Abstract:
A process for producing L-aspartyl-L-phenylalanine alkyl or aryl esters wherein L-aspartic acid and L-phenylalanine which is esterified with an alkyl group having at least two carbon atoms, an aryl group or an aralkyl group, are contacted in an aqueous medium with particular microorganisms or enzyme fractions from these microorganisms and the condensed product crystallizes out due to insolubility in the aqueous medium.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a process for producing L-aspartyl-L-phenylalanine alkyl, aryl or aralkyl esters. 
     2. Description of the Prior Art 
     L-aspartyl-L-phenylalanine esters are peptides which have been noted as sweeteners in recent years. L-aspartyl-L-phenylalanine methyl ester (abbreviated as APM hereinafter) is a well known example of such a sweetener. 
     It is well known that the process for production of APM includes both chemical and enzymatic synthetic processes. 
     The chemical synthetic process for the production of APM comprises condensing N-protected L-aspartic acid anhydride and L-phenylalanine methyl ester (abbreviated as PM hereinafter) to obtain N-protected APM from which the protective group is subsequently removed. The enzymatic synthetic process comprises allowing a protein-decomposing enzyme to act on an N-protected L-aspartic acid and PM to obtain N-protected APM, or the PM adduct of N-protected APM, and then removing the protective group to form APM. However, both processes require the complicated steps of introducing and removing protective groups. 
     A process is also known for producing APM without using protective groups (see Japanese Patent Kokai No. 126796/1983, &#34;Digests of the Publications at the Annual Meeting of the Agricultural Chemical Society of Japan&#34; in 1983, p. 42). This process is a microbiological synthetic process which uses a microorganism selected from one of the following genuses: Pseudomonas, Torulopsis, Rhodotorula, and Sporobolomyces. This process, however, is not always suitable for the industrial production of APM because it produces extremely low yields. 
     The present inventors have previously found that by employing microorganisms, one can bring about the direct and effective formation of APM from L-aspartic acid and PM (see Japanese Patent Application No. 75559/1983). 
     However, a major drawback associated with processes for producing aspartyl-phenylalanine esters using as a starting material unprotected L-aspartic acid is the fact that the reaction forming APM from L-aspartic acid and PM is an equilibrium reaction, and this equilibrium prevents the substrates from being converted efficiently to aspartyl-phenylalanine ester products. 
     Therefore, a need continues to exist for a process providing for the efficient conversion of unprotected L-aspartic acid in reaction with L-phenylalanine esters to produce L-aspartyl-L-phenylalanine ester products in good yields. 
     SUMMARY OF THE INVENTION 
     Accordingly, it is an object of the present invention to provide a process for the production of L-aspartyl-L-phenylalanyl esters in good yields. 
     It is another object of the present invention to provide a process for the microbial production of L-aspartyl-L-phenylalanyl esters in good yields. 
     It is another object of the present invention to provide a process for the microbial extract or enzymatic production of L-aspartyl-L-phenylalanine esters in good yields. 
     It is another object of the present invention to provide a process for the production of L-aspartyl-L-phenylalanyl esters in good yields, where an unprotected L-aspartic acid starting material is used. 
     It is another object of the present invention to provide a process for the production of L-aspartyl-L-phenylalanyl esters, where a L-phenylalanine ester starting material is used. 
     It is another object of the present invention to provide a process for the production of L-aspartyl-L-phenylalanyl esters, where the product formed has a low solubility in the reaction medium. 
     These and other objects of the present invention have been realized by contacting an unprotected L-aspartic acid and a L-phenylalanine ester with a microorganism or an enzyme-containing fraction of the microorganism. 
     The L-phenylalanine ester used in accordance with the present invention comprises L-phenylalanine esterified with a moiety containing at least 2 carbon atoms. The esterifying moiety may be a straight, branched or cyclic alkyl group containing at least 2 carbon atoms. The esterifying moiety may also be an unsubstituted or substituted aryl group containing at least 5 carbon atoms, or an unsubstituted or substituted aralkyl group containing at least 6 carbon atoms. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The abbreviation PR is used in this application to represent phenylalanine esterified with alkyl, aryl or aralkyl alcohols where the alkyl moiety has two or more carbon atoms. The substituents on the aryl group are not specifically limited but, for example, may include one or more halogens, alkyl groups of 1-10 carbon atoms, nitro groups and cyano groups. 
     Furthermore, the L-phenylalanine ester or the L-aspartic acid may be in the form of free bases or salts of any of various acids, such as, for example, hydrochloric acid, p-toluenesulfonic acid, trifluoroacetatic acid, hydrobromic acid, formic acid or acetic acid. 
     APR itself is expected not only to be used as a sweetener but also to be used as a raw material to synthesize APM by ester exchange or any other method. 
     Accordingly, this invention is directed to a process for the production of APR, characterized by contacting L-aspartic acid and PR with at least one microorganism selected from one of the following genuses: Corynebacterium, Candida, Cryptococcus, Escherichia, Flavobacterium, Geotrichum, Micrococcus, Pachysolen, Saccharomyces, Trichosporon, Xanthomonas, Kluyveromyces, Endomyces, Arthrobacter, Cellulomonas and Brevibacterium. 
     The process for converting L-aspartic acid and PR to APR by conducting the condensation in an aqueous medium through the action of microorganisms selected from the above-mentioned genuses can be carried out by contacting L-aspartic acid and PR with microorganism cells, culture solutions or microorganism cell-treating materials of the above-mentioned microorganisms. 
     The following are examples of the microorganisms which have the ability to convert L-aspartic acid and PR to APR by condensation in this invention: 
     Corynebacterium sp.: ATCC 21251 
     Corynebacterium xerosis: ATCC 373 
     Candida intermedia: FERM-BP508 
     Cryptococcus neoformans: IFO 4289 
     Escherichia coli: FERM-BP477 
     Flavobacterium sewanens: FERM-BP476 
     Geotrichum candidum: IFO 4599 
     Micrococcus luteus: ATCC 4698 
     Pachysolen tannophilus: IFO 1007 
     Trichosporon capitatum: IFO 1197 
     Xanthomonas campestris: FERM-BP507 
     Kluyveromyces thermotolerans: IFO 0662 
     Endomyces ovetencis: IFO 1201 
     Saccharomyces cerevisiae: IFO 2003 
     Arthrobacter citreus: ATCC 11624 
     Cellulomonas flavigena: ATCC 8183 
     Brevibacterium linens: ATCC 8377 
     The cells of these microorganisms can be obtained by using ordinary culture media. 
     L-aspartic acid and PR may be added at the beginning or in the process of cultivation of these microorganisms. 
     The culture media to be used for the microorganisms of this invention are ordinary ones containing usual carbon and nitrogen sources and inorganic ions in addition to L-aspartic acid and PR. Moreover, the addition of trace amounts of organic nutritive substances such as vitamins and amino acids often brings about desirable results. 
     The carbon sources suitable for use in this invention include carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols and the like. The nitrogen sources suitable for use herein include ammonia gas, aqueous ammonia, ammonium salts, and the like. The inorganic ions may be magnesium ion, phosphoric acid ion, potassium ion, iron ion, and the like. 
     The cultures are conducted under aerobic conditions at pH 4-8 at suitable temperatures 
     controlled within the range of 25°-40° C., and for 1-10 days to obtain desirable results. 
     The microorganisms to be used in this invention include the whole culture solutions obtained after completion of the cultivation thereof, the microorganisms separated from the culture solutions, or washed microorganisms. Also, the microorganisms to be used in this invention may be freeze-dried, acetone-dried, contacted with toluene, surfactants, etc., treated with lysozyme, exposed to ultrasonic waves, mechanically ground or treated in any manner which results in obtaining an enzyme protein fraction having enzyme activity which can change L-aspartic acid and PR to APR. The fixed cells of these microorganisms, insolubilized materials obtained from cell-treating materials, etc. may be used. 
     As aqueous media, there can be used those containing water, buffers, and organic solvents such as ethanol. Moreover, nutritive elements needed for the growth of microorganisms, anti-oxidants, surfactants, coenzymes, hydroxylamine and metallic ions, etc. can be added to the aqueous media if necessary. 
     When the cells of the above-mentioned microorganisms are grown in aqueous media and simultaneously contacted with L-aspartic acid and PR so as to exert action thereon, the aqueous media should contain L-aspartic acid, PR, and nutritive elements such as carbon sources, nitrogen sources, and inorganic ions, etc. needed for the growth of microorganisms as described above. Further, the addition of trace amount of organic nutritive elements such as vitamins and amino acids often brings about desirable results. 
     When the whole culture solutions, culture cells or cell-treating materials of the above-mentioned microorganisms are contacted with L-aspartic acid and PR to exert action thereon, the aqueous media are prepared by dissolving or suspending L-aspartic acid, PR and the culture solutions, microorganism culture cells, or microorganism cell-treating materials. The media are then maintained at proper temperatures of 10°-70° C., at a pH of 4-8, and are allowed to stand or are stirred. As a result, a great deal of APR is produced and accumulated in the aqueous media after 5-100 hours. 
     The APR thus produced can be separated and purified by known processes for separation. The APR obtained may be characterized with an amino-acid analyzer. 
    
    
     The invention now being generally described, the same will be better understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof. 
     EXAMPLE 1 
     Into a 500 ml flask was introduced 50 ml of a medium (pH 7.0) containing 2.0 g/dl of glucose, 0.5 g/dl of (NH 4 ) 2  SO 4  0.1 g/dl of KH 2  PO 4 , 0.1 g/dl of K 2  HPO 4 , 0.1 g/dl of MgSO 4 .7H 2  O, 0.05 g/dl of FeSO 4 .7H 2  O, 1 mg/dl of MnSO 4 .4H 2  O, 1.0 mg/dl of yeast extract, 0.5 g/dl of malt extract, and 4.0 g/dl of calcium carbonate, which was sterilized at 120° C. for 15 minutes. 
     Each one of the thus prepared media was inoculated, using a platinum loop with Flavobacterium sewanens FERM-BP476 or Arthrobacter citreus ATCC 11624, incubated in a bouillon-agar medium at 30° C. for 24 hours, and cultured at 30° C. for an additional 20 hours. The cells were harvested from this culture solution by centrifugation, washed once with the same amount of physiological saline as that of the culture solution and collected. 
     These microorganism cells were added to Reaction Solution A shown in Table 1 to equal 5 g/dl (final conditions, pH 5.4, 5 ml), and allowed to react at 37° C. for 16 hours. The resulting APR was determined with an amino acid analyzer to give the results in Table 2. 
     
                       TABLE 1______________________________________Reaction Solution A*______________________________________L-aspartic acid       10 g/dlL-phenylalanine ester or its                 15 g/dlhydrochloride shown in Table 2______________________________________ *The substrates are included in 0.1 M phosphoric acid buffer (final pH 5.4). 
    
     
                                           TABLE 2__________________________________________________________________________                 APR formed (mg/dl)Phenylalanine ester or its                 Flavobacterium sewanens                             Arthrobacter citreushydrochloride starting material                 FERM-BP476  ATCC 11624__________________________________________________________________________Phenylalanine methyl ester hydrochloride                 590         583Phenylalanine ethyl ester hyorochloride                 710         702Phenylalanine n-propyl ester hydrochloride                 812         810Phenylalanine iso-propyl ester hydrochloride                 1020        1005Phenylalanine n-butyl ester hydrochloride                 920         911Phenylalanine isobutyl ester hydrochloride                 880         865Phenylalanine sec-butyl ester hydrochloride                 850         841Phenylalanine tert.-butyl ester hydrochloride                 821         809Phenylalanine cyclohexyl ester hydrochloride                 807         799Phenylalanine amyl ester hydrochloride                 792         785Phenylalanine hexyl ester hydrochloride                 774         762Phenylalanine heptyl ester hydrochloride                 772         760Phenylalanine octyl ester hydrochloride                 763         740Phenylalanine nonyl ester hydrochloride                 751         729Phenylalanine decyl ester hydrochloride                 748         715Phenylalanine benzyl ester hydrochloride                 729         699Phenylalanine p-nitrophenyl ester                 703         689Pnenylalanine phenyl ester                 699         678__________________________________________________________________________ 
    
     EXAMPLE 2 
     Cells of the microorganisms, grown and washed in a manner similar to example 1 and shown in table 4, were added to Reaction Solution B shown in Table 3 to equal 5 g/dl (final condition, pH 5.4, 5 ml), and kept at 37° C. for 16 hours. The resulting aspartyl-phenylalanine isopropyl ester was determined with an amino acid analyzer to give the results in Table 4. 
     
                       TABLE 3______________________________________Reaction Solution B*______________________________________L-aspartic acid      10 g/dlL-phenylalanine isopropyl ester                15 g/dlhydrochloride______________________________________ *The substrates are included in 0.1 M phosphoric acid buffers (final pH 5.4). 
    
     
                       TABLE 4______________________________________               Aspartyl-phenylalanine               isopropyl ester formed inMicroorganisms      Reaction Solution (mg/dl)______________________________________Corynebacterium sp.       ATCC 21251  862Corynebacterium       ATCC 373    324xerosisCandida intermedia       FERM-BP508  425Cryptococcus       IFO 4289    189neoformansEscherichia coli       FERM-BP477  1172Flavobcterium       FERM-BP476  1050sewanensGeotrichum  IFO 4599    204candidumMicrococcus luteus       ATCC 4698   915Pachysolen  IFO 1007    148tannophilusTrichosporon       IFO 1197    163capitatumXanthomonas FERM-BP507  372campestrisKluyveromyces       IFO 0662    135thermotoleransEndomyces ovetencis       IFO 1201    399Saccharomyces       IFO 2003    127cerevisiaeArthrobacter citreus       ATCC 11624  1018Cellulomonas       ATCC 8183   795flavigenaBrevibacterium linens       ATCC 8377   939______________________________________ 
    
     EXAMPLE 3 
     Into 100 ml of Reaction Solution B was introduced 5 g of Flavobacterium sewanens FERM-BP476, grown and washed in a manner similar to Example 1, and the reaction was carried out at 37° C. for 24 hours. 
     The resulting reaction solution was spotted on a TLC plate for development in the form of a belt, and developed with a solvent system consisting of n-butanol: acetic acid: water=2:1:1. Part of the product aspartyl phenylalanine isopropyl ester was taken out and extracted with distilled water. Then, the resulting reaction product was crystallized to obtain 1023 mg of crystals. The obtained crystals were characterized as to optical rotation, melting point, and specific rotatory power, and the product obtained from Reaction Soluton B was identical to an authentic aspartyl-phenylalanine isopropyl ester specimen. 
     EXAMPLE 4 
     Into 100 ml of Reaction Solution B was introduced 5 g of Arthrobacter citreus ATCC 11624, grown and washed in a manner similar to Example 1, and the reaction was carried out at 37° C. for 24 hours. 
     The resulting reaction solution was spotted on a TLC plate for development in the form of a belt, and developed with a solvent system consisting of n-butanol: acetic acid: water=2:1:1. Part of the product aspartyl-phenylalanine isopropyl ester was taken out and extracted with distilled water. Then, the resulting reaction product was crystallized to obtain 1005 mg of crystals. The obtained crystals were characterized as to optical rotation, melting point, and specific rotatory power, and the product obtained from Reaction Solution B was identical to an authentic aspartyl-phenylalanine isopropyl ester specimen. 
     EXAMPLE 5 
     Into the culture solution of Escherichia coli FERM-BP477, maintained at 30° C. for 12 hours in the same medium used in Example 1, was poured under sterile conditions 10 ml of aqueous solution (adjusted to pH 5.4) containing 5 g/dl of L-aspartic acid and 10 g/dl of L-phenylalanine isopropyl ester, and the cultivation was further continued for 120 hours after the solution was adjusted under sterile conditions to pH 5.4. It was maintained at a pH of 5.4 by adjustments at intervals of 2 hours during incubation. 
     The resulting product in this culture solution was determined with an amino-acid analyzer and 489 mg/dl of aspartyl-phenylalanine isopropyl ester was formed. 
     EXAMPLE 6 
     Into the culture solution of Brevibacterium linens ATCC 8377, maintained at 30° C. for 12 hours in the same medium used in Example 1, was poured under sterile conditions 10 ml of aqueous solution (adjusted to pH 5.4) containing 5 g/dl of L-aspartic acid and 10 g/dl of L-phenylalanine isopropyl ester, and the cultivation was further continued for 10 hours after the solution was adjusted under sterile conditions to pH 5.4. It was maintained at a pH of 5.4 by adjustments at intervals of 2 hours during incubation. 
     The resulting product in this culture solution was determined with an amino-acid analyzer and 479 mg/dl of aspartyl-phenylalanine isopropyl ester was formed. 
     EXAMPLE 7 
     Flavobacterium sewanens FERM-BP476, grown and washed in a manner similar to example 1, was added to Reaction Solution A (phenylalanine n-butyl ester was used as the phenylalanine ester) to equal 5 g/dl (final conditions, pH 5.4, 5 ml) and allowed to react at 37° C. for 16 hours. 
     One liter of resulting enzyme reaction solution was left still for 24 hours at 0° C. and 3 g of crystals which separated out were filtered. Part of the product was taken out and measured by high-speed liquid chromatography (column:silicon ODS; eluent:methanol-water) to show that the product contained 1.67 g of L-aspartyl-L-phenylalanine n-butyl ester. these crystals were added to a mixed solution consisting of 3.8 g of 35% hydrochloric acid, 1.0 g of methanol and 2.0 g of water and the solution was mixed continuously for 7 days at 15° C. The resulting product was characterized with an amino-acid analyzer, infrared spectrum, acid titration, hydrochloric acid titration with the result that 0.38 g of the product L-aspartyl-L-phenylalanine methyl ester hydrochloride were obtained. The yield of L-aspartyl-L-phenylalanine butyl ester is 23%. 
     EXAMPLE 8 
     Arthrobacter citreus ATCC 11624, grown and washed in a manner similar to example 1, was added to Reaction Solution A (phenylalanine n-butyl ester was used as the phenylalanine ester) to equal 5 g/dl (final conditions, pH 5.4, 5 ml) and allowed to react at 37° C. for 16 hours. 
     One liter of resulting enzyme reaction solution was left still for 24 hours at 0° C. and 3 g of crystals which separated out were filtered. Part of the product was taken out and measured by high-speed liquid chromatography (column: silicon ODS; eluent: methanol-water) to show that the product contained 1.65 g of L-aspartyl-L-phenylalanine n-butyl ester. These crystals were added to a solution containing 3.8 g of 35% hydrochloric acid, 1.0 g of methanol and 2.0 g of water and the solution was mixed continuously for 7 days at 15° C. The product was filtered and dried to obtain 0.39 g of dried white crystals. The resulting product was characterized with an amino-acid analyzer, infrared spectrum, acid titration, and hydrochloric-acid titration with the result that 0.36 g of the product L-aspartyl-L-phenylalanine methyl ester hydrochloride was obtained. The yield of L-aspartyl-L-phenylalanine butyl ester is 22%. 
     To determine if any other specific microorganism, which is a member of the previously specified genuses, is suitable for the purposes of this invention, one would follow the procedures outlined in the examples given above and analyze the resulting product as described above. A suitable microorganism is one which is capable of bringing about condensation of aspartic acid and PR. 
     Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.