and further on an agar plate with a Medium having the same composition as the above minimal medium, with the exception that instead of (NH _{4 I 2} SO ₄ 0.2 g / dl L-serine is present, streaked out.

From the strains that show no growth on the latter medium, but on the agar plate with the former medium, d. H. the minimal medium, will grow stems that lead to the formation of L-serine in good yields in the lags are selected. This includes the strain Nocardia butanica KY 7985, NRRL 11 189.

The mutants listed in Table 11 are obtained using Nocardia butanica KY 7985, NRRL 11189 as starting strain in the following manner, are microorganisms cells of the parent strain at a concentration of about 10 ⁸ cells per ml in 0.1 M Tris-maleate Buffercr suspended at pH 6.0. The suspension is crosslinked with N-methyl-N'-nitro-N-nitrosoguanidine in a concentration of 0.5 mg / ml. The suspension was 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 minimum medium given above, with the exception that the antagonist is present in the concentration given in Table II, and is kept at 30 ° C. for 2 to 10 days incubated.
In this way, the mutants listed in Table II are isolated from the colonies which grow on the medium. The mutants differ from the parent strain in that they are resistant to the metabolic antagonists.

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

A selected strain is grown in a nutrient medium that does not contain either glycine or L-serine in order to confirm that the strain forms k ^ in L-serine. Then the strain is in a nutrient medium, which contains L-serine and no glycine, cultivated to determine the ability to utilize L-serine. is after the end of cultivation, the amount of L-serine remaining in the fermentation liquid is greater than that of the Initially rigid.m, the strain has no or a reduced ability to utilize L-serine.

In other procedures, cells from the

Logarithmic phase, cells from the stationary phase, cell extracts or broken cells all in one aqueous medium is incubated with a cold of L-serine for a sufficiently long time. Then the Determined amount of L-serine in the mixture and with the amount of L-serine present at the beginning of the incubation compared.

The experiments described below show that Nocardia butanica reduced KY 7,985 together Has the ability to utilize I -serine.

Attempt I.

The strain Nocardia butanica KY 7 985, NRRLIl 189, is dated on an agar slant pH who; 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 g / dl agar cultured for 2 days at 30 ° C.

The cultivation culture obtained is placed on ml of a cultivation medium with a pH value 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 · 7 H ₂ O ₁ 03 g / dl urea, 50 μg / dl biotin, 0.5 g / dl yeast extract and g / dl peptone in a large test tube (20x190 mm) inoculated. The cultivation is carried out with shaking at 30 ^° C. for 24 hours.

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

glucose

(NH ₄ I ₂ SO ₄

urea

ΚΗ, ΡΟι

K ₂ HPO ₄

MgSO ₄ · _{7H 2} O

NaCl

FeSO ₄ · _{7H 2} O

MnSO ₄ · η H ₂ O

Biotin

Yeast extract

L-serine

CaCO ₃

(PH value

5 g / dl 0.2 g / dl

02 g / dl 0.15 B / dl 0.05 g / dl 0.05 g / dl 0.01 g / dl 0.001 g / dl 0.001 g / dl 50 μg / liter 0.1 g / dl

0.5 g / dl

3 g / dl 7.2)

glucose

(NH ₄ I ₂ SO ₄

Glycine

L-serine

urea

Yeast extract

Biotin

KH ₂ PO ₄

K ₂ HPO ₄

MgSO ₄ · 7 H ₂ O

NaQ

FeSO ₄ · _{7H 2} O

MnSO ₄ · π H ₂ O

CaQ ₂ · 2 H ₂ O

(PH value

Breeding turns 40 Shaking done 5OmI of this

20th

25th

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

The quantitative determination of L-serine is carried out by paper chromatography using Toyo filter paper No. 50 using a mixture of n-butanol, acetone, water and diethyl-j5 amine (10: 10: 5: 2 parts by volume) carried out as the mobile phase.

The decreased amount of L-serine is 1.0 mg / ml. As a control, the procedure described above is used Procedure repeated using Nocardia butanica ATCC 197. The amount of L-serine is 4.6 mg / ml.

Attempt 2

The cultivation of Nocardia butanica KY 7,985, NRRL 189, is carried out according to Experiment 1. I ml the The culture culture obtained is poured into 20 ml of a fermentation medium of the following composition, which is in a 300 ml Erlenmeyer flask, inoculated:

Culture fluids are centrifuged to obtain the microorganism cells. The microorganism cells are washed with isotonic sodium chloride solution and then ^{suspended in 0.1 M pyrophosphate buffer with a pH of 9.0 containing 5} × 10 5 M pyridoxalphosphoric ^{acid, 5 × 10 4} M EDTA and ΙΟ ⁴ M mercaptoethanol.

To break up the cells, the suspension obtained is treated for 30 minutes at 20 KHz with an ultrasonic grinder. 0.5 ml 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 14 hours left to stand at 37 ° C.

According to experiment 1, the reduced amount of L-serine is determined by paper chromatography to be 1.6 mg / ml. In a control attempt using of Nocardia butanica ATCC 2i i97 the amount of L-serine is 4.2 mg / ml.

According to the invention, when cultivating a mutant in a nutrient medium containing glycine for the formation of L-serine in the fermentation liquid, the productivity of L-serine can be further increased by adding the cultivation medium either at the beginning of cultivation or during the growth phase of the cells Phosphate in a concentration of 0.037 m Phosphatfe ^ -nen (PO ₄ - -) added.

The influence of high phosphate ion concentrations in the growth medium on productivity L-Serine when using Nocardia butanica KY 7 988 is being investigated. The results are in Experiment 3 shown.

Attempt 3

Nocardia butanica KY 7 988, NRRL 11 059, is added to 5 ml of a fermentation medium with a content of 5 g / dl glucose, 1 g / dl (NH ₄ I ₂ SO ₄ , 2.5 g / dl glycine, 0.05 g / dl K ₂ HPO ₄ (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 · 7 H ₂ O, 0.001 g / dl FeSO ₄ 7 H ₂ O, 0.001 g / dl MnSO ₄ η H ₂ O, 1 g / dl peptone, 3 g / dl CaCO ₃ and 0 to 2 g / dl MgXPO ₄ J ₂ 8 H ₂ O (pH value 7.2) in 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.

Table III

5 g / dl

02 g / dl

03 g / dl lg / dl 02 g / dl O ^ g / dl

50 μg / LiteΓ 0.15 g / dl 0.05 g / dl 0.05 g / dl 0.01 g / dl 0.001 g / dl 0.001 g / dl 0.001 g / dl 72)

Hours obtained at 30 ⁰ C with fermentation

50

60

65

Addition to Total concentration yield Mg, 'PO ₄ ) ₂ 8 H ₂ O of phosphate ions of L-serine (g / dl wt.) (m) (mg / ml)

55

0.014 1.1 0.019 2.0 0.024 2.9 0.029 3.2 0.034 3.5 0.039 3.9 0.044 44 0.048 5.0 0.053 5.2 0.058 54 0.063 6.3 0.089 7.3 0.112 7.0

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

The above-mentioned process is repeated, be used with the modification that 0 to 2 g / dl MgSO * · 7 H ₂ O ϊ instead of Mg ₃ (PO ₄ J ₂ x 8 H2O. It has been found that increasing amounts of MgSO ₄ · 7 H ₂ O ke'i-ϊη contribute to the good yields of L-serine.

Examples of phosphate compounds that are used in the inven- tion Proper procedures that can be used are:

NaH ₂ PO ₄ , Na ₂ HPO ₄ , Na ₃ PO ₄ , KH ₂ PO ₄ , K ₂ HPO ₄ , K ₃ PO ₄₁ NH ₄ · H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , (NH ₄ ) ₃ PO ₄ , Mg (H ₂ PO ₄ ) ₂ , MgHPO ₄ , Mg ₃ (PO ₄ ) J, Ca (H ₂ PO ₄ J ₂ , CaHPO ₄ , Ca ₃ (PO ₄ ) J, Mn (H ₂ PO ₄ ) j, MnHPO ₄ , Mn ₃ (PO ₄ J ₂ , ZnHPO ₄ , Zn ₃ (PO ₄ ) ,, FeHPO ₄ , Fe ₃ (PO ₄ J _2. Co ₃ (PO ₄ Jj, K (NH ₄ ) HPO ₄ and Na (NHiJ ₂ POi and hydrates of these salts.

Orthophosphates are generally used as phosphates. Of course you can too Meta-, pyrol- and polyphosphates can be used.

Furthermore, materials with a content of phosphates, such as phosphate ores, industrial chemicals, such as phosphate ion-adsorbing ion exchange resins and phosphate ion-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 im generally more than 0.037 mol / liter and preferably 0.05 to 0.4 mol / liter.

In the inventive implementation of glycine to L-serine, the productivity of L-serine can be increased by adding at least one additive from the group of hydrocarbons, Alcohols, ketones, ethers, esters, polyalcohols and derivatives of polyalcohols are added.

When the mutant is grown in a 4N medium containing glycine to accumulate L-serine in fermentation liquor, the productivity of L-serine by adding the additives to the medium at any time during the cultivation of the Microorganisms are increased. In this case, the additive is generally added when the The microorganism has stopped growing. It can also be added to the medium containing glycine or glycine can be added after the addition of the additive is complete.

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

Table IV «

Hydrocarbons: ligroin, petroleum benzine, "petroleum spirit", petroleum ether, cyclohexane, hexane, Kerosene, hexadecane;

Alcohols: methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol, n-amyl alcohol, cyclohexanol, furfuryl alcohol;

Ketones: acetone, methyl ethyl ketone, diethyl ketone

clay, ethyl ether, isopropyl ether, n-butyl ether, 1,2-propylene oxide, 1,4-dioxane, furan, furfural, tetrahydrofuran;

Ester:

60

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

Polyalcohols (multi-valued Alcohols):

Derivatives of Polyalcohols;

Ethylene glycol, propylene glycol, 1,3-butanediol, glycerin;

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) used.

Any fermentation media for cultivating the mutant can be used in the process according to the invention synthetic or natural media are used that contain appropriate carbon sources, nitrogen sources, inorganic constituents and trace amounts Contain nutrients necessary for the growth of the particular mutant.

The nutrient medium can contain any carbon and nitrogen sources as long as they are supported by the Microorganism can be recycled. 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 corresponding 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 and nitrogenous organic products such as peptone, NZ amine, meat extract, yeast extract, Corn steep liquor, casein hydrolyzate, fish meal or its hydrolysates and chrysalis hydrolyzate.

Examples of inorganic constituents 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 growth of the mutants, these must of course be included in the nutrient medium. However, it is it is not necessary that these are added separately to the medium, provided that they are added together with the the above-mentioned components of the nutrient media are supplied. Certain natural products thus supply the special growth factors in an appropriate manner.

The cultivation is carried out under aerobic conditions, for example with shaking or with aeration and agitation. The cultivation temperature is generally from 20 to 40 ⁰ C, preferably maintaining the pH value of the medium during the culturing in the near neutral to high yields

to achieve. However, compliance with these temperature and pH conditions is essential for practical implementation of the method according to the invention is not absolutely necessary. The cultivation time is generally 1 to 7 days.

When the cultivation is complete, the fermentation liquid is centrifuged to obtain the microorganism cells, filtered or subjected to an appropriate treatment. The microorganism cells are direct used or initially with suitable devices, for example an ultrasonic shredding device, broken up.

If the cultivation of the mutant with the formation of L-serine in the fermentation liquid in process 1 in glycine is carried out containing nutrient medium, the same fermentation medium as it is above for the extraction of the microorganism cells is indicated, with the exception that im Medium glycine is included. The cultivation conditions can be the same as those given above correspond.

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

If the implementation is carried out according to the procedure. II carried out, the cells in the phosphate buffer solution of pH 7.0 with a content of 0.5 to 20 percent Glycine in a concentration of 5 to 200 mg / ml, based on the dry matter, suspended. Afterward the reaction is carried out for 5 to 30 hours at room temperature to convert L-serine in the aqueous Enrich medium.

If immobilized cells are used as microorganism cells in the method according to the invention, by conventional methods, for example by entrapment, such as entrapment in gel or Microcapsules, produced by adsorption, or by covalent bonding, can be converted into L-serine advantageously produce on an industrial scale.

When immoboilizing the cells by enclosing them in gel, a suspension of microorganism cells with a monomeric 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 N, N, N ', N'-tetramethylethylenediamine, added. A suspension polymerization is then carried out at 0 to 40 ^° C. for 1 hour. During immobilization by enclosing in microcapsules, a substance that leads to the formation of a semipermeable! Membrane capable of, such as ethyl cellulose or polystyrene, in a water-immiscible organic solvent whose boiling point is lower than water, such as benzene or cyclohexane, is then dissolved into a suspension containing a protective colloid such as gelatin or polyvinyl alcohol Stirring is given, resulting in a second emulsion. The solvent is removed from the second emulsion, whereby microcapsules are formed.

The contact of glycine with the immobilized cells obtained in this way can intermittently, or continuously, 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 gicin solution. The suspension 5 is converted to 30 hours with stirring at 20 to 4O ⁰ C, to transform glycine into L-serine. To

-ι completed the reaction, the mixture obtained filtered or centrifuged to obtain a filtrate or a supernatant containing L-serine. The glycine concentration in the glycine solution is preferably 5 to 50 g / liter, based on the

ι «Total volume of glycine solution and immobilized cells. The immobilized cells are removed from the Separated reaction mixture and can be used again.

When using columns, it will be 5 to 50

i> g / liter of glycine solution via an immobilized Cells packed column, whereby L-serine is formed in the solution.

In the fluidized bed process, the immobilized cells in the reactor are using air and

- '<> a phosphate buffer with a content of 5 to 50 g / liter glycine and 0.5 to 3 g / dl of an inorganic one Compound, such as magnesium sulphate, manganese sulphate and phosphate, kept as a fluidized bed, creating in the Solution L-Serine is formed. The fluidized bed reaction

»J is carried out at a pH of 5 to 9 and temperatures of 20 to 40 ^° C. After the reaction has ended, the microorganism cells and the precipitate are removed from the mixture or the fermentation liquid by conventional methods. Afterward

ίο 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 cultivation culture, which was cultivated for ² days at 30 ° C. on an agar plate with a content of 0.5 g / d) 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, 7 ml of a culture medium with a content of 4 g / dl

4-, glucose, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ ■ 7 H ₂ O, 03 g / dl urea, 50 μg / Liters of 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) inoculated. The cultivation is 24 hours

-> o carried out with shaking at 30 ° C. 1 ml of the resulting culture is added to 20 ml of a fermentation medium with a content of 3 g / dl glucose, 1 g / dl (NHi) ₂ SO ₄ , 0.2 g / dl yeast extract, 1 g / dl glycine, 0.001 g / dl FeSO ₄ · 7H ₂ O, 0.001 g / dl MnSO ₄ - π H ₂ O, 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 ₃ (pH 7.0), which is housed in a 300-ml ErlenmeyerkoIben inoculated culturing is 4 days at 30 ⁰ C. carried out 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 used to remove microorganism cells and The precipitate is centrifuged. The supernatant obtained is poured into 400 ml of a strongly acidic cation exchange resin (H + form) packed column to adsorb L-serine thereon. After washing the

Synthetic resin with 1.5 liters of water is eluted with 0.5 η aqueous ammonia. The fractions containing L-serine are collected and concentrated to a volume of 15 ml. 0.1m 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 m with citric acid, the solution obtained is transferred to a 3.2 × 85 cm column packed with strongly acidic cation exchange resin in the Na + form, which has been equilibrated with the citrate buffer , given. The L-serine is adsorbed on this column. Elution is carried out with the above citrate buffer of pH 3.41. The column is kept at a temperature of 37.5 ° C. during the elution. the

riauuiicii collected with a vjcnaii au i ^ -aciui nciucn and passed through a column packed with 385 ml of 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 Nocardia butanica ATCC 21197 is used. L serine is obtained in a yield of 2.0 mg / ml.

Mg ₃ (PO ₄ ); ■ 8 H ₂ O (pH 8.2), which is in a 300 ml L'rienmeyer flask, inoculated. Cultivation is carried out for 5 days with shaking at 30 ° C. During this process, 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 b / w. 6.0 mg / ml.

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

After the cultivation of Nocardia butanica NRRL 11059 is finished, it is purified in a manner similar to that in Example I. 5.1 g of crystalline L-serine are obtained.

Example 4

TI II- \ t U _ "_"

I OWIIW T OllgVglUllllll

forming mutants used. For comparison, Nocardia butanica ATCC 21197 is used.

0.25 ml of a seed culture which has been obtained by culturing the microorganisms given in Table V according to Example 3 are added to 5 ml of a fermentation medium of pH 6.1 with a content of 5 g / dl glucose, 1 g / dl (NH ₄ I ₂ SO ₄ , 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 · 7 H ₂ 0, 0.01 g / dl NaCl, 1 mg / dl FeSO ₄ · 7 H ₂ O, 1 mg / dl MnSO ₄ · η H ₂ O, 1 g / dl peptone and 3 g / dl CaCO ₃ , which is in a large The test tube is inoculated in. Cultivation is carried out for 4 days with shaking at 30 ° C. The yields of L-serine are given in Table V.

Table V Example 2

The fermentation procedure of Example 1 is repeated with the modification that a medium with a content of 8 g / dl glucose and 2.5 g / dl glycine instead of 3 g / dl glucose and 1 g / dl glycine as Fermentation medium is used and that 50 hours after inoculation 1% (volume / volume) propylene glycol is added. Breeding is carried out for 73 hours. L-serine is formed in an amount of 94 mg / ml

Example 3

The L-serine-forming mutants Nocardia butanica IE-36, KY 7988, NRRL 11059 and Nocardia butanica KY 7985, NRRL 11189, are used. The mutants are added to 7 ml of a culture medium with a pH of 7.2 and a content of 4 g / dl glucose, 03 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 - 7 H ₂ O, 0.5 g / dl Hefeexirakt 2 g / dl peptone, and 50 μ ^ / ϋίβΓ biotin, which in a 50 ml "> comprehensive large test tube (χ 20,190 now) is inoculated the cultivation is carried out for 24 hours at 30 ⁰ C. 2 ml of the seed culture obtained are added to 20 ml of a fermentation medium with a content of 5 g / dl glucose, 0.5 g / dl NH ₄ CI, 2 g / dl glycine, 0.15 g / di KH ₂ PO ₄ , 0.05 g / dl K ₂ HFO ₄ , 0.05 g / dl MgSO ₄ 7 H ₂ 0.0.01 g / dl NaCI, 1 mg / dl FeSO ₄ 7 H ₂ O, 1 mg / dl MnSO ₄ π H ₂ O, 1 g / dl peptone, 2 g / dl

Microorganism
10 yield
of L-serine (mg / ml) Nocardia butanica KY 7983,
,. NRRL 11187 4.1 Nocardia butanica KY 7984,
NRRL 11188 4.1 Nocardia butanica KY 7986,
NRRL 11190 2.9 ⁵⁰ Nocardia butanica KY 7987,
NRRL 11191 2.8 Nocardia butanica KY 7985,
NRRL 11189 2.0 55 Nocardia butanica ATCC 21197
(Parent strain) 0.5

Example 5

The microorganisms shown in Table VT are used. The procedure of Example 3 is followed repeated, with the modification that 0.25 ml of one The culture culture obtained according to Example 3 can be inoculated onto 5 ml of the fermentation medium from Example 3. The yields of L-serine are in Table VI specified.

Table VI

Microorganism yield
of L-serine (mg / m!) Nocardia butanica KY 7989,
NRRL 11192 7.0 Nocardia butanica KY 7990,
NRRL 11193 9.5 Nocardia butanica KY 7991,
NRRL 11194 9.0 Nocardia butanica KY 7985,
NRRL 11189 6.0 Nocardia butanica ATCC 21197 4.5 Example 6

glucose 5 g / dl (NH ₄ I ₁ SO ₄ lg / dl Glycine 2.5 g / dl KH ₂ PO ₄ 0.15 g / dl (0.011 m phosphate ions) 4o K ₂ HPO ₄ 0.05 g / dl (0.003 m phosphate ions) Mg ₃ (PO ₄ ) J · 8 H ₂ O 3 g / dl (0.147 m phosphate ions) MgSO ₄ · 7 H ₂ O 0.05 g / dl 45 FeSO ₄ · _{7H 2} O 0.001 g / dl MnSO ₄ · π H ₂ O 0.001 g / dl Peptone lg / dl CaCO ₃ 3 g / dl (PH value 7.0) so

Culturing is 5 days under shaking at 3O ⁰ C conducted There 8.4 mg / ml L-serine are formed.

For comparison, the above procedure is repeated, with the modification that a fermentation medium without Mg ₃ (PO ₄ I ₂ · 8 H ₂ O, which has 0.014 m of phosphate ions, is used. 2.5 mg / ml of L-serine are formed After the end 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.

Example 7

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

55

w>

Medium I.

10

15th

It is Nocardia butanica IE-36, KY 7988, FERM-P 3768, NRRL 11059 A loop used by a 2-day culture at 30 ⁰ C in a yeast broth slant culture containing 0.5 g / dl yeast extract, 1 g / dl peptone, 1 g / dl meat extract, 0.5 g / dl NaCl and 2 g / dl agai (pH 7.2) are added to 7 ml of a growth 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 · 7 H ₂ O, 0.5 g / dl yeast extract and 2 g / dl peptone (pH value 7.2) extending (in a 50 ml large test tube 2 O x 190 ram) inoculated is culturing is 24 hours at 30 ⁰ C carried out 1 ml of the seed culture obtained is applied to 20 ml of a fermentation medium having the following composition, which is in a 250 ml Erlenmeyer flask is inoculated: J5

Glocose 5 g / dl

(NH ₄ I ₂ SO ₄ 0.4 g / dl

KH ₂ PO ₄ 0.15 g / dl

K-HPO ₄ 0.05 g / dl

MgSO ₄ · 7 H ₂ O 0.05 g / dL

FeSO ₄ · 7 H ₂ O 0.001 g / dl

MnSO ₄ · a H ₂ O 0.001 g / dl

Glycine 2 g / dl

Peptone 1 g / dl

CaCO ₃ 3 g / dl

pH 7.0

This medium is 0.014 m in phosphate ions. Medium II

This medium has the same composition as medium I, with the addition of those in Table VIl specified amounts of phosphate are present.

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

Table VII

Phosphate addition (2) Total con yield (2) centering of L-serine (2) on Phos (2) phations (%, W / v) (2) (m) (mg / ml) Mg- (PO ₄ ): · 8H ₂ O (2) 0.112 7.0 MgHPO ₄ • _{3H 2} O 0.129 7.5 Ca ₃ (PO ₄ ): 0.143 2.5 Mg ₂ (P ₂ O ₇ ) • 3 H ₂ O 0.159 1.8 Znj (PO ₄ ) ₂ ■ 4 H ₂ O 0.102 2.0 K ₃ PO ₄ · ηΗ, Ο 0.089 *) 6.0

+ Mg ₂ SO ₄ 7H ₂ O (3)

(none) 0.014

1.0

*) K ₃ PO ₄ · nH ₂ 0 calculated as trihydrate

Example 8

Nocardia butanica KY 7990, NRRL 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 ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ · 7 H ₂ 0.0.5 g / dl yeast extract and 2 g / dl peptori (pH 7.2), which is in a 300 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 poured onto 30 ml of a fermentation medium with a content of 8 g / dl glucose, 1 g / dl (NH _{4 I 2} SO ₄ , 1 g / dl peptone, 3 g / dl Mg ₃ (PO _{4 I 2} · 8 H ₂ O, 0.15 g / dl KH ₂ PO ₄ , 0.05 g / dl K ₂ HPO ₄ , 0.05 g / dl MgSO ₄ · 7 H ₂ O, 0.001 g / dl FeSO ₄ · 7 H ₂ O and 0.001 g / dl MnSO ₄ · 4 HaO ibeirimpft (pH 7.2), located in 15 equipped with baffle plate !! 300-ml Erlenmeyer flask, (. the cultivation is 24 hours under shaking at 30 ⁰ C 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 a further 16 hours. The fermentation liquids obtained are combined. The mixture is centrifuged to obtain the microorganism cells. The cells obtained are in 0 , Suspended 2 m phosphate buffer with a content of 2.5 g / dl glycine

030 127 / 34-

Suspension has a content of 60 mg / ml, based on the dryness of the cells. The suspension is then poured into a 300 ml Erlenraeyer's 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. 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 JiSO ₄ , ZS g / dl glycine, 0.15 g / dl KH ₂ PO ₊ , 0.05 g / dl K ₂ HPO ₊ , 0.05 g / dl MgSO ₄ · 7 H ₂ O, 0.001 g / dl FeSO ₄ 7 H ₂ O. 0.001 g / dl MnSO ₊ π H ₂ O, 1 g / dl peptone and 3 g / dl Mg ₃ (PO ₊ J ₂ 8 H ₂ O (pH 7, 0), which is in a 300 ml Erlenmeyer flask, inoculated. The cultivation is carried out for 48 hours with shaking at 30 ^° C. The glucose concentration after the cultivation is less than 1 g / dL 0.5 ml of the fermentation liquid is mixed with 0.5 ml of 6 η hydrochloric acid in order to dissolve the insoluble constituents present in the fermentation liquid with the exception of the microorganism cells Suspension measured at a wavelength of 660 μm with a spectrophotometer, giving a value of 0.27, which corresponds to the value obtained 2 hours earlier in the same way e has been measured

The additives given in Table VIII are then added in the given concentrations added. Cultivation is continued for 72 hours. Yields of L-serine are shown in Table VIII specified.

After the end of the cultivation, 1 liter of the using acetone as an additive in the cultivation The fermentation liquor obtained is purified. The purification is carried out carried out according to Example 1. 6.2 g of crystalline L-serine are obtained.

Example 10

Table VIII Conc yield additive tralion of L-serine (%. VoIVVoI.) (mg / ml) 3 13.0 Ligroin 3 10.4 Cyclohexane 1 10.8 Methanol 1 9.0 Ethanol 0.5 11.9 acetone 1 9.5 Methyl ethyl ketone 0.5 10.5 Ethyl acetate I. 10.0 Methyl acetate 0.3 10.0 Citric acid tributyl ester 2 9.5 1,4-dioxane I. 9.8 Tetrahydrofuran 0.5 10.0 Propylene glycol 0.5 9.5 1,3-butanediol (1.13 9.0 Ethylene glycol mono-lithium iither 0.13 9.0 Ethylene glycol monomethyl iilheracetat 8.2 no addition

The cultivation culture is carried out according to Example 8 100 ml of the cultivation culture are added to 3 liters of the fermentation medium according to Example 8, which additionally contains 0.01 g / dl CaCl ₂ . 2 H ₂ O and is in a 5-liter fermenter, inoculated Culturing is 48 hours under aeration and agitation at 30 ⁰ C. The fermentation liquor is then carried out

ίο centrifuged the microorganism cells obtained are washed twice with 0.1 M phosphate buffer of pH 7.0 and stored frozen in phosphate buffer 5 g of ethyl cellulose (50 to 100 cp) are in a solvent mixture of 72.5 g

is benzene and 25.5 g of n-hexane containing Sorbitan monolaurate dissolved as a dispersant

In addition, 5 g of frozen microorganism cells (dry weight) are placed in 50 ml of 0.1 M phosphate buffer of pH 7.0 with a content of 100 mg Mg (H ₂ PO ₄ ) ₂ and 100 mg of Mg ₃ (PO ^ suspended The resulting suspension is added to the ethyl cellulose solution. The mixture is stirred thoroughly to form a first emulsion men) polyethylene glycol solution (average Mo Lekulargewicht 6000) from pH 8 to 9 and cooled with ice to 10 to 15 ° C. The mixture is under Formation of a second emulsion stirred. This emulsion is continuously stirred at a rate of 10 to 15 ml / min to n-hexane (5 to 10 ° C) cooled with ice water to remove the benzene and the To crystallize ethyl cellulose. After 1 hour, immobilized cells enclosed in ethyl cellulose are obtained Cells. 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 entrapped cells is 300 to 350 mg cells / ml microcapsules. 100 ml of the immobilized cells are placed in a 1 liter Reaction vessel (90 mm 150 mm α) kept in a fluidized bed using air and 0.1 M phosphate buffer solution with a pH of 6.1. The phosphate buffer solution contains 0.2 g / ml glycine, 5 g / ml Mg (H ₂ POt) ₂ and 5 g / ml Mgj (PO ₄ ) 2. This solution goes to the reactor supplied with a residence time of 15 to 20 hours. An overflow solution containing L-serine is continuously applied for 50 hours formed from 10 to 13 mg / ml

B e i s ρ i e I 11

There are 500 ml of cyclohexane as a dispersion medium and 4 g of sorbitan monolaurate mixed as a dispersant and mixed with ice-cooling and under nitrogen as Protective gas stirred at 10 to 15 ° C. Then will

. ') the mixture with 19.5 g of acrylamide and 03 g of N.N-bisacrylamide in 100 ml of 0.1 M phosphate buffer solution of pH 6.1 is dissolved or suspended, and with 10 g of frozen cells (based on the dry components) which have been prepared according to Example 10

bo are offset. The resulting mixture is 10 ml 2.5 percent (igem (Gewichl / Völümen) Ν, Ν, Ν ', Ν'-Tetramethylethylenediamine as a polymerization stabilizer and 125 ml of 2.5 percent (weight / volume) ammonium persulfate added as a polymerization initiator. the

H'. The polymerization reaction is carried out for 1 hour, whereby immobilized cells are formed which are entrapped in gel and are JOO to 400 u.

The activity of the immobilized cells is about 30 to 40 percent of the cells before immobilization. The amount of cells enclosed in the gel is 50 to 70 mg cells / ml gel, ·

Then 100 liters of gel and 800 ml of a glycine solution with a content of 25 mg / ml are poured into a 1 liter reactor. The reaction is carried out for 25 hours at 3O ⁰ C batchwise in the reaction mixture is L-serine is in a concentration of I2j5 mg / ml. The gel is then recovered from the reaction mixture. The above procedure is repeated twice using the recovered gel. The results are shown in Table IX

Table Tue Conc
tration of the
Glycine
(mg / ml) concentration
of L-serine
(mg / ml) Implementation
degree of efficiency
(%) number of
Use
fertilize 25th
25th
25th 12.5
11.8
10.5 50.0
47.2
42.0 1
2
3

1 liter of the first reaction mixture is filtered to remove the immobilized cells. The pH of the transferring filtrate is adjusted with hydrochloric acid adjusted to 2.0 then the filtrate is over a column packed with a strongly acidic cation exchange resin (Diaion SK-IB). The elution is carried out with 2 η aqueous ammonia solution. The eluate is evaporated under reduced pressure 8.5 g of L-serine are obtained.

Claims (11)

Patent claims: 1. A process for the preparation of L-serine by a microbiological route, characterized in that glycine is in an aqueous Medium in the presence of microorganism cells of a mutant of the species Nocardia butanica which are used for Conversion of glycine to L-Seriu is able and the no or a reduced ability to Utilization of L-serine and / or resistance to at least one metabolic antagonist of glycine, serine, methionine, glutamine, histidine, Leucine, isoleucine, valine, purine, pyrimidine and / or Has folic acid, converts to L-serine, L-serine im Enriched medium and isolated from it 2. The method according to claim 1, characterized in that one Nocardia butanica NRRL 11 059, Nocardia butanica NRRL11 187, Nocardia butanica NRRL11 188, Nocardia butanica NRRL11 189, Nocardia butanica NRRL11 190, Nocardia butanica NRRL11 191, Nocardia butanica NRRL1M92, Nocardia butanica NRRL 11 193 or Nocardia butanica N RRL 11 194 used 3. The method according to claim 1, characterized in that the conversion of glycine to Carries out L-serine with cultivation of the mutant in a nutrient medium containing glycine 4. The method according to claim 3, characterized in that the nutrient medium at least Contains 0.037 mol / liter of phosphate ions 5. The method according to claim 3, characterized in that the nutrient medium 0.05 to 0.4 mol / liter Contains phosphate ions 6. The method according to claim 3, characterized in that the cultivation is carried out ^{at temperatures of 20 to 40 0 C for 1 to 7 days} 7. The method according to claim 1, characterized in that the reaction is carried out in an aqueous Medium carries out the glycine and microorganism cells obtained by culturing the mutant contains 8. The method according to claim 7, characterized in that one glycine in a concentration of 0.5 to 20 percent used 9. The method according to claim 1, characterized in that the conversion of glycine to L-serine in the presence of at least one additive from the group of hydrocarbons, alcohols, Carries out ketones, esters, ethers, polyalcohols and derivatives of polyhydric alcohols 10. The method according to claim 9, characterized in that the additive in one concentration from 0.01 to 5 percent by volume based on the volume of the medium 11. The method according to claim 7, characterized in that there is used as microorganism cells immobilized cells used