GB1592499A - Process for production of l-serine - Google Patents

Description

(54) PROCESS FOR PRODUCTION OF L-SERINE (71) We, KYOWA HAKKO KOGYO CO., LIMITED, a company existing under the laws of Japan, of Ohtemachi Building Ohtemachi, Chiyoda-ku, Tokyo, Japan, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: Background of the invention The present invention relates to a process for the production of L-serine from glycine.

L-serine is one of the amino acids well known in the art and is in great demand as a medicament.

Heretofore, L-serine has been prepared using various methods. For example, it has been obtained by hydrolysis of proteins.

As for processes for the production of L-serine from glycine by fermentation, a process using a microorganism belonging to the genus Nocardia is known and is described in Japanese Patent Publication No. 9391/76. However, the yield of L-serine is poor.

On page 80 of the published summary of the lectures given during the Congress of Fermentation Technology, Japan 1975, it was reported that increased yields of L-serine are obtained by culturing a mutant belonging to the species Corynebacterium glycinophilum and having both the ability to convert glycine to L-serine and a lowered ability to decompose L-serine.

As other prior methods of producing L-serine by fermentation, processes of culturing a strain belonging to the genus Arthrobacter, Corynebacterium, Brevibacterium, Escherichia, Micrococcur, Pichia, or Candida in a nutrient medium or in a nutrient medium containing glycine are known.

However, processes which have a high yield of L-serine are in demand for utilization in industrial practice.

The present invention is based on the discovery that mutants capable of producing L-serine from glycine and belonging to the genus Nocardia and having no or lowered ability to decompose L-serine as compared to the parent strain, when cultured in a nutrient medium containing glycine, produce remarkable amounts of L-serine in the culture liquor.

Further, it has been found that mutants capable of producing L-serine from glycine and belonging to the genus Nocardia, and having a resistance to at least one antagonist having antagonism towards one or more of the following: glycine, L-serine, L-methionine, L-glutamine, L-histidine, L-leucine, L-isoleucine, L-valine, purine, pyrimidine and L-folic acid, when cultured in a nutrient medium containing glycine, also produce remarkable amounts of L-serine in the culture liquor.

Further, it has been discovered that the improved yields of L-serine are obtained even when such processes are carried out on an industrial scale.

In accordance with the present invention, therefore, there is provided a process for producing L-serine which comprises converting glycine to L-serine in an aqueous medium containing glycine in the presence of microbial cells of a microorganism belonging to the genus Nocardia and capable of converting glycine to L-senne, wherein there are used microbial cells of a mutant strain having no or a lowered ability to decompose L-serine as compared to the parent strain from which the mutant is derived and/or a resistance to at least one metabolic antagonist having antagonism towards one or more of the following: glycine, L-serine, L-methionine, L-glutamine, L-histidine, L-leucine, L-isoleucine, Lvaline, purine, pyrimidine and L-folic acid.

As the strain used in the present invention, any mutant belonging to the genus Nocardia and capable of converting glycine to L-serine, and having no or a lowered ability to decompose L-serine, as compared with the parent strain and/or a resistance to at least one metabolic antagonist having antagonism towards one or more of the following: glycine, L-serine, L-methionine, L-glutamine, L-histidine, L-leucine, L-isoleucine, L-valine, purine, pyrimidine and L-folic acid may be used.

Such a strain may be obtained by endowing a strain of microorganisms belonging to the genus Nocardia and capable of converting glycine to L-serine with no or a lowered ability to decompose L-serine as compared with the parent strain and/or a resistance to at least one of the metabolic antagonists mentioned above.

Alternatively, the strain may be obtained by endowing a strain of microorganisms belonging to the genus Nocardia and having no or a low ability to decompose L-serine and/or resistance to at least one of the metabolic antagonists mentioned above with the ability to convert glycine to L-serine.

Furthermore, the strain to be used in the present invention may have any other property for contributing to the L-serine productivity than the properties mentioned above.

Examples of metabolic antagonists used for induction of mutants are shown in Table 1.

Example of Antagonist Glycine glycine-hydroxamate, amino methylsulfonic acid, chloromethyl-glycine, glycidic acid, N-methyl-glycine L-Serine serine-hydroxamate, D-serine, homoserine L-Methionine ethionine, tri-fluoro-methionine, a-methylmethio nine, methionine-hydroxamate, selenomethionine L-Glutamine methionine-sulfone, methionine sulfoximine, azaserine, alanosine, duazomycin L-Histidine thiazole-alanine, triazole-alanine, aminomethyl triazole, a-methyl-histidine, histidine-hydroxamate L-Leucine norleucine, tri-fluoroleucine, azaleucine, leucine hydroxamate L-lsoleucine thiaisoleucine, O-methyl-threonine, isoleucine hydroxamate, D-isoleucine, norleucine L-Valine norvaline, valine-hydroxamate, D-valine norleucine Purine 6-mercaptopurine, 8-azaguanine, 2-fluoroadenine Pyrimidine 5-fluorouracil, 2-thiouracil, 5-fluorocitosine, 6-azauracil L-Folic acid amethopterin, aminopterin, trimethoprim, pyrimethamine In order to obtain mutants for use in the present invention, standard procedures for inducing mutation may be followed such as irradiation with ultraviolet light, X-ray or Co 60, or treatment with mutation inducing chemicals such as N-methyl-N'-nitro-Nnitrosoguanidine.

A simple method to select a strain having no or a lowered ability to decompose L-serine from the colonies obtained by mutation is as follows: A colony is selected from the colonies obtained by mutation and is cultured in a medium containing L-serine as the only carbon source.

The strain that does not grow or grows less than the parent strain in the medium is selected as the strain having no or a lowered ability to decompose L-serine.

Nocardia butanica KY 7985 is an example of a mutant having a lowered ability to decompose L-serine, which is obtained by mutating the L-serine-producing parent strain Nocardia butanica ATCC 21197 by the method described later.

Further, the strains listed below in Table 2 are examples of mutants having resistance to metabolic antagonists, which are obtained by mutating Nocardia butanica KY 7985 by methods described later.

The microbiological properties of the parent strain of Nocardia butanica (ATCC 21197) are described in Japanese Patent Publication No. 48673/72. KY 7985 differs from the parent strain merely in its reduced ability to decompose L-Serine.

The above-noted mutants have been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Chiba-ken, Japan.

The strain KY-7985 is deposited under FERM-P No. 3782 and the other strains are deposited under FERM-P Nos. shown in Table 2.

These mutants have also been deposited with the Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois.

The strain KY 7985 has been accorded accession number NRRL 11189 and the other strains have been accorded accession numbers shown in Table, 2.

TABLE 2 Private FERM-P NRRL Antagonist No. No. No.

20IMP-14 3764 11187 trimethoprim (0.35) KY 7983 20MP-24 3765 11188 6-mercaptopurine (0.5) KY 7984 21SX-2 3766 11190 serine hydroxamate (1) KY 7986 21GX-1 3767 11191 glycine hydroxamate (1) KY 7987 IE-36 3768 11059 glycine hydroxamate (1) KY 7988 ethionine (5), norleucine (1) IAU-3 3769 11192 glycine hydroxamate (1) KY 7989 6-azauracil (0.5) 36MSF-2 3770 11193 glycine hydroxamate (1) KY 7990 ethionine (5), methionine sulfone (5) 36TRA-1 3771 11194 glycine hydroxamate (1) KY 7991 ethionine (5), 1,2,4-triazole-1,3 alanine (1) Note: Figure in the parentheses means concentration (mg/ml) of metabolic antagonist in the medium used for isolation of mutant.

Strains KY 7983, 7984 and 7986-91 differ from the parent strain (ATCC 21197) in their reduced ability to decompose L-serine and their resistance to the antagonist noted.

The strain KY 7985, NRRL 11189 is obtained in the following manner. Microbial cells of the parent strain, i.e. Nocardia butanica ATCC 21197, are suspended at a concentration of about 108 cells per ml, in 0.1 M-Tris-maleate buffer solution (pH 6.0). To Che suspension is added 0.5 mg/ml of N-methyl-N'-nitro-N-nitrosoguanidine and the mixture is allowed to stand at room temperature for 30 minutes. Then the resultant suspension is smeared on an agar plate of a nutrient medium comprising 0.5 g/dl glucose, 0.5 g/dl yeast extract, 1 g/dl peptone, 1 g/dl meat extract, 0.5 g/dl NaCI and 2 g/dl agar (pH 7.2) and incubated at 30"C for 2 days to form colonies. Cells of the resulting colonies are smeared on an agar plate of a minimum medium comprising 0.5 g/dl glucose, 0.2 g/dl (NH4)2SO4, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4.7H2O, 0.01 g/dl NaCI, 0.001 g/dl FeSO4.7H2O, 0.001 g/dl MoSO4. 5nH2O, 0.001 g/dl CaCl2.2H2O, 100 Fg/l biotin, 1 mg/l thiamine hydrochloride, and 2 g/dl agar (pH 7.2) and also on an agar plate of a medium having the same composition as that of the minimum medium described above but containing 0.2 g/dl L-serine instead of (NH4)2S04 The strains having an ability to produce L-serine in a good yield are selected from those strains which do not grow on the latter medium, but do grow on the agar plate of the former medium, i.e. minimum medium.

The strain, Nocardia butanica KY 7985, NRRL 11189 is one of them.

The mutants shown in Table 2 are obtained in the following manner from the strain, Nocardia butanica KY 7985, NRRL 11189 as the parent strain. Microbial cells of the parent strain, i.e. Nocardia butanica KY 7985, NRRL 11189 are suspended at a concentration of about 108 cells per ml, in 0.1M Tris-maleate buffer solution (pH 6.0). To the suspension is added N-methyl-N'-nitro-N-nitrosoguanidine at a concentration of 0.5 mg/ml and the suspension is allowed to stand at room temperature for 30 minutes.

Then the suspension is diluted and then smeared on to an agar plate of a medium having the same composition as that of the above-mentioned minimum medium except that it also contains the antagonist at a concentration shown in Table 2. The plate is then incubated at 30"C for 2 - 10 days.

The mutants shown in Table 2 are selected from those colonies which grow on the medium. The mutants are distinguished from the parent strain by possession of a resistance to the metabolic antagonists.

Whether a mutant has a lowered ability to decompose L-serine as compared with that of the parent strain may be determined as follows: A selected strain is cultured in a nutrient medium containing neither glycine nor L-serine in order to confirm that the strain does not produce L-serine. Then the strain is cultured in a nutrient medium containing not glycine but L-serine in order to determine the ability to decompose L-serine. After the completion of culturing, if the amount of L-serine remaining in the culture liquor is larger than that of the parent strain, the strain has no or a lowered ability to decompose L-serine.

Alternative methods will be apparent to those skilled in the art.

It is understood from the following experiments that Nocardia butanica KY 7985 has a lowered ability to decompose L-serine.

Experiment I The strain of Nocardia butanica KY 7985, NRRL 11189 is cultured on an agar slant of a medium (pH 7.2) containing 0.5 g/dl glucose, 0.5 g/dl yeast extract, 2 g/dl peptone, 0.5 g/dl NaCI, and 2 g/dl agar at 30"C for 2 days.

One loopful of the resulting seed culture is inoculated into 7 ml of a seed medium (pH 7.2) containing 4 g/dl glucose, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4.7H2O, 0.3 g/dl urea, 50 llg/dl biotin, 0.5 g/dl yeast extract and 2 g/dl peptone in a large test tube (20 mm x 190 ml). Culturing is carried out with shaking at 300C for 24 hours.

0.25 ml of the resulting seed culture is inoculated into 5 ml of medium containing L-serine having the following composition in a large test tube: glucose 5 g/dl (NH4)2SO4 0.2 g/dl urea 0.2 g/dl KH2PO4 0.15 g/dl K2HPO4 0.05 g/dl MgSo4.7H20 0.05 g/dl NaCl 0.01 g/dl FeSO4.7H20 0.001 g/dl MnSO4.4H20 0.001 g/dl biotin 501gel yeast extract 0.1 g/dl L-serine 0.5 g/dl CaC03 3 g/dl (pH 7.2) Culturing is carried out with shaking at 300C for 3 days.

The quantitative determination of L-serine is carried out by paper chromatography using Toyo filter paper No. 50 with developing solvent consisting of n-butanol; acetone; water and diethyl amine [10 : 10 : 5 : 2 by volume].

The reduced amount of L-serine is 1.0 mg/ml. As a control, the same procedures described above were repeated using Nocardia butanica ATCC 21197. The amount of L-serine produced was 4.6 mg/ml.

Experiment 2 The seed culturing of Nocardia butanica KY 7985, NRRL 11189 is carried out in the same manner as described in Experiment 1 and one ml of the resulting seed culture is inoculated into 20 ml of a fermentation medium having the following composition in a 300 ml Erlenmeyer flask: glucose 5 g/dl (NH4)2S04 0.2 g/dl glycine 0.5 g/dl L-serine 1 g/dl urea 0.2 g/dl yeast extract 0.5 g/dl biotin 50 g/l KH2PO4 0.15 g/dl K2HPO4 0.05 g/dl MgS04.7H20 0.05 g/dl NaCl 0.01 g/dl FeSO4.7H20 0.001 g/dl MnSO4-4H20 0.001 g/dl CaCL2.2H20 0.001 g/dl (pH 7.2) Culturing is carried out with shaking at 30"C for 40 hours.

50 ml of the culture broth resulting from the fermentation is subjected to centrifugation to recover the microbial cells: The microbial cells are washed with isotonic-sodium chloride solution and then are suspended in 0.1 M pyrophosphate buffer solution (pH 9.0) containing 5 x 10-5 M of pyridoxal phosphoric acid, 5 x 10-4 M of EDTA and 10-4 M of 2-mercapto ethanol.

The resulting suspension is subjected to disruption with an ultra sonic disintegrator for 30 minutes and then to centrifugation to obtain a supernatant solution. 0.5 ml of the supernatant solution (concentration of protein: 6.0 mg/ml) and 0.5 ml of 0.1 M pyrophosphate buffer solution (pH 9.0) mentioned above containing 1.4 g/dl L-serine are combined and the mixture is allowed to stand at 370C for 14 hours.

The amount of L-serine produced is determined by paper chromatography described in Experiment 1 and is 1.6 mg/ml. As a control, when Nocardia butanica ATCC 21197 is used in the same manner, the amount of L-serine produced is 4.2 mg/ml.

In the present invention, when the mutant is cultured in a nutrient medium containing glycine to produce L-serine in the culture liquor, the productivity of L-serine can be further enhanced by including phosphate at a concentration of more than 0.037 A based on the phosphate ion in the fermentation medium either at the start of the fermentation or during the growth phase of the cells.

The influence on the productivity of L-serine by the presence of a high concentration of phosphate ion in the culture medium was studied by using Nocardia butanica KY 7988 and the result are shown in the following Experiment 3.

Experiment 3 Nocardia butanica KY 7988, NRRL 11059 is inoculated into 5 ml of a fermentation medium containing 5 g/dl glucose, 1 g/dl (NH4)2SO4, 2.5 g/dl glycine, 0.05 g/dl K2HPO4 (correspond to 0.003 M of phosphate ion), 0.15 g/dl KH2PO4 (correspond to 0.011 M of phosphate ion), 0.05 g/dl MgSO4.7H2O, 0.001 g/dl FeSO4.7H2O, 0.001 g/dl MnSO4.4H2O, 1 g/dl peplcne, 3 g/dl CaCO3 and 0 - 2 g/dl Mg3(PO4)2.8H2O (pH 7.2) in a large test tube.

Culturing is carried out with shaking at 28"C for 5 days, whereby L-serine is produced in a yield shown in Table 3.

TABLE 3 Supplemented Total concentration Yield of L-serine Mg3(PO4)2.8H2O of phosphate ion (g/dl W/V) (M) (mg/ml) 0 0.014 1.1 0.1 0.019 2.0 0.2 0.024 2.9 0.3 0.029 3.2 0.4 0.034 3.5 0.5 0.039 3.9 0.6 0.044 4.5 07 0.048 5.0 0:8 0.053 5.2 09 0.058 5.5 1.0 0.063 6.3 1.5 0.089 7.3 2.0 0.112 7.0 As is apparent from Table 3, the yield of L-serine is increased with the increasing concentration of phosphate ion (max: 6 times).

The same procedures as mentioned above are repeated using 0-2 g/dl MgSO4.7H2O instead of Mg > ,(PO4)2.8H2O. As a result, it is found that increasing the amount of MgSO4.7H2O does not contribute towards the yield of L-serine.

Examples of phosphates which may be used in the present invention are NaH2PO4, Na2HPO4, Na3PO4, KH2PO4, K2HPO4, K3PO4, NH4.H2PO4, (NH4)2HPO4, (NH4)3PO4, Mg(H2PO4)2, MgHPO4, Mg3(PO4)2, Ca(H2PO4)2, CaHPO4, Ca3(PO4)2, Mn(H2PO4)2, MnHPO4. Mn3(PO4)2, ZnHPO4, Zn > ,(PO4)2, FeHPO4, Fe3(PO4)2, Co3(PO4)2, K(NH4)HPO4 and Na(NH4)2PO4 and the hydrates thereof.

As the phosphate, ortho-phosphate is usually used and, of course, meta-, pyro- and poly-phosphate may be used.

Further, phosphate containing substances, such as phosphate ore, industrial chemicals, ion exchange resins containing adsorbed phosphate ion and activated carbon containing adsorbed phosphate ion may also be used.

These phosphates may be used alone or as mixtures of two or more thereof.

The concentration of phosphate in the medium, which promotes the conversion of glycine to L-serine, is usually more than 0.037 mole/l, preferably, 0.05 - 0.4 mole/1.

In the present invention, it has also been discovered that the productivity of L-serine can be further enhanced by using an aqueous medium containing one or more of the following: a hydrocarbon, a monohydric alcohol, a ketone, an ether, an ester, a polyhydric alcohol or an ester or ether derivative of a polyhydric alcohol. When not initially present as a carbon source in the nutrient medium, the additive may be added to the medium at any time during culturing the microorganism but will usually be added at the time when the microorganism has completed its growth and before or after the addition of the glycine in the production phase.

Examples of productivity enhancing additives used in the present invention are shown in Table 4.

TABLE 4 Hydrocarbons: ligroin, petroleum benzine, petroleum spirit, petroleum ether, cyclohexane, hexane, kerosene, hexadecane Monohydric methanol, ethanol, n-propanol, iso-propanol, Alcohols: n-butanol, iso-butanol, sec-butanol, tert butanol, n-amylalcohol, cyclohexanol, furfuryl alcohol Ketones: acetone, methyl ethyl ketone, diethyl ketone Ethers: ethyl ether, isopropyl ether, n-butyl ether, 1,2-propylene oxide, 1,4-dioxane, furan, furfural, tetrahydrofuran Esters: ethyl formate, methyl acetate, ethyl acetate tributyl citrate, dioctyl phthalate, ethyl propionate, ethyl benzoate Polyhydric ethylene glycol, propylene glycol, Alcohols: 1,3-butanediol, glycerol Ether and ester Diethylene glycol, triethylene glycol, ethylene derivatives of glycol monomethyl ether, ethyl Cellosolve polyhydric alcohols: (Registered Trade Mark), ethylene glycol monomethyl ether acetate, ethylene glycol monobutyl ether, glycerol triacetate, glycerol 1-methyl ether These additives are usually used at a concentration of 0.01 - 5 % (V/V).

As to the fermentation medium employed in the present process for culturing the mutant, any synthetic or natural medium can be employed, so long as it contains a carbon source, a nitrogen source, inorganic materials, and trace amounts of nutrients necessary for the specific mutant.

Any carbon source and nitrogen source can be used in the medium, so long as they can be utilized by the microorganism. For example, carbohydrates such as glucose, fructose, sucrose, maltose and mannose; sugar alcohols such as sorbitol and mannitol; glycerol; starch; starch hydrolyzate liquor and molasses may be used. Further, various organic acids such as pyruvic acid, lactic acid, acetic acid, fumaric acid and gluconic acid and lower alcohols such as methanol and ethanol, glycols such as ethylene glycol and hydrocarbons such as ethane, propane, butane, n-paraffins and kerosene may also be used.

As a nitrogen source, the following substances are appropriate: 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 nitrogen-containing materials; and nitrogenous organic materials such as peptone, NZ-amine, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or its digested product and chrysalis hydrolyzate.

As inorganic materials, monopotassium dihydrogen phosphate, dipotassium monohydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganous sulfate and calcium carbonate may be used.

If other nutrients are necessary for the growth of the mutants, they must, of course, be present in the medium. However, it is not necessary that they be separately added to the medium so long as they are supplied to the medium together with the other components as described above. That is, certain natural ingredients may adequately supply the specific growth promoting factors.

Culturing is carried out under aerobic conditions e.g. by shaking or aeration-agitation. A suitable culturing temperature is from 20 to 40"C. It is desirable to keep the pH of the medium around neutrality throughout culturing in order to obtain a high yield, but these temperature and pH conditions are not essential for the practice of the present invention.

Culturing is usually carried out for 1 to 7 days.

After the completion of the culturing, the resultant culture broth is subjected to centrifugation, filtration or the like to obtain the microbial cells. The microbial cells may be obtained as they are or the cells may first be disrupted by suitable means such as by ultrasonic disintegration.

When culturing the mutant to obtain L-serine in Process (I) the same fermentation medium is used as that described above in obtaining the microbial cells except that glycine is added to the medium. The same culturing conditions as those described above may also be used.

Usually in the L-serine production phase the glycine will be present in the nutrient medium at a concentration of 0.1 - 5 % (W/V) either at the start of the fermentation or during the growth phase of the cells.

When the conversion is carried out according to Process (II), the cells are suspended in a phosphate buffer solution (pH 7.0) containing 0.5 - 20% glycine at a concentration of 5 - 200 mg/me based on dry weight. Then the conversion is carried out at room temperature for 5 30 hours to accumulate L-serine in the aqueous medium.

According to the present invention, when immobilized cells prepared by a known method such as by an entrapment method for example, gel entrapment or microcapsule entrapment or by an adsorption method or by a covalent bonding method L-serine is produced in high yield.

In the immobilization of cells by the gel entrapment method, to the suspension of microbial cells are added a monomer such as acrylic acid amide, N,N'-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'-tetramethylethylene diamine and suspension polymerization is carried out at 0 40"C for about one hour.

In the immobilization by the microcapsule entrapment method, a substance capable of forming semipermeable membrane such as ethylcellulose or polystyrene is dissolved in an organic solvent immiscible with water and having lower boiling point than that of water, such as benzene or cyclohexane. Then, to the solution are added microbial cells to form first a water-in-oil emulsion. Then the emulsion is added to a suspension containing a protective colloid substance such as gelatin or polyvinylalcohol with stirring to form the second emulsion. The organic solvent is then removed from the second emulsion whereby microcapsules are formed.

The contact of glycine with the thus obtained immobilized cells may be carried out in batch systems or continuous systems such as the column method or by a fluidization method.

In the batch systems, the immobilized cells are suspended at a concentration of 5 - 15 g/de in a glycine solution. The suspension is subjected to reaction to convert glycine to L-serine with stirring at 20 - 40"C, for 5 - 30 hours.

After the completion of the reation, the resultant mixture is subjected to filtration or centrifugation to obtain a filtrate or supernatant containing L-serine.

The preferred glycine concentration of the glycine solution is 5 - 50 g/e based on the total volume of glycine solution and immobilized cells.

The immobilized cells separated from the reaction mixture may be reused.

In the column method, 5 - 50 g/e glycine solution is passed through the column packed with the immobilized cells to produce L-serine in the solution.

In the fluidization method, the immobilized cells are fluidized in a reactor by using air and phosphate buffer solution containing 5 - 50 glP glycine, 0.5 - 3 g/de inorganic compounds such as magnesium sulfate, manganese sulfate or phosphate to produce L-serine in the solution. The fluidization reaction is carried out at 20-40"C and at a pH of 5 - 9.

After the completion of conversion, the microbial cells and precipitates are removed from the mixture or culture liquor by conventional methods. Then L-serine is recovered from the resultant solution by known methods, such as an ion exchange resin treatment.

The practice of certain specific embodiments of the invention is illustrated by the following representative examples.

EXAMPLE 1 Fermentation Nocardia butanica KY 7985, FERM-P 3782, NRRL 11189 is used. One loopful of the seed culture obtained by culturing at 300C for 2 days in an agar plate of the medium containing 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 (pH 7.2) is inoculated into 7 ml of a seed medium containing 4g/dl glucose 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4.7H2O, 0.3 g/dl urea, 50 Zg/l.biotin 0.5 g/dl yeast extract and 2 g/dl peptone (pH 7.2) in a large test tube (20 mm x 190 mm). Culturing is carried out with shaking at 30"C for 24 hours. One ml of the resulting seed culture is inoculated into 20 ml of a fermentation medium containing 3 g/dl glucose, lg/dl (NH4)2SO4, 0.2 g/dl yeast extract, lg/dl glycine, 0.001 g/dl FeSO4.7H2O, 0.001 g/dl MnSO4.4H2O, 0.05 g/dl MgSO4.7H2O, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4 and 3 g/dl CaCO3 (pH 7.0) in a 300 ml Erlenmeyer flask.

Culturing is carried out with shaking at 300C for 4 days, whereby L-serine is produced in a yield of 3.5 mg/ml.

Purification After the completion of the culturing, one liter of the culture broth resulting from the fermentation is subjected to centrifugation to remove the microbial cells and precipitates; and the resulting supernatant is passed through a column packed with 400 ml of a strongly acidic cation exchange resin, Diaion SK-IA H+ form, manufactured by Mitsubishi Kasei Kogyo K.K., Japan) to adsorb L-serine thereon.

After the resin is washed with 1.5 P of water, the resin is subjected to elution with 0.5 N aqueous ammonia and then the fractions containing L-serine are collected and concentrated to 15 ml.

0.1 M citrate buffer solution having a pH of 3.41 is prepared by dissolving 21.01 g of citric acid in 200 ml of 1 N caustic soda, adding 110 ml of 1 N hydrochloric acid and 5 ml of 2-Mercapto ethanol and making the resultant solution up to 1 P with water.

After the pH of the concentrate is adjusted to 3.41 with citric acid, the resulting solution is passed through a column (3.2 cm x 85 cm) packed with Diaion SK-1A (Na+ form) eauilibrated with the citrate buffer solution to adsorb L-serine thereon and elution is carried out with

EXAMPLE 4 In this example, L-serine producing mutants shown in Table 5 are used.

As a control, Nocardia butanica ATCC 21197 is also used.

0.25 ml of seed culture obtained by culturing microorganisms shown in Table 5 in the same manner as described in Example 3 is inoculated into 5 ml of a fermentation medium (pH 6.1) containing 5 g/dl glucose, 1 g/dl (NH4)2SO4, 2 g/dl glycine, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4.7H2O, 0.01 g/dl NaCl, 1 mg/dl FeSO4.7H2O, 1 mg/dl MnSO4.4H2O, 1 g/dl peptone and 3 g/dl CaCO3 in a large test tube.

Culturing is carried out with shaking at 30 C for 4 days. L-serine is produced in a yield shown in Table 5.

TABLE 5 Microorganism Yield of L-serine (mg/ml) Nocardia KY 7983, NRRL 11187 4.1 butanica KY KY 7984, NRRL 11188 4.1 KY KY 7986, NRRL 11190 2.9 KY KY 7987, NRRL 11191 2.8 KY KY 7985, NRRL 11189 2.0 ATCC 21197 0.5 EXAMPLE 5 In this example, the microorganisms shown in Table 6 are used. The same procedures as described in Example 3 are repeated except 0.25 ml of the seed culture obtained by culturing in the same manner as described in Example 3 is inoculated into 5 ml of the same fermentation medium as described in Example 3. L-serine is produced in the yields shown in Table 6.

TABLE 6 Microorganism Yield of L-serine (mg/ml) Nocardia KY 7989, NRRL 11192 7.0 butanica " KY 7990, NRRL 11193 9.5 " KY 7991, NRRL 11194 9.0 " KY 7985, NRRL 11189 6.0 ATCC 21197 4.5 EXAMPLE 6 Nocardia butanica IE-36, KY 7988, FERM-P No. 3768, NRRL 11059 is used. One loopful of the seed culture obtained by culturing at 300C for 2 days in a yeast bouillon slant containing 0.5 g/dl yeast extract, 1 g/dl peptones, 1 g/dl meat extract, 0.5 g/dl NaCl and 2 g/dl agar (pH 7.2) is inoculated into 7 ml of a seed medium containing 4 g/dl glucose, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4.7H2O, 0.5 g/dl yeast extract and 2 g/dl peptones (pH 7.2) in a 50 ml - large test tube (20 mmx 190 mm). Culturing is carried out at 306C for 24 hours.

One ml of the resulting seed culture is inoculated into 20 ml of a fermentation medium having the following composition in a 250 ml Erlenmeyer flask: lucose 5 g/dl (NH4)2SO4 1 g/dl glycine 2.5 g/dl KH2PO4 0.15 g/dl (5).011 M as phosphate ion K2HPO4 0.05 g/dl 0.003 M as phosphate ion) Mg3(PO4)2.8H2O 3 g/dl (0.147 M as phosphate ion) MgSO4.7H2O 0.05 g/dl FeSO4.7H2O 0.001 g/dl MnSO4.4H2O 0.001 g/dl peptone 1 g/dl CaCO3 3 g/dl (pH 7.0) Culturing is carried out with shaking at 30"C for 5 days. L-serine is produced in a yield of 8.4 mg/ml.

As a control, the same procedures described above are repeated except that the Mg3(PO4)2.8H2O which contains 0.014 M of the phosphate ion was omitted from the fermentation medium. L-serine is produced in a yield of 2.5 mg/ml. After the completion of the culturing, 1 e of culture broth is subjected to the similar purifying procedures as described in Example 1 and 4.0 g of L-serine crystals is obtained.

EXAMPLE 7 In this example, Nocardia butanica KY 7988 NRRL 11059 is used. The same seed culture procedures as described in Example 6 are repeated. 0.25 ml of the resulting seed culture is inoculated into 5 ml of fermentation mediums having the following compositions (Medium I and II) in a 50 ml - large test tubes.

Medium I glucose 5 g/dl (NH4)2S04 0.4 g/dl KH2PO4 0.15 g/dl K2HPo4 0.05 g/dl MgSO4.7H2O 0.05 g/dl FeSO4.7H2O 0.001 g/dl MnSO4.4H2O 0.001 g/dl glycine 2 g/dl peptone 1 g/dl CaCO3 3 g/dl pH 7.0 This medium contains 0.014 M of phosphate ion.

Medium 11 The same composition as Medium I except that phosphate is added as shown in Table 6.

Culturing is carried out in the same manner as in Example 6. L-serine is produced in the yields shown in Table 7.

TABLE 7 Supplemented Total concentra Phosphate tion of phosphate Yield of L-serine % (w/v) ion (M) (mg/ml) Mg3(PO4)2.8H2O (2) 0.112 7.0 MgHPO4.3H2O (2 0.129 7.5 Ca3(PO4)2 (2) 0.143 2.5 Mg2(P2O7).3H2O (2) 0.159 1.8 Zn(PO4)2.4H20 (2l 0.102 2.0 K3Po4.nH2o (2) 0.089* 6.0 + Mg2SO4 7H2O (3) (none) 0.014 1.0 * K3PO4.nH2O is calculated as tri-hydrate.

EXAMPLE 8 In this example, Nocardia buranica KY 7990 NRRL 11193 is inoculated into 30 ml of a seed medium containing 4 g/dl glucose, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4.7H2O, 0.5 g/dl yeast extract, and 2 g/dl peptone (pH 7.2) in a 300 ml Erlenmeyer flask. Culturing is carried out with shaking at 300 for 24 hours. One ml of the resulting seed culture is inoculated into each of fifteen 30 ml quantities of a fermentation medium containing 8 g/dl glucose, 1 g/dl (NH4)2SO4, 1 g/dl peptone, 3 g/dl Mg3(PO4)2.8H2O, 0.15 gldl KH2PO4, 0.05 g/dl K2HPO4 0.05 g/dl MgSO4.7H2O, 0.001 g/dl FeSO4.7H2O, and 0.001 g/dl MnSO4.4H2O (pH 7.2) contained in fifteen 300 ml Erlenmeyer flasks provided with baffles.

Culturing is carried out with shaking at 30"C for 24 hours.

Then, 2 ml of 2 gldl glycine solution is added to the medium and culturing is carried out for 16 hours. The resulting culture broths are combined and the mixture is subjected to centrifugation to obtain microbial cells. The resulting cells are dispersed in 0.2 M phosphate buffer solution containing 2.5 g/dl glycine to obtain a suspension containing 60 mg/ml of cells on a dry weight basis. Then, the suspension is Poured into a 300 ml Erlenmeyer flask and the conversion is carried out with shaking at 300C for 20 hours. L-serine is produced in a yield of 12 mg/ml.

EXAMPLE 9 In this example, Nocardia butanica KY 7988, NRRL 11059 is used.

The same seed culture procedures as described in Example 6 are repeated.

Two ml of the resulting seed culture is inoculated into 20 ml of a fermentation medium containing 5 g/dl glucose, 1 g/dl (NH4)2SO4, 2.5 g/dl glycine, 0.15 g/dl KH2PO4, 0.05 g/dl K2HPO4, 0.05 g/dl MgSO4.7H2O, 0.001 g/dl FeSO4.7H2O, 0.001 g/dl MnSO4.4H2O, 1 g/dl peptone and 3 g/dl Mg3(PO4)2.8H2O, (pH 7.0) in a 300 ml Erlenmeyer flask. Culturing is carried out with shaking at 30"C for 48 hours. The concentration of glucose is less than 1 g/dl after culturing.

At this time, it is understood from the following results that the growth of microorganism has completed.

That is, 0.5 ml of 6N-hydrochloride is added to 0.5 ml of culture liquor to dissolve the insoluble material other than microbial cells in the culture liquor. Then, the optical density of the suspension obtained by adding 24 ml of water to the resultant mixture, is measured at a wave length of 660 my with spectrophotometer (101 type, produced by Hitachi, Ltd.) to provide 0.27. This value is the same with that measured 2 hours before in the same manner as described above.

Then, the additives listed in Table 8 are added at a concentration shown in Table 8 and culturing is continued for 72 hours. As a result, L-serine is produced in the yields shown in Table 8.

After the completion of the culturing, 1 e of culture liquor obtained by culturing using acetone as the additive is subjected to purification.

The similar purifying procedures as described in Example 1 are repeated and 6.2 g of L-serine crvstals is obtained.

TABLE 8 Additive Concentration Yield of L-serine (% v/v) (mg/ml) ligroin 3 13.0 cyclohexane 3 10.4 methanol 1 10.8 ethanol 1 9.0 acetone 0.5 11.9 methylethylketone 1 9.5 ethyl acetate 0.5 10.5 methyl acetate 1 10.0 tributyl citrate 0.3 10.0 1,4-dioxane 2 9.5 tetrahydrofuran 1 9.8 propylene glycol 0.5 10.0 1,3-butanediol 0.5 9.5 ethyl Cellosolve 0.13 9.0 ethylene glycol mono- 0.13 9.0 methyl ether acetate no additive - 8.2 EXAMPLE 10 In this example, the same seed culture procedures as described in Example 8 are repeated. 100 me of the seed culture is inoculated into 3 e of the fermentation medium having the same composition as described in Example 8 except that it additionally contains 0.01 gldt of CaCt2.2H2O in a 5 e jar fermenter. Culturing is carried out with aeration and agitation at 30"C for 48 hours. The culture liquor is subjected to centrifugation to obtain microbial cells and the cells are washed twice with 0.1M phosphate buffer solution (pH 7.0) and preserved in phosphate buffer solution in a frozen state. 5 g of ethylcellulose (50 - 100 cps) is dissolved in a mixed solvent consisting of 72.5 g of benzene and 25.5 g of n-hexane containing Span-20 (sorbitan monolaurate as a dispersing agent. ("Span" is a Registered Trade Mark) On the other hand, 5 g of frozen microbial cells (by dry weight) is suspended in 50 me of 0.1M phosphate buffer solution (pH 7.0) containing 100 mg of Mg(H2PO4)2 and 100 mg of Mg3(PO4)2. The resultant suspension is added to the ethylcellulose solution and the mixture is stirred vigorously to form a first emulsion. Then, the emulsion is added to 300 me of 1% (V/W) polyethyleneglycol (average molecular weight: 6,000) solution having a pH of 8 - 9 and cooled with ice 010 - 15"C), and the mixture is stirred to form a second emulsion. To the emulsion is continuously added the n-hexane cooled with ice water (5 - 10 C) at a rate of 10 - 15 me/min to remove benzene and to crystallize ethylcellulose.

After an hour, the immobilized cells entrapped in ethylcellulose having a particle diameter of 5 - 100 11 are obtained.

The activity of the immobilized cells is about 50 - 60% of that of the cells before immobilization and the amount of the entrapped cells is 300 - 350 mg cells/mt microcapsule. 100 mt of the immobilized cells is fluidized in a 1reactor (90 mm x 150 mm) by using air and 0.1M phosphate buffer solution (pH 6.1). The phosphate buffer solution contains 0.2 g/me of glycine, 5 g/me of Mg(H2PO4)2 and 5 g/mb of Mg3(PO4)2, and is fed to the reactor at a retention time of 15 - 20 hours. As a result, L-serine is produced continuously in the overflow solution at a concentration of 10 - 13 mg/me for 50 hours.

EXAMPLE 11 In this example, 500 me of cyclohexane as dispersing medium and 4 g of Span-20 as the dispersing agent are mixed and stirred at 10 - 15"C under ice cooling and nitrogen gas atmosphere. Then, to the mixture are added 19.5 g of acrylamide and 0.5 g of N,N'-bis-acrylamide dissolved or suspended in 100 me of 0.1M phosphate buffer solution (pH 6.1) and 10 g of frozen cells (dry weight) prepared as in the same manner as in Example 10. To the resultant mixture are added 10 me of 2.5% (W/V) N,N,N',N'tetramethylethylenediamine as a polymerization stabilizer and 125 me of 2.5% (W/V) of ammonium persulphate as a polymerization. Polymerization is then carried out for one hour to form immobilized cells entrapped in gel having a diameter of 300 - 400 .

The activity of the immobilized cells is about 30 - 40% of that of the cells before immobilization and the amount of cells entrapped in gel is 60 - 70 mg cell/mt gell.

Then, 100 me of gel and 800 me of 25 mg/me glycine solution is poured into a 1 e reactor and the reaction is carried out at 300C for 25 hours in a batch system to produce L-serine in the reaction mixture at a concentration of 12.5 mg/me. The gell is recovered from the reaction mixture and the above reaction procedure is repeated twice using the recovered gell. The results are shown in Table 9.

TABLE 9 Number Concentration of Concentration of Conversion of use glycine (mg/me) L-serine (mglmb) ratio (%) 1 25 12.5 50.0 2 25 11.8 47.2 3 25 10.5 42.0 One e of the first reaction mixture is subjected to filtration to remove the immobilized cells and the pH of the supernatant filtrate is adjusted to 2.0 with hydrochloride. Then, the filtrate is passed through the column packed with strongly acidic cation exchange resin Diaion SK-1B (trademark), and elution is carried out with 2N-aqueous ammonia. The eluate is concentrated in vacuo to obtain 8.5 g of L-serine.

WHAT WE CLAIM IS: 1. A process for producing L-serine which comprises converting glycine to L-serine in an aqueous medium containing glycine in the presence of microbial cells of a microorganism belonging to the genus Nocardia and capable of converting glycine to L-serine, wherein there are used microbial cells of a mutant strain having no or a lowered ability to decompose L-serine as compared to the parent strain from which the mutant is derived and/or a resistance to at least one metabolic antagonist having antagonism towards one or more of the following: glycine, L-serine, L-methionine, L-glutamine, L-histidine, Lleucine, L-isoleucine, L-valine, purine, pyrimidine and L-folic acid.

2. A process according to claim 1, wherein said cells are of a mutant strain belonging to the species Nocardia butanica.

3. A process according to claim 1, wherein said cells are of a mutant strain selected from Nocardia butanica NRRL 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 and Nocardia butanica NRRL 11194.

4. A process according to claim 1, wherein the conversion of glycine to L-serine is carried out by aerobically culturing the said mutant strain in a culture medium capable of supporting the growth of the microorganism and containing glycine.

5. A process according to claim 4, wherein the culture is effected at a temperature in the range 20C - 40"C.

6. A process according to claim 4 or 5, wherein the culture medium contains at least 0.037 mol/l of phosphate ion.

7. A process according to claim 6, wherein the amount of phosphate ion in the culture medium is from 0.05 to 0.4 mol/l.

8. A process according to claim 4 or 5, wherein the culture medium contains at least one

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (27)

**WARNING** start of CLMS field may overlap end of DESC **. solution contains 0.2 g/me of glycine, 5 g/me of Mg(H2PO4)2 and 5 g/mb of Mg3(PO4)2, and is fed to the reactor at a retention time of 15 - 20 hours. As a result, L-serine is produced continuously in the overflow solution at a concentration of 10 - 13 mg/me for 50 hours. EXAMPLE 11 In this example, 500 me of cyclohexane as dispersing medium and 4 g of Span-20 as the dispersing agent are mixed and stirred at 10 - 15"C under ice cooling and nitrogen gas atmosphere. Then, to the mixture are added 19.5 g of acrylamide and 0.5 g of N,N'-bis-acrylamide dissolved or suspended in 100 me of 0.1M phosphate buffer solution (pH 6.1) and 10 g of frozen cells (dry weight) prepared as in the same manner as in Example 10. To the resultant mixture are added 10 me of 2.5% (W/V) N,N,N',N'tetramethylethylenediamine as a polymerization stabilizer and 125 me of 2.5% (W/V) of ammonium persulphate as a polymerization. Polymerization is then carried out for one hour to form immobilized cells entrapped in gel having a diameter of 300 - 400 . The activity of the immobilized cells is about 30 - 40% of that of the cells before immobilization and the amount of cells entrapped in gel is 60 - 70 mg cell/mt gell. Then, 100 me of gel and 800 me of 25 mg/me glycine solution is poured into a 1 e reactor and the reaction is carried out at 300C for 25 hours in a batch system to produce L-serine in the reaction mixture at a concentration of 12.5 mg/me. The gell is recovered from the reaction mixture and the above reaction procedure is repeated twice using the recovered gell. The results are shown in Table 9. TABLE 9 Number Concentration of Concentration of Conversion of use glycine (mg/me) L-serine (mglmb) ratio (%)

1 25 12.5 50.0

2 25 11.8 47.2

3 25 10.5 42.0 One e of the first reaction mixture is subjected to filtration to remove the immobilized cells and the pH of the supernatant filtrate is adjusted to 2.0 with hydrochloride. Then, the filtrate is passed through the column packed with strongly acidic cation exchange resin Diaion SK-1B (trademark), and elution is carried out with 2N-aqueous ammonia. The eluate is concentrated in vacuo to obtain 8.5 g of L-serine.

WHAT WE CLAIM IS: 1. A process for producing L-serine which comprises converting glycine to L-serine in an aqueous medium containing glycine in the presence of microbial cells of a microorganism belonging to the genus Nocardia and capable of converting glycine to L-serine, wherein there are used microbial cells of a mutant strain having no or a lowered ability to decompose L-serine as compared to the parent strain from which the mutant is derived and/or a resistance to at least one metabolic antagonist having antagonism towards one or more of the following: glycine, L-serine, L-methionine, L-glutamine, L-histidine, Lleucine, L-isoleucine, L-valine, purine, pyrimidine and L-folic acid.

2. A process according to claim 1, wherein said cells are of a mutant strain belonging to the species Nocardia butanica.

3. A process according to claim 1, wherein said cells are of a mutant strain selected from Nocardia butanica NRRL 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 and Nocardia butanica NRRL 11194.

4. A process according to claim 1, wherein the conversion of glycine to L-serine is carried out by aerobically culturing the said mutant strain in a culture medium capable of supporting the growth of the microorganism and containing glycine.

5. A process according to claim 4, wherein the culture is effected at a temperature in the range 20C - 40"C.

6. A process according to claim 4 or 5, wherein the culture medium contains at least 0.037 mol/l of phosphate ion.

7. A process according to claim 6, wherein the amount of phosphate ion in the culture medium is from 0.05 to 0.4 mol/l.

8. A process according to claim 4 or 5, wherein the culture medium contains at least one

of the following: a hydrocarbon, a ketone, an ester, an ether, a monohydric alcohol, a polyhydric alcohol or an ether or ester derivative of a polyhydric alcohol.

9. A process according to claim 8, wherein said additive is present in the culture medium in an amount of from 0.01 - 5% on a volume basis.

10. A process according to claim 1, wherein the conversion is carried out in an aqueous medium containing glycine and using previously cultured cells of said mutant strain.

11. A process according to claim 10, wherein the medium contains 0.5 - 20% glycine.

12. A process according to claim 10 or 11, wherein said conversion is carried out using immobilised cells.

13. A process according to claim 1, wherein the conversion is carried out using a mutant strain having, as compared to the parent strain from which the mutant is derived, a lowered ability or no ability to decompose L-serine.

14. A process according to claim 13, when carried out under the conditions required by claim 6 or 7.

15. A process according to claim 13, when carried out under the conditions required by claim 8 or 9.

16. A process according to claim 14 or 15, when carried out under the conditions required by claim 4 or 5.

17. A process according to claim 13, when carried out under the conditions required by claim 10,11 or 12.

18. A process according to claim 1, wherein the conversion is carried out using a mutant strain having a resistance to a metabolic antagonist having antagonism towards one or more of the following: glycine, L-serine L-methionine, L-glutamine, L-histidine, L-leucine, L-isoleucine, L-valine, purine, pyrimidine and L-folic acid.

19. A process according to claim 18, when carried out under the conditions required by claim 6 or 7.

20. A process according to claim 18, when carried out under the conditions required by claim 8 or 9.

21. A process according to claim 19 or 20, when carried out under the conditions required by claim 4 or 5.

22. A process according to claim 18, when carried out under the conditions required by claim 10, 11 or 12.

23. A process according to claim 1, substantially as hereinbefore described in Example 1 or 2.

24. A process according to claim 1, substantially as hereinbefore described in Example 3, 4 or 5.

25. A process according to claim 1, substantially as hereinbefore described in Example 6 or 7.

26. A process according to claim 1, substantially as hereinbefore described in Example 8 or 9.

27. A process according to claim 1, substantially as hereinbefore described in Example 10 or 11.