Abstract:
A process for producing L-serine by the combination of chemical synthesis and enzyme chemical synthesis is disclosed. In this process L-serine is biochemically produced from 2-oxo-axazolidine-4-carboxylic acid or a salt thereof.

Description:
This application is a Continuation of application Ser. No. 813,557, filed on Dec. 26, 1985, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention: 
     The present invention relates to a process for biochemically producing L-serine from 2-oxo-oxazolidine-4-carboxylic acid or a salt thereof (hereinafter abbreviated as OOC) in a high yield. 
     2. Discussion of the Background: 
     L-Serine is an amino acid found in proteins. It is important as a medical or foodstuff additive or as a raw material for cosmetics. 
     L-Serine is widely found in nature as a component for constructing proteins. L-serine has been heretofore produced by hydrolyzing silk yarn, flocks, sericin, human hair, swine hair, etc., which all contain relatively large amounts of L-serine. The liberated L-serine is separated from other amino acids and purified. However, the yield is low in this process. And the process is not necessarily advantageous or economical because of restrictions in the supply of raw materials, etc. 
     Some processes for the chemical synthesis of L-serine are also known. However, their product is the optically inactive DL form of serine. Separating L-serine from a DL mixture involves complicated optical resolution, etc. which cannot be said to be an industrially feasible process for the production of L-serine. 
     As a process for producing L-serine by fermentation, there is known a process for producing it from glycine by using microorganisms of the genus Pseudomonas, etc. However, this process is not advantageous from point of view of yield, economical considerations, etc. 
     On the other hand, as a process for producing L-serine utilizing enzymes, there exists a process which utilizes serine hydroxymethyl transferase derived from animals and microorganisms. However, this process suffers the disadvantage that expensive tetrahydrofolate must be incorporated, in addition to glycine and formaldehyde. Furthermore, the process is disadvantageous in that the yield is poor. 
     Thus there is a strongly felt need for a process which readily provides optically pure L-serine in high yields and in an economically advantageous manner. 
     SUMMARY OF THE INVENTION 
     Accordingly, it is an object of this invention to provide a process for the ready production of L-serine. 
     It is another object of this invention to provide a process for the economical production of L-serine. 
     It is another object of this invention to provide a process for the facile production of L-serine in high yields. 
     Accordingly, the present invention provides a novel and efficient process for producing L-serine by the combination of chemical synthesis and enzyme chemical synthesis. This process satisfies all of the above objects of the invention. The present inventors have now surprisingly discovered that L-serine is readily produced by a process in which a cell treated product of a microorganism capable of producing L-serine from 2-oxo-oxazolidine-4-carboxylic acid or a salt thereof is reacted with 2-oxo-oxazolidine-4-carboxylic acid or salt thereof to produce L-serine. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present inventors have investigated various processes for producing L-serine by the combination of chemical synthesis and enzyme chemical synthesis and have thus discovered the present novel process for producing L-serine. 
     The present invention is directed to a process for producing L-serine by chemical synthesis in a simple and inexpensive manner. This process comprises reacting a cell treated product of microorganisms capable of producing L-serine from OOC or a salt thereof, with OOC or a salt thereof to produce L-serine. This invention also relates to a process for producing L-serine by reacting a microorganism capable of racemizing OOC or a salt thereof and a cell treated product of a microorganism capable of producing L-serine from OOC or a salt thereof, with OCC or a salt thereof to produce L-serine. The L-serine is then readily isolated. 
     Examples of the microorganisms capable of racemizing OOC or a salt thereof, and which can be used in the present invention include: 
     
         ______________________________________Agrobacterium radiobacter                  AJ 2782                  ATCC 6466Alcaligenes marchallii AJ 2147                  ATCC 21030Arthrobacter citreus   AJ 1423                  ATCC 11624Bacillus licheniformis AJ 3290                  ATCC 21417Beijerinchkia indica   AJ 2821                  ATCC 9037Brevibacterium ammoniagenes                  AJ 1443                  ATCC 6871Corynebacterium acetoacidphilum                  AJ 1550                  ATCC 13870Escherichia coli       AJ 2621                  ATCC 13070Mycobacterium ammoniaphilum                  AJ 1997                  ATCC 15354Micrococcus flavus     AJ 1021                  ATCC 400Pseudomonas oleovorans AJ 2058                  ATCC 8062Saricina lutea         AJ 1217                  ATCC 272Serratia marcescens    AJ 2686                  ATCC 14225______________________________________ 
    
     and the like. 
     Other bacteria than those described above may be used in the present invention, as long as they are microorganisms capable of racemizing OOC. 
     Next, examples of the microorganisms capable of producing L-serine by hydrolysis of OOC or a salt thereof, and which can be used in the present invention include: 
     
         ______________________________________Alcaligenes faecalis   AJ 2541                  FERM-P 8030,                  FERM-BP 940Arthrobacter grobiformis                  AJ 1422                  ATCC 8010Bacillus subtilis      AJ 1992                  ATCC 13952Cellulomonas flavigena AJ 1568                  ATCC 491Corynebacterium hydrocarboclastus                  FERM-P 1097Flavobacterium aquatile                  AJ 2135                  ATCC 8375Jensenia canicruria    AJ 3147                  ATCC 11048Mycobacterium ammoniaphilum                  AJ 1997                  ATCC 15354Micrococcus roseus     AJ 1006                  ATCC 9815Rhodococcus erythropolis                  AJ 9126                  ATCC 4277Pseudomonas testosteroni                  AJ 2270                  ATCC 17409Pseudomonas acidovorans                  AJ 3117                  ATCC 15668Candida zeylanoides    AJ 4677                  IFO 0719Citeromyces matritensis                  AJ 4287                  CBS 2764Cryptococcus laurentii AJ 5225                  IFO 0609Debaryomyces hansenii  AJ 4179                  IFO 0023Endomycopsis oventensis                  AJ 5062                  CBS 2508Geotricum fragrans     AJ 14298                  CBS 15225Hansenula californica  AJ 5573                  IFO 0800Kluyveromyces marxianus                  AJ 4074                  IFO 0219Nadosonia falvescens   AJ 5332                  IFO 0666Rhodotorula marina     AJ 5014                  IFO 0879Torulopsis famata      AJ 4342                  IFO 0623Trichosporon fermentans                  AJ 5152                  IFO 1199 Wickerhamia fluorescens                  AJ 4285                  IFO 1116Achromobacter viscosus ATCC 12448Aeromonas salmonicida  ATCC 14174Agrobacterium radiobacter                  ATCC 6466Azotobacter vinelandii ATCC 9046Brevibacterium pusillum                  ATCC 19096Escherichia coli       ATCC 13071Klebsiella penumonia   ATCC 8329Kluyvera non-citrophila                  FERM-P 3150Kurthina zophii        ATCC 6900Mycoplana dimorpha     ATCC 4279Proteus rettgeri       FERM-P 8196,                  FERM-BP 941                  AJ 2770Salmonella schottmuelleri                  ATCC 8759Serratia marcescens    ATCC 14225Streptomyces humifer   FERM-P 2347Vibrio-tyrogenes       ATCC 7085Xantomonas canpestris  ATCC 7381Pichia membranaefaciens                  IFO 0460Saccharomyces fermentati                  IFO 0422Tremella brasiliensis  IFO 9289Alternaria cucumerina  IFO 7417Curvularia geniculata  ATCC 6671Fusarium nivale        ATCC 42308Helminthosporium gramineum                  ATCC 6695Phoma destructiva      ATCC 24636Sclerotium bataticola  ATCC 12265Cochliobolus miyabeanus                  IFO 5844Mucor circinelloides   ATCC 8770Aspergillus repens     ATCC 5817Penicillium decumbens  ATCC 10436Gromerella cingulata   ATCC 11326Septoria glycines      ATCC 38699Pseudoplea trifolii    IFO 7252Stemphylium astragali  IFO 7244Diplodia natalensis    ATCC 34643Stachylidium bicolor   ATCC 12672Eupenicillium alutaceum                  ATCC 18542Anixiella reticulata   ATCC 34511Arachniotus flavoluteus                  ATCC 18430Byssochlamys fulva     ATCC 10099Coniochaeta tetraspora ATCC 22275Golasinospora longispora                  ATCC 18493Gymnoascus umbrinus    IFO 8358Microascus cinereus    ATCC 16594Microthecium retisporum                  ATCC 22184Sordaria humana        ATCC 22796Sporormiella isomera   ATCC 24341Toxotrichum cancellatum                  ATCC 15316Melanospora zamiae     ATCC 12340______________________________________ 
    
     and the like. 
     Alcaligenes faecalis (FERM-P 8030) was originally deposited on Dec. 24, 1984, and Proteus rettgeri (FERM-P 8196) was originally deposited on Apr. 25, 1985 at the Fermentation Research Institute, Agency of Industrial Sciences and Technology, Ministry of International Trade and Industry (FRI), 1-3, Higashi 1-Chome, Yatabo-machi, Tsukuba-gun, Ibaragi-ken 305, Japan, and were accorded the FERM-P numbers indicated above. The microorganisms deposited were then converted into deposits under the Budapest Treaty on Nov. 24, 1985, and were accorded the corresponding FERM-BP numbers. 
     Microorganisms other than the bacteria described above may be used as the bacteria in the present invention, as long as they are microorganisms capable of producing L-serine through the decomposition of OOC. 
     Ordinary nutrient media may be appropriately used as the media for culturing the microorganisms capable of racemizing OOC and the microorganisms capable of producing L-serine by hydrolyzing OOC as described above. As carbon sources, there may be used, for example, sugars such as glucose, sucrose, glycerol, molasses, etc.; organic acids such as fumaric acid, acetic acid, etc.; alcohols such as ethanol, methanol, etc. As nitrogen sources, there may be used ammonium sulfate, ammonium chloride, etc. and as organic nutrient sources, there may be used yeast extract, peptone, meat extract, corn steep liquor, etc. As inorganic ions, there may be used ions of magnesium, iron, manganese, potassium, sodium, phosphoric acid, etc. and as vitamins, pyridoxine, pyridoxal phosphate, etc. 
     Incubation may be carried out in a conventional manner. For example, the pH of the medium is adjusted to 6 to 9 and the bacteria of the present invention are aerobically cultured at 20° to 40° C. for 1 to 3 days. Upon incubation, the culture or cell product having a high capability of hydrolysis or racemization may be obtained sometimes by incorporating a small quantity of OOC in the medium. 
     The cell treated product used in the present invention refers to a product which possesses activity of racemizing OOC. Examples of such cell treated products include a culture solution per se, a solution obtained by separating cells from the culture solution, separated cells, decomposition products of the separated cells, purified decomposition products, etc. These cell treated products may be used as they are, or may be subjected to treatments such as freeze drying, drying with acetone, etc. Alternatively, the products may be subjected to immobilization, etc. 
     The concentration of a substrate in enzyme reaction may vary depending upon batch system or continuous system, but generally it is from 0.1 to 30% in an aqueous medium, preferably 0.5 to 10%, in the batch system; in the continuous system, it is preferred that the concentration be somewhat lower than the above ranges. 
     The reaction is carried out generally in an aqueous medium, at 15° to 60° C., preferably at about 30° to about 40° C., at pH of 4 to 10, preferably about 7. The reaction time is not the same since it varies depending upon means of settling, stirring, flowing, etc. and mode or titer of enzyme standard. However, in the batch system, the reaction time is generally for about 10 minutes to about 72 hours. 
     In case that cells of the above-described microorganisms are brought into contact with OOC while culturing the cells in an aqueous medium, the aqueous medium containing OOC and further containing nutrient sources required for growth of the microorganisms such as carbon sources, nitrogen sources, inorganic ions, etc. are used. In addition, the incorporation of organic trace nutrients such as vitamins, amino acids, etc. often give desired results. 
     As carbon sources, carbohydrates such as glucose, sucrose, etc., organic acids such as acetic acid, etc., alcohols and the like may be appropriately used. As nitrogen sources, ammonia gas, ammonia water, ammonium salts and the like may be used. As inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions and the like may be appropriately used depending upon necessity. 
     The incubation is carried out while controlling the conditions within appropriate ranges at pH or 4 to 8 at temperatures of 25° to 40° C. under aerobic conditions, whereby desired results can be obtained. 
     On the other hand, in case that the culture solution of the above-described microorganisms are reacted with OOC as they are, or the cultured cells or cell treated products are reacted by bringing them into contact with OOC, OOC and the culture solution, or an aqueous medium in which the cultured cells or cell treated products are dissolved or suspended may be settled or stirred for a while while controlling the temperature to a suitable range between 15° to 60° C. and keeping the pH at 4 to 10. Thus after 10 minutes to 72 hours passes, large quantities of racemic compounds or hydrolytic products of OOC are produced and accumulated in the aqueous medium. 
     A quantitative analysis of the D-form and the L-form was run by liquid chromatography using a resin for optical resolution to determine whether or not the OOC was racemized by the enzyme reaction in the present invention. Further NMR spectrum, X-ray diffraction pattern, liquid chomatography, quantitative assay data for bioassay, specific rotary power data, etc. was obtained from the serine crystals obtained in the examples later described to determine whether or not L-serine was produced by the hydrolysis of OOC through the enzyme reaction. 
     Other features of the invention would become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof. 
     EXAMPLE 1 
     A 500 ml volume flask was charged with 50 ml of medium (pH 7.0) containing 2% of glycerol, 0.5% of yeast extract, 0.5% of peptone, 0.25% of NaCl, 0.2% of DL-OOC and 4.0% of calcium carbonate (separately sterilized) and sterilized at 120° C. for 15 minutes. A microorganism shown in Table 1 cultured at 30° C. for 24 hours in bouillon-agar medium was inoculated on the medium. After culturing at 30° C. for 24 hours, the cells were centrifuged, washed and collected. The cells were added to acetate buffer (0.1M at the end; terminal pH, 4.0) or phosphate buffer (0.1M at the end terminal pH, 7.0) or tris buffer (0.1M at the end; terminal pH, 8.5) containing 1% of D-OOC in a 5% concentration calculated as bacterial cells. The mixture was settled at 30° C. for 24 hours to react them. After completion of the reaction, L-OOC produced was quantitatively determined by liquid chromatography using a resin for optical resolution. The results are shown in Table 1. 
     
                       TABLE 1______________________________________Amount of L-OOC Produced by VariousMicroorganisms at pH of 4.0, 7.0 or 8.5            Amount of L-OOC            Produced [mg/dl]Microorganism      pH 4.0   pH 7.0  pH 8.5______________________________________Agrobacterium radiobacter               0        45     171ATCC 6466Alcaligenes marchallii              18       128     264ATCC 21030Arthrobacter citreus              16        25      85ATCC 11624Bacillus licheniformis              43       479     362ATCC 21417Beijerinchkia indica               0       158     341ATCC 9037Brevibacterium ammoniagenes              25       417     477ATCC 6871Corynebacterium acetoacidphilum              29       262     265ATCC 13870Escherichia coli   41        68      0ATCC 13070Mycobacterium ammoniaphilum               0       174     314ATCC 15354Micrococcus flavus  0       245     315ATCC 400Pseudomonas oleovorans              18       164     207ATCC 8062Saricina lutea      0       158      70ATCC 272Serratia marcescens              76        29      0ATCC 14225______________________________________ 
    
     EXAMPLE 2 
     In 50 ml of medium similar to Example 1 charged in a 500 ml flask, Bacillus licheniformis (ATCC 21417) was cultured at 30° C. for 16 hours. Into the culture solution was sterilizingly poured 10 ml of an aqueous solution (adjusted pH to 7.0) containing 500 mg of D-OOC. After adjusting the pH of the culture solution to 7.0 under sterilized conditions, incubation was carried out for an additional 10 hours. During the incubation, the pH was sterilizingly adjusted to 7.0 every two hours. 
     A part of the culture solution was withdrawn to quantitatively determine L-OOC by liquid chromatography using a resin for optical resolution. L-OOC was formed in an amount of 406 mg/dl. 
     EXAMPLE 3 
     Bacillus licheniformis (ATCC 21417) was inoculated on 50 ml of medium similar to Example 1 charged in a 500 ml flask. After culturing at 30° C. for 16 hours, the cells were centrifuged, washed and collected. The cells were added to phosphate buffer (0.1M at the end; terminal pH, 7.0) containing 1% of L- or D-OOC in a concentration of 5% calculated as bacteria followed by reaction by settling at 30° C. for 18 hours. After completion of the reaction, D-OOC or L-OOC in the reaction solution by liquid chromatography using a resin for optical resolution was quantitatively determined. The results are shown in Table 2. 
     
                       TABLE 2______________________________________Amount of L- or D-OOC Produced WhenL- or D-OOC is Used as SubstrateAmount of OCC Prior to   Amount of OOCReaction [mg/dl]         Reaction [mg/dl]______________________________________L-OOC     1000           L-OOC:   509                    D-OOC:   473D-OOC     1000           L-OOC:   492                    D-OOC:   471______________________________________ 
    
     EXAMPLE 4 
     In 50 ml of medium similar to Example 1 charged in a 500 ml flask, Bacillus licheniformis (ATCC 21417) was cultured at 30° C. for 16 hours. After adding 5 ml of a 4% sodium alginate solution to 5 ml of a suspension of the cells in physiological saline in a concentration of 20 g/dl to mix them, the mixture was slowly dropwise added to a 15 g/dl calcium chloride solution to prepare bead-like immobilized cells. The total amount of the immobilized cells were poured into phosphate buffer (0.1M at the end; terminal pH, 7.0) containing 1% D-OOC followed by reacting at 30° C. for 16 hours. As a result, D-OOC was racemized and 432 mg/dl of L-OOC was formed in the reaction solution. 
     EXAMPLE 5 
     In a 500 ml volume flask was charged 50 ml of medium (ph 7.0) containing 2% of glycerol, 0.5% of yeast extract, 0.5% of peptone, 0.25% of NaCl, 0.2% of DL-OOC and 4.0% of calcium carbonate (separately sterilized) followed by sterilization at 120° C. for 15 minutes. 
     A platinum earpick of microorganism shown in Table 3 cultured at 30° C. for 24 hours in bouillon-agar medium was inoculated on the medium. After culturing at 30° C. for 20 hours, the cells were collected from the culture solution by centrifugation. The cells were washed with an equivalent amount of physiological saline to that of the culture solution. The cells were collected and added to a reaction solution containing 1% of L-OOC, pH of which had been adjusted to 7 or 8.5 in a 5% concentration calculated as cells. The mixture was settled at 30° C. for 48 hours to react them. After completion of the reaction, L-serine was quantitatively determined by bioassay. The results are shown in Table 3. 
     
                       TABLE 3______________________________________Amount of L-Serine Accumulated in VariousMicroorganisms at pH 7.0 or pH 8.5             Amount of L-Serine             Accumulated [mg/dl]Microorganism       pH 7.0   pH 8.5______________________________________Alcaligenes faecalis               20       58AJ 2541, FERM-BP 940Arthrobacter grobiformis                4       10ATCC 8010Bacillus subtilis   10       13ATCC 13952Cellulomonas flavigena               41       31ATCC 491Corynebacterium hydrocarboclastus                4       14FERM-P 1097Flavobacterium aquatile               26       23ATCC 8375Jensenia canicruria 18       23ATCC 11048Mycobacterium ammoniaphilum               66       42ATCC 15354Micrococcus roseus  124      26ATCC 9815Rhodococcus erythropolis               26       23ATCC 4277Pseudomonas testosteroni               368      132ATCC 17409Pseudomonas acidoverans               328      142ATCC 17409Candida zeylanoides 28       36IFO 0719Citeromyces matritensis               15       23CBS 2764Cryptococcus laurenti               15       39IFO 0609Debaryomyces hansenii               13       29IFO 0023Endomycopsis oventensis               30       33CBS 2508Geotricum fragrans  20       26CBS 15225Hansenula californica               17       26IFO 0800Kluyveromyces marxianus               20       33IFO 0219Nadosonia falvescens               15       19IFO 0666Rhodotorula marina  20       54IFO 0879Torulopsis famata   20       28IFO 0623Trichosporon fermentans               24       33IFO 1199Wickerhamia fluorescens               28       36IFO 1116Achromobacter viscosus               95       121ATCC 12448Aeromonas salmonicida               58        0ATCC 14174Agrobacterium radiobacter               32       94ATCC 6466Azotobacter vinelandii                5        0ATCC 9046Brevibacterium pusillum               36        9ATCC 19096Escherichia coli    14        2ATCC 13071Klebsiella penumonise               12       26ATCC 8329Kluyvera non-citrophilia                0       36FERM-P 3150Kurthina sophii     22        7ATCC 6900Mycoplana dimorpha  35       30ATCC 4279Proteus-rettgeri    31        4AJ 2770, FERM BP-941Salmonella shottmuelleri               51       43ATCC 8759Serratia marcescens 57       25ATCC 14225Streptomyces humifer                0       31FERM-P 2347Vibrio tyrogenes    27       52ATCC 7085Xanthomonas campestris               17       20ATCC 7381Pichia membranaefaciens               48       37IFO 0460Saccharomyces fermentati               63       42IFO 0422Tremella brasiliensis               47       33IFO 9289Alternaria cucumerina               98       12IFO 7417Curvularia geniculata               195      79ATCC 6671Fusarium nivale     117      54ATCC 42308Helminthosporium gramineum               183      71ATCC 6695Phoma destructiva   103      86ATCC 24636Sclerotium bataticola               155      140ATCC 12265Cochliobolus miyabeanus               264      71IFO 5844Mucor circinelloides               113      48ATCC 8770Aspergillus repens  192      76ATCC 5817Penicillium decumbens               102      24ATCC 10436Gromerella cingulata               31        0ATCC 11326Septoria glycines   51        3ATCC 38699Pseudoplea trifolii 77       32IFO 7252Stemphylium astragali                8        0IFO 7244Diplodia natalensis 42        0ATCC 34643Stachylidium bicolor               70        6ATCC 12672Eupenicillium alutaceum               35        7ATCC 18542Anixiella reticulata               102      35ATCC 34511Arachniotus flavoluteus               84        0ATCC 18430Byssochlamys fulva  51       16ATCC 10099Coniochaeta tetraspora               36        0ATCC 22275Golasinospora longispora               45        3ATCC 18493Gymnoascus umbrinus 102      25IFO 8358Microascus cinereus 50        8ATCC 16594Microthecium retisporum               100      23ATCC 22184Sordaria humana     171      43ATCC 22796Sporormiella isomera               152      14ATCC 24341Toxotricum cancellatum               35         0ATCC 15316Melanospora zamiae  81       10ATCC 12340______________________________________ 
    
     EXAMPLE 6 
     In 50 ml of medium similar to Example 1 charged in a 500 ml flask, Pseudomonas acidovorans (ATCC 15668) was cultured at 30° C. for 12 hours. Into the culture solution was sterilizingly poured 10 ml of an aqueous solution (adjusted pH to 7.0) containing 500 mg of DL-OOC. After adjusting pH of the culture solution to 7.0 under sterilized conditions, incubation was carried out for further 10 hours. During the incubation, the pH was sterilizingly adjusted to 7.0 in every two hours. 
     A part of the culture solution was withdrawn and appropriately diluted to quantitatively determine L-serine by bioassay. L-OOC was formed in an amount of 288.3 mg/dl. 
     EXAMPLE 7 
     Pseudomonas testosteroni (ATCC 17409) was inoculated on 50 ml of medium similar to Example 5 charged in a 500 ml flask. After culturing at 30° C. for 16 hours, the culture solution was centrifuged, washed and freeze dried. The cells were suspended in 1 liter of an enzyme reaction solution in a 5% concentration. The enzyme reaction solution comprised 1% of L-OOC and 1% of KH 2  PO 4 . The reaction was performed by settling at pH of 7.0 at 30° C. for 48 hours. After completion of the reaction, the reaction mixture was centrifuged. The supernatant was taken. A part of the supernatant was appropriately diluted and L-serine produced was quantitatively assayed by bioassay. 
     L-Serine accumulated in an amount of 3.42 mg/dl (molar yield, 45%). 
     On the other hand, after completion of the reaction, the reaction mixture was centrifuged to remove the bacteria. After the supernatant was obtained, 5 g of activated charcoal was added. The mixture was heated and filtered to obtain 990 ml of the supernatant. After the supernatant was concentrated under reduced pressure, the pH was adjusted to 3.0 and passed through a column packed with 500 ml of cationic ion exchange resin Dia Ion SK-lB. After washing with 2000 ml of distilled water, elution was performed with 2N ammonia water to collect serine fractions. After pH of the concentrate was adjusted to 5.7, approximately 2-fold amount of methanol was slowly added thereto at low temperature to precipitate L-serine crystals. The system was allowed to stand for further a day at 10° C. The precipitated crystals were separated by filtration, washed with methanol and dried to obtain 1.2 g of the crystals. 
     EXAMPLE 8 
     In 50 ml of medium similar to Example 1 charged in a 500 ml flask, Pseudomonas testosteroni (ATCC 17409) was cultured at 30° C. for 16 hours. The cells were suspended in physiological saline in a concentration of 20 g/dl and 5 ml of a 4% sodium alginate solution was added to 5 ml of the suspension. After mixing them, the mixture was slowly dropwise added to a 15 g/dl calcium chloride solution to prepare bead-like immobilized cells. The whole amount of the immobilized cells was poured into phosphate buffer (0.1M at the end) terminal pH, 7.0) containing 1% of L-OOC followed by reacting at 30° C. for 16 hours. As a result, 354 mg/dl of L-serine was formed. 
     EXAMPLE 9 
     Bacillus licheniformis (ATCC 21417) was inoculated on medium similar to Example 1. After culturing at 30° C. for 16 hours, the cells were centrifuged, washed and collected. The cells were added to phosphate buffer (0.1M at the end; terminal pH, 7.0) containing 1% of D-OOC in a 5% concentration calculated as cells. The system was settled at 30° C. for 48 hours to react them. After completion of the reaction, a 5% concentration as cells of Pseudomonas testosteroni (ATCC 17409) cultured under the same conditions was added to the reaction solution. The mixture was settled at 30° C. for further 48 hours to react them. After completion of the reaction, L-serine was quantitatively determined by bioassay, whereby 359 mg/dl of L-serine was formed. On the other hand, when only viral cells of Pseudomonas testosteroni (ATCC 17409) was reacted, no formation of L-serine was observed. 
     Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.