Abstract:
A process for the production of methylsuccinic acid represented by the formula (I): ##STR1## wherein the symbol * represents an asymmetric carbon atom, comprising the steps of culturing Candida in a medium containing squalene and producing methylsuccinic acid.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a process for the production of an optically active methylsuccinic acid from squalene. The optically active methylsuccinic acid produced by the present invention is a chiral compound having a methyl group on the asymmetric carbon. atom, and is useful as a starting material for synthesis . of pharmaceuticals, agrochemicals and other physiologically active substances, and as a starting material for liquid crystal polymers. 
     2. Description of the Related Art 
     Currently, as processes for the production of optically active methylsuccinic acid, there is known a process wherein itaconic acid is asymmetrically reduced with rhodium complex having a chiral phosphine as a ligand, but the optical purity of the product is as low as about 50%, and even in a current modified process, the optical purity is at most about 90%. Also, rhodium is expensive. 
     SUMMARY OF THE INVENTION 
     Accordingly, the present invention provides a process for the production of an optically active methylsuccinic acid having a very high optical purity. The optically active methylsuccinic acid prepared by the present invention has an at least 95%, preferably at least 98%, and most preferably 100% optical purity. 
     Namely, the present invention provides a process for the production of an optically active methylsuccinic acid represented by the formula (I): ##STR2## wherein the symbol * represents the asymmetric carbon atom, comprising culturing a microorganism belonging to the genus Candida and capable of converting squalene into an optically active methylsuccinic acid, to thereby convert squalene to an optically active methylsuccinic acid, and recovering the optically active methylsuccinic acid. 
    
    
     BRIEF EXPLANATION OF THE DRAWINGS 
     FIG. 1 shows an infrared absorption (IR) spectrum of (R)-(+)-methylsuccinic acid obtained by the present process; and 
     FIG. 2 shows a  13  C-nucleomagnetic resonance (NMR) spectrum of (R)-(+)-methylsuccinic acid obtained by the present process. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Microorganisms belonging to the genus Candida and used in the present invention can be obtained, for example, by the following procedure. Namely, a naturally occurring source is added to a culture medium containing squalene as a sole carbon source, and culturing is carried out to isolate microorganisms capable of assimilating squalene as a carbon source from the culture. 
     As the culture medium, any conventional medium can be used as long as it contains squalene as a sole carbon source; i.e., contains components necessary for the growth of the microorganism, such as a nitrogen source, inorganic salts, and if necessary, other minor nutrients such as vitamins, amino acids, and nucleotides and the like. The squalene as a sole carbon source is contained in an amount of 0.1 to 100 g, preferably 1 to 10 g per liter. 
     As the nitrogen source, for example, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea and the like are used alone or in combination. The amount of nitrogen source used varies, depending on the nature of the nitrogen source, and is usually 0.1 to 10 g, preferably 1 to 3 g, per liter of the medium. The inorganic salts, potassium phosphate, sodium phosphate, magnesium sulfate, ferric sulfate, ferric chloride, calcium chloride and the like are used alone or in combination. The amount of inorganic salts used varies, depending on the nature of the inorganic acid, and is usually 0.001 to 10 g, preferably 0.01 to 5 g, per liter. 
     If necessary, minor nutrients, such as vitamins, yeast extract, pepton, corn steep liquor and the like can be used alone or in combination. 
     The amount added depends on the nature of additive, and is usually up to 10 g, for example 0.05 to 10 g, and preferably 0.1 to 5 g per liter. A pH value of a culture medium is 2 to 10, and preferably 3 to 6. 
     Further, an antibiotic, biocide or the like is preferably included in the medium, to allow a selective growth of a particular microorganism. For example, for a selective growth of yeast, streptomycin is added to the culture medium in an amount of 0.1 to 100 mg, preferably 10 to 50 mg/liter, to inhibit the growth of bacteria. 
     The sources from which microorganisms are isolated may be any naturally occurring materials, such as soil, sewerage, or spoiled fruit. The source is added to a medium in an amount of 1 to 100 g, preferably 30 to 50 g, per liter. The culture temperature is 15° to 80° C., preferably 20° to 35° C., and the culturing is carried out for 1 to 30 days, preferably 4 to 7 days, and then a part of the culture is inoculated to a fresh medium and the culturing is carried out for 1 to 30 days, preferably 4 to 7 days; this procedure is repeated 2 to 5 times. The culture is carried out under an aerobic condition provided by a conventional means such as aeration or stirring, or a combination thereof. 
     The growth of microorganisms is determined by a measurement of the turbidity or by a microscopical observation. Preferably, the isolation of microorganisms is carried out after the turbidity reaches OD 610  =0.1 to 10, more preferably 0.2 to 1. For the isolation, a conventional isolation method such as a plate culture method is preferably used. The plate culture medium contains 0.1 to 10% agar and is, for example, a yeast extract/malt extract medium containing 2% agar (YM agar medium). 
     As an example of a microorganism isolated according to the above-mentioned procedure and able to be used for the present process, a yeast strain SQL349 is mentioned. This strain SQL349 was deposited with the Fermentation Research Institute Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan, as FERM P-11653 on Aug. 9, 1990, and transferred to an international deposition under the Budapest Treaty as FERM BP-3580 on Sep. 26, 1991. 
     The microorganism SQL349 has the following taxonomical properties. 
     a. Morphology 
     Vegetable cell: spherical-elliptic, proliferating by multi-poler budding 
     Liquid culture: forming precipitate and surface film layer (25° C., 3 days) 
     Pseudomycelium: formed (25° C., 3 days) 
     True mycelium : formed (25° C., 6 days) 
     Ascospore: not formed on Adams medium, Gorodkowa medium, malt medium, YM medium, V-8 medium, and a potato-dextrose medium 
     
         ______________________________________b.    Physiological properties(1)        Fermentation of sugars    Glucose -        Sucrose   -    Maltose -        Lactose   -    Raffinose            -        Galactose -(2)        Assimilation of:    Nitrate -        Inositol  -    Erythritol            +        Sucrose   -    Glucose +        Trehalose -    Cellobiose            -        Lactose   -    Maltose -    Raffinose            -(3)        Growth temperature    25° C.            +        30° C.                               +    37° C.            -(4)        DBB colorination reaction                       -(5)        Degradation of fat                       +______________________________________ 
    
     According to the above result, the strain SQL349 was identified as Candida lipolytica. Note, Candida lipolytica is taxonomically the same as the imperfect generation of Saccharomycopsis lipolytica identified at the Institute of Fermentation Osaka (IFO) and the imperfect generation of Yarrowia lipolytica identified at the American Type Culture Collection (ATCC). 
     In addition to the above-mentioned microorganism, according to the present invention, for example, Candida lipolytica IFO 0746 (ATCC 20114), IFO 1209 (ATCC 8662), IFO 1542 (ATCC 20306), IFO 1632, IFO 1741, IFO 10073 (ATCC 48436), IFO 1742 (ATCC 9773), IFO 1195, IFO 1548 (ATCC 18942), IFO 1549 (ATCC 18945), IFO 1550 (18943), IFO 1746 ATCC 20237, ATCC 20255, ATCC 20362, ATCC 20363, ATCC 20460, ATCC 20461, ATCC 20496, ATCC 22421, ATCC 22422, ATCC 22423, ATCC 34922, ATCC 44601, ATCC 46330, ATCC 46482, ATCC 46483, ATCC 46484, and the like can be used, and among them, strains marked IFO can be obtained without limitation from the Institute of Fermentation Osaka, 17-85, Juso-honmachi 2-chome, Yodogawa-ku, Osaka 532, Japan; and strains marked ATCC can be obtained without limitation from the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Md. 20852, USA. 
     The medium used to produce methylsuccinic acid according to the present process contains components necessary for the growth of a producer microorganism, i.e., a carbon source, nitrogen source, inorganic salts, and if necessary, minor components such as vitamins, amino acids, and nucleotides and the like. Although squalene can be used as a sole carbon source, if necessary sugars such as glucose or erythritol, hydrocarbons such as n-paraffins, or alcohols such as ethanol or propanol, etc., may be used. The amount of carbon source used varies, depending on the nature of carbon source, and is usually up to 100 g, for example, 0.1 to 100 g, preferably 0.5 to 10 g, per liter. As the nitrogen source, for example, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea and the like are used alone or in combination. The amount of nitrogen source varies, depending on the nature of the nitrogen source, and is usually 0.1 to 10 g, preferably 1 to 3 g, per liter of medium. 
     As the inorganic acids, potassium phosphate, sodium phosphate, magnesium sulfate, ferric sulfate, ferric chloride, calcium chloride and the like are used alone or in combination. The amount of inorganic salts depends on the nature of the inorganic salt, and is usually 0.001 to 10 g, preferably 0.01 to 5 g, per liter. If necessary, vitamins, nucleotides, yeast extract, pepton, corn steep liquor and the like are used alone or in combination. The amount of the additive depends on the nature of the additive, and is usually up to 10 g, for example, 0.05 to 10 g, preferably 0.1 to 5 g, per liter. 
     A pH value of a medium is usually adjusted to 2 to 11, preferably 3 to 6. If the pH value of the medium is lowered during the culturing, the pH value is controlled if necessary by adding an alkali such as an NaOH aqueous solution. In addition to the above-mentioned medium, other conventional media such as a bouillon medium, yeast extract/malt extract medium (YM medium) and the like may be used. 
     As a precursor of the methylsuccinic acid, squalene is added to a culture medium. The squalene may be added to the medium before the onset of culturing, or may be added continuously or periodically during the culturing. The total amount of squalene used is, for example, 0.1 to 100 g, preferably 2 to 20 g, per liter. 
     To produce a large amount of the desired product, a large scale culture is necessary, and accordingly, an inoculum culture is preferably prepared prior to the production culture. For the inoculum culture the same medium as described above for the production culture can be used, except that squalene is not necessary in the inoculum culture medium. 
     For the inoculum culture and production culture (main culture), culturing is carried out at 15° to 80° C., preferably 20° to 35° C., for 1 to 20 days, preferably 2 to 5 days, with shaking, stirring, agitation and/or aeration. 
     According to another embodiment of the present invention, once a producer microorganism is cultured, and the cultured cells brought into contact with squalene in an aqueous medium such as phosphate buffer under an aerobic condition, for example, under the same condition as that used for the culturing as described above, to produce the desired product, i.e., methylsuccinic acid. 
     The method of recovering the desired product from a cultured broth or reaction mixture is not critical. For example, first a cultured broth or reaction mixture is adjusted to a pH of 1 to 6, preferably a pH of 1 to 3, and then extracted with a solvent to obtain an extract containing the desired product, i.e., methylsuccinic acid. 
     Any solvent which dissolves the desired product, for example, an organic solvent such as carbon tetrachloride, trichloroethylene, toluene, benzene, dichloromethane, chloroform, diethyl ether or ethyl acetate, may be used, but preferably dichloromethane, chloroform, diethyl ether or ethyl acetate is used. 
     To isolate and purify the desired product from the extract, conventional procedures such as adsorption, elution, distillation and the like may be used. For example, an extract is evaporated to remove a solvent, and from the resulting residue, the desired product is purified by adsorption chromatography using silica gel, active carbon, stylene-divinylbenzene copolymer resin or the like; and ion exchange chromatography using strong anion-exchange resin; weak anion-exchange resin or the like. 
     Alternatively, the products can be methylesterified by a conventional methyl-esterification method, for example, by refluxing with methanol in the presence of an acid catalyst, and the resulting ester mixture distilled to isolate methylsuccinate methyl ester. 
     In the present invention, the identification and determination of the optical purity for the optically active methylsuccinic acid were carried out as follows. The confirmation of the desired product methylsuccinic acid was carried out by IR spectrum,  1  H-NMR spectrum,  13  C-NMR spectrum, and mass spectrum. 
     The absolute configuration and optical purity of the present compound were determined by a comparison of a specific rotation of the present compound with that described in literature, and a  13  C-NMR spectrum analysis of a diastereomer of the present compound with L-menthol. Namely, the absolute configuration and optical purity of the product were obtained by comparing a specific rotation thereof with a specific rotation of (R)-(+)-methylsuccinic acid, [α] 22   D  =+16.88° (c=2.1 g/100 ml, ethanol) described in the literature (Justus Liebigs Ann. Chem., 538, 1, 1939). Further, the product was reacted with L-menthol in the presence of p-toluenesulfonic acid to synthesize diastereomer, and the  13  C-NMR spectrum thereof was then measured. Next, the optical purity was calculated from a ratio of signal areas of a carbon atom at the 3-position of the R-form and S-form of the methylsuccinic acid moiety. 
     The present compound has carboxyl groups, and therefore, can form salts, for example, salts of alkaline metals such as lithium, sodium and potassium, alkaline earth metals such as calcium and magnesium, and ammonium, and the like. These salts can be obtained according to a conventional procedure from free compound. For example, a free compound can be neutralized with an appropriate base to obtain a corresponding salt. 
     Since the present compound has carboxyl groups, it can be converted to an aldehyde or alcohol by reducing same with, for example, lithium alminium hydride. 
     Therefore, according to the present invention, an optically active, highly pure methylsuccinic acid can be produced. 
     EXAMPLES 
     Next, the present invention is explained in more detail with reference to Examples. 
     EXAMPLE 1 
     First, 50 ml each of the medium shown in Table 1 was put into 500 ml-Sakaguchi flasks and autoclaved at 121° C. for 20 minutes, to the medium was added 1 g of a soil sample (from Miura, Kanagawa, Japan), and then shaking culture was carried out at 30° C. for 5 days. Among these cultures, those in which microbial growth was observed were selected, a portion of the culture was plated on YM agar medium shown in Table 2, and a strain SQL349 (FERM BP-3580) was isolated. 
     This strain was cultured in YM liquid medium at 30° C. for 24 hours, with shaking, and a portion of the culture broth was inoculated into 50 ml of the medium having the composition shown in Table 1, at an inoculum ratio of 1% by volume, and culturing was carried out at 30° C. for 4 days. The culture broth was adjusted to pH of 2 with hydrochloric acid, and extracted with diethyl ether, and after phase separation, the organic phase was dried on anhydrous sodium sulfate, and the solvent then distilled off. The resulting solid residue was applied to a silica gel column, and then stepwise eluted with hexane/diethyl ether (100:10), hexane/diethyl ether (80:20), and hexane/diethyl ether (60:40), in this order. 
     Fractions of hexane/diethyl ether (60:40) were combined, the solvent was distilled off, and the resulting residue was adsorbed on a column containing HP-2MG resin (Mitsubishi Chemicals, Japan) and eluted with water to obtain a fraction containing the desired compound (R)-(+)-methylsuccinic acid, which was obtained in the form of a white solid in an amount of 170 mg per liter of culture broth. 
     This compound was confirmed to be methylsuccinic acid by IR spectrum,  1  H-NMR spectrum,  13  C-NMR spectrum and MASS spectrum. IR spectrum and  13  C-NMR spectrum of the resulting methylsuccinic acid are shown in FIGS. 1 and 2 respectively. 
     The absolute configuration and optical purity of the obtained compound were determined by a comparison of specific rotation thereof with a reported value, and a  13  C-NMR spectrum analysis of a diastereomer of the product with L-menthol. Namely, a specific rotation of the product [α] 22   D  =+16.80° (c=2.16 g/100 ml, ethanol) was compared with a reported value [α] 22   D  =+16.88° (c=2.16 g/100 ml, ethanol) of (R)-(+)-methylsuccinic acid(Justus Liebigs Ann. Chem., 538, 1, 1939), and the configuration and optical purity of the product were determined as R-form and 99.5% respectively. 
     Further, the product was reacted with L-menthol in the presence of p-toluenesulfonic acid, to synthesize a diastereomer, and the  13  C-NMR spectrum thereof was obtained. The enantiomer excess obtained from a ratio of signal areas of carbon atom at a 3-position of the R-form and S-form of methylsuccinic acid was at least 98% e.e. 
     
                       TABLE 1______________________________________Composition        Amount added______________________________________Squalene               2.0 g/LNH.sub.4 NO.sub.3      2.5 g/LKH.sub.2 PO.sub.4      1.5 g/LNa.sub.2 HPO.sub.4     1.5 g/LMgSO.sub.4.7H.sub.2 O  0.5 g/LFeSO.sub.4.7H.sub.2 O  0.01 g/LCaCl.sub.2.2H.sub.2 O  0.01 g/LYeast extract          0.2 g/L          pH 5.0______________________________________ 
    
     
                       TABLE 2______________________________________Composition         Amount added______________________________________Yeast extract           3.0 g/LMalt extract            3.0 g/LPepton                  5.0 g/LGlucose                 10.0 g/LAgar                    20.0 g/L           pH 6.0______________________________________ 
    
     EXAMPLE 2 
     Strain SQL349 (FERM BP-3580) was cultured in a YM liquid medium at 30° C. for 16 hours, with shaking, and the culture broth was inoculated to 3 l of a medium in a 5 l jar fermenter. The medium had a composition shown in Table 1 but contained 10.0 g/l squalene and 1.0 g/l glucose as carbon sources. The culturing was carried out at 30° C., 500 rpm and 0.8 VVM aeration for 64 hours, and the produced (R)-(+)-methylsuccinic acid was purified according to the same procedure as described in Example 1 to obtain (R)-(+)-methylsuccinic acid at a yield of 1.7 g per liter of culture broth. 
     The product was confirmed to be (R)-(+)-methylsuccinic acid by IR spectrum,  1  H-NMR spectrum,  13  C-NMR spectrum, MASS spectrum, specific rotation, and  13  C-NMR of diastereomer with L-menthol, and the optical purity was at least 98%. 
     EXAMPLE 3 
     A strain SQL349 (FERM BP-3580) was cultured in 10 ml of a YM medium at 30° C. for 72 hours, and the culture was centrifuged to obtain cultured cells. The cells were washed with 0.1 M phosphate buffer (pH 7) and suspended in 10 ml of the same buffer, 10 mg of glucose and 20 mg of squalene were added therein, and the mixture was shaken at 30° C. for 48 hours. The product (R)-(+)-methylsuccinic acid was purified according to the same procedure as described in Example 1, and as a result, (R)-(+)-methylsuccinic acid was obtained at a yield of 240 mg/l of reaction mixture. 
     The product was confirmed to be (R)-(+)-methylsuccinic acid by IR spectrum,  1  H-NMR spectrum,  13  C-NMR spectrum, MASS spectrum, specific rotation, and  13  C-NMR spectrum of diastereomer with L-menthol, and the optical purity was at least 98%. 
     EXAMPLE 4 
     Microbial strains shown in Table 3 were separately cultured according to the same method as described in Example 2, to produce optically active methylsuccinic acid. The products thereof were purified and the optical activity determined as described in Example 1. 
     
                       TABLE 3______________________________________             Amount of    Culture  methylsuccinic                          Steric Optical    time     acid produced                          Configu-                                 purityStrain   (hr)     (g/L)        ration (%)______________________________________IFO 10073    72       2.2          R      &gt;99IFO 1209 99       1.3          R      &gt;99IFO 1741 90       1.4          R      &gt;99IFO 1742 163      0.6          R      &gt;99IFO 0746 100      0.3          R      &gt;99______________________________________