Process for preparing optically active tryptophanes
This application is a continuation of Ser. No. 276,057, June 22, 1981, abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a process for preparing optical active tryptophanes by biochemical asymmetric hydrolysis of DL-tryptophane amides, in which an amidase derived from a microorganism is utilized. 
     2. Description of the Prior Art 
     It is well-known in the art that L-tryptophanes and D-tryptophanes are optically active and have different properties and uses. For instance, L-tryptophane is an important compound as an essential amino acid and is suitable for use in an amino acid infusion solution, animal food stuff additives, hypnotizing agent and the like. On the other hand, D-tryptophane is useful as, for example, dentifrice additives. 
     Many organic synthesis methods for preparing tryptophanes have been heretofore proposed. However, when the tryptophanes are prepared by organic synthesis methods, the products are obtained in the form of mixtures of D-tryptophanes and L-tryptophanes, i.e. racemic mixtures. Therefore, the effective optical resolution of DL-tryptophanes is an extremely important problem to be solved in the art. 
     Proposed optical resolution methods of DL-tryptophanes are only those in which hydantoin or N-acyl derivatives of DL-tryptophanes are used as a substrate and in which microorganisms capable of producing hydantoinase or acylase are interacted with the substrate. Thus, the development of the substrate and the microorganisms used in the field of the biochemical optical resolution of DL-tryptophanes are extremely limited. 
     SUMMARY OF THE INVENTION 
     Accordingly, an object of the present invention is to obviate the afore-mentioned problems in the prior arts and to provide a novel biochemical optical resolution of DL-tryptophanes in which L-tryptophanes and D-tryptophanes can be separated from each other at a high yield. 
     Another object of the present invention is to selectively hydrolyze L-tryptophane amides, whereby optical active L-tryptophanes are prepared at a high yield. L-tryptophanes are prepared at a high yield. 
     A further object of the present invention is to hydrolyze D-tryptophane amides after the separation of L-tryptophanes from DL-tryptophane amides, whereby optical active D-tryptophanes are prepared at a high yield. 
     Other objects and advantages of the present invention will be apparent from the description set forth hereinbelow. 
     In accordance with the present invention, there is provided a process for preparing optically active L-tryptophanes having a general formula ##STR1## wherein X represents hydrogen, hydroxyl group, halogen atoms such as fluorine, chlorine, bromine and iodine, lower alkyl groups, preferably having 1 to 4 carbon atoms, or lower alkoxy groups, preferably having 1 to 4 carbon atoms, comprising the step of interacting DL-tryptophane amides having a general formula. ##STR2## wherein X is as defined above with the culture product of a microorganism capable of producing amidase or the treated product thereof, whereby the asymmetric hydrolysis of L-tryptophane amides is effected. 
     In accordance with the present invention, there is also provided a process for preparing optically active D-tryptophanes having a general formula ##STR3## wherein X is as defined above, comprising the steps of: 
     (a) interacting DL-tryptophane amides having a general formula, ##STR4## wherein X is as defined above with the culture product of a microorganism capable of producing amidase or the treated product thereof, whereby the asymmetric hydrolysis of L-tryptophane amides is effected; 
     (b) separating the resultant L-tryptophanes obtained from the asymmetric hydrolysis of L-tryptophane amides, and; 
     (c) hydrolyzing the resultant D-tryptophane amides. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Biochemical asymmetric hydrolysis of DL-tryptophane amides in which an enzyme derived from animal visceral organs is employed is reported [see: Bull. Agr. Chem. Soc. Japan, Vol. 21, No. 1, P. 62-66 (1957)]. However, the biochemical asymmetric hydrolysis of DL-tryptophane amides in which there is employed an enzyme derived from microorganisms has not been reported. 
     According to the present invention, new substrates are provided in the biochemical optical resolution of DL-tryptophanes in which an enzyme derived from microorganisms is employed and the variety of the microorganisms which can be utilized in the optical resolution is widened. Thus, the present invention is industrially very valuable in this field. 
     The DL-tryptophane amides employed as a substrate in the practice of the present invention can be readily synthesized by, for example, reacting the esters of DL-tryptophanes with ammonia in a conventional manner. 
     The microorganisms which can produce the amidase used in the practice of the present invention are those which belong to any systematic group, so long as they have the capability of selectively hydrolyzing only L-tryptophane amides in the racemic mixtures of DL-tryptophane amides. Examples of the genus names of these microorganisms are listed in the following Table, in which the typical species name of the microorganism belonging to each genus is also listed. However, it should be noted that the microorganisms which can be employed in the practice of the present invention are not limited to these specific examples. All the exemplified microorganisms are known and also readily available from the depositories of JFCC (Japanese Federation of Culture Cellections of Microorganisms) such as IFO (Institute for Fermentation, Osaka, Japan) and IAM (Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan). 
     
                       TABLE                                                       
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NAME OF MICROORGANISMS                                                    
                      DEPOSIT NO.                                         
______________________________________                                    
(1)   Genus Rhizopus      IFO-4768                                        
     Rhizopus Chinensis                                                   
(2)  Genus Absidia        IFO-4011                                        
     Absidia Orchidis                                                     
(3)  Genus Penicillium    IFO-5692                                        
     Penicillium frequentans                                              
(4)  Genus Pullularia     IFO-4464                                        
     Pullularia pullulans                                                 
(5)  Genus Fusarium       IFO-5421                                        
     Fusarium roseum                                                      
(6)  Genus Gibberella     IFO-5268                                        
     Gibberella fujikuroi                                                 
(7)  Genus Trichoderma    IFO-4847                                        
     Trichoderma viride                                                   
(8)  Genus Gliocladium    IFO-5422                                        
     Gliocladium roseum                                                   
(9)  Genus Cunninghamella IFO-4441                                        
     Cunninghamella elegans                                               
(10) Genus Actinomucor    IFO-4022                                        
     Actinomucor repens                                                   
(11) Genus Geotrichum     IFO-6454                                        
     Geotrichum candidum                                                  
(12) Genus Saccharomyces  IFO-0505                                        
     Saccharomyces rouxii                                                 
(13) Genus Shizosaccharomyces                                             
                          IFO-0346                                        
     Shizosaccharomyces pombe                                             
(14) Genus Pichia         IFO-0195                                        
     Pichia polimorpha                                                    
(15) Genus Hansenula      IFO-0117                                        
     Hansenula anomala                                                    
(16) Genus Debariomyces   IFO-0023                                        
     Debariomyces hansenii                                                
(17) Genus Nadsonia       IFO-0665                                        
     Nadsonia elongata                                                    
(18) Genus Sporobolomyces IFO-0376                                        
     Sporobolomyces pararoseus                                            
(19) Genus Cryptococcus   IFO-0378                                        
     Cryptococcus albidus                                                 
(20) Genus Torulopsis     IFO-0768                                        
     Torulopsis candida                                                   
(21) Genus Brettanomyces  IFO-0642                                        
     Brettanomyces anomalus                                               
(22) Genus Candida        IFO-0396                                        
     Candida utilis                                                       
(23) Genus Tricosporon    IFO-0598                                        
     Tricosporon beigelii                                                 
(24) Genus Rhodotorula    IFO-0412                                        
     Rhodotorula minuta var texensis                                      
(25) Genus Mycobacterium  IFO-3158                                        
     Mycobacterium phlei                                                  
(26) Genus Nocardia       IFO-3424                                        
     Nocardia asteroides                                                  
(27) Genus Streptomyces   IFO-3356                                        
     Streptomyces griseus                                                 
(28) Genus Enterobacter   IFO-3320                                        
     Enterobacter aerogenes                                               
(29) Genus Alcaligenes    IAM-1517                                        
     Alcaligenes viscolactis                                              
(30) Genus Flavobacterium IAM-1100                                        
     Flavobacterium arborescens                                           
(31) Genus Bacillus       IFO-3026                                        
     Bacillus subtilis                                                    
(32) Genus Agrobacterium   IFO-13262                                      
     Agrobacterium tumefaciens                                            
(33) Genus Micrococcus    IFO-3242                                        
     Micrococcus flavus                                                   
(34) Genus Sarcina        IFO-3064                                        
     Sarcina aurantiaca                                                   
(35) Genus Arthrobacter   IFO-3530                                        
     Arthrobacter simplex                                                 
(36) Genus Brevibacterium  IFO-12071                                      
     Brevibacterium ammoniagenes                                          
(37) Genus Pseudomonas    IFO-3081                                        
     Pseudomonas fluorescens                                              
(38) Genus Lactobacillus  IFO-3322                                        
     Lactobacillus casei                                                  
(39) Genus Streptococcus  IFO-3434                                        
     Streptococcus lactis                                                 
(40) Genus Clostridium    IFO-3346                                        
     Clostridium acetobutyricum                                           
(41) Genus Klebsiella     IFO-3317                                        
     Klebsiella Pneumonia                                                 
     (as Aerobacter aerogenes)                                            
(42) Genus Achromobacter  IAM-1013                                        
     Achromobacter cycloclastes                                           
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     Among these microorganisms, microorganisms belonging to genera Trichoderma, Rhodotorula, Nocardia, Mycobacterium, Bacillus, Rhizopus, Candida, Hansenula, Streptomyces, Aerobacter, Arthrobacter, Pseudomonas, Gibberella, Torulopsis and Enterobacter are especially useful in the practice of the present invention. 
     In the practice of the present invention, the above mentioned microorganisms can be interacted with the DL-tryptophane amides in the form of either the culture products obtained from the cultivation of cells in liquid culture media or the treated products thereof. Examples of the treated products are the cells isolated from the culture solutions, the enzyme (i.e. amidase) isolated from the culture products or the strains according to any conventional enzyme isolating technique (e.g. the crude enzyme, the purified enzyme, the enzyme-containing extracts or the concentrated solutions thereof). The cells or enzymes can be immobilized on carriers in the practice of the present invention. 
     Examples of the carriers are natural products such as alginic acid, carrageenan, collagen, cellulose, acetylcellulose, agar-agar, cellophane, collodion and the like, and synthetic polymer substances such as polyacrylamide, polystyrene, polyethylene glycol, polypropylene glycol, polyurethane, polybutadiene and the like. The immobilization of the cells or enzymes on the carrier can be carried out in a conventional manner under moderate conditions so that the activity of amidase is not impaired. 
     The suitable reaction temperature of the asymmetric hydrolysis according to the present invention can be within the range of from 20° to 50° C. However, in order to minimize the decrease in the enzymatic activity, the use of the reaction temperature of from 25° to 40° C. is economically advantageous. 
     The suitable reaction time of the asymmetric hydrolysis according to the present invention can be within the range of from 5 to 50 hours. However, the reaction time can be shortened by raising the reaction temperature or by increasing the amount of the enzymes used. Furthermore, the reaction can be generally carried out under a pH of 5 through 10, more preferably 7 through 9. 
     The amount of the microorganisms employed in the practice of the present invention is preferably in a weight ratio of from 0.01 to 2, in terms of the freeze dried cells, based on the weight of the DL-tryptophane amides. In the case where the cultivation mixtures of the microorganisms, treated products thereof, or the immobilized products thereof are employed, the amount thereof can be determined in terms of the amount of the freeze dried cells. The suitable concentration of the substrate, i.e. DL-tryptophane amides in the reaction mixture is generally within the range of from 1 to 40% by weight, more preferably 5 to 30% by weight. 
     According to the present invention, the asymmetric hydrolysis reaction is stopped after the hydrolysis of L-tryptophane amide proceeds at the conversion rate of almost 100%, and, then, L-tryptophanes and D-tryptophane amides are separately isolated from the reaction mixture. This separation can be readily carried out by using any conventional separation techniques, such as fractional crystallization and solvent extraction. 
     D-tryptophane amides are not affected by the action of the microorganisms in the above mentioned asymmetric hydrolysis and, therefore, almost all D-tryptophane amides can be recovered from the racemic mixture. The D-tryptophane amides thus recovered can be readily hydrolyzed by using any conventional technique, for example, by heating in the presence of an aqueous acid or alkaline solution. Thus, optically active D-tryptophane can be obtained at a high yield. The D-tryptophane amides recovered above can also be subjected to a racemization and reused as a substrate in the asymmetric hydrolysis of the present invention. 
     The optical purity and yield of the L-tryptophanes as well as the D-tryptophanes are equal to or more than those obtained from known biochemical optical resolution. 
     EXAMPLES 
     The present invention will now be further illustrated by, but is by no means limited to, the following Examples wherein the yield of L- or D-tryptophanes is calculated from the following equation. 
    
    
     EXAMPLES 1 THROUGH 15 
     100 ml of a culture medium having a pH of 7.0 and containing 5% by weight of glycerol, 5% by weight of corn steep liquor, 0.5% by weight of ammonium sulfate and 1 ml of a mixture of inorganic salts was charged into a shaking flask. The inorganic salt mixture was prepared by dissolving 20 g of MgSO 4 .7H 2  O, 5 g of FeSO 4 .7H 2  O, 2 g of CaCl 2 , 0.2 g of MnCl 2 .4H 2  O, 0.1 g of NaMoO 4 .2H 2  O and 0.1 g of NaCl in 1000 ml of distilled water. After sterilizing the content of the flask, 2 loopful of each of the microorganisms listed in Table 1 below were inoculated from an agar slant and, then, the reciprocal shaking culture (or incubation) was carried out at a temperature of 30° C. for 65 hours. 
     Thereafter, 2 g of DL-tryptophane amide was added to the flask and, then, the reciprocal shaking culture was carried out for 48 hours at a temperature of 30° C. The cells was removed from the reaction mixture by centrifugation or filtration. The pH of the culture filtrate was adjusted to 6.0 and, then, the filtrate was vacuum concentrated until the volume of the filtrate became 5 through 10 ml. The crystallized L-tryptophane was collected by filtration after cooling. 
     The results are shown in the following Table 1. 
     
                       TABLE 1                                                     
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                     Formed                                               
                     L-Tryptophane                                        
                           Iso-    Optical Ro-                            
                           lation  tation [α] .sub.D.sup.20         
Exam-                      Yield   (C = 0.5,                              
ple No.                                                                   
      Microorganism as used                                               
                           (%)     H.sub.2 O)                             
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1     Enterbacter aerogenes                                               
                      IFO-3317 52    -31.5°                        
2     Bacillus Subtilis                                                   
                      IFO-3026 47    -30.5°                        
3     Candida utilis  IFO-0396 44    -31.2°                        
4     Rhodotorula glutinis                                                
                      IFO-0413 54    -33.0°                        
      var. rebescens                                                      
5     Rhizopus chinensis                                                  
                      IFO-4768 51    -32.0°                        
6     Trichoderma Viride                                                  
                      IFO-4847 27    -30.0°                        
7     Nocardia asteroides                                                 
                      IFO-3424 29    -29.8°                        
8     Mycobacterium phlei                                                 
                      IFO-3158 25    -31.0°                        
9     Streptomyces griseus                                                
                      IFO-3356 30    -29.5°                        
10    Klebsiella pneumoniae                                               
                      IFO-3320 32    -30.0°                        
      (as Aerobacter aerogenes)                                           
11    Arthrobacter simplex                                                
                      IFO-3530 37    -31.8°                        
12    Pseudomonas fluorescens                                             
                      IFO-3081 28    -32.0°                        
13    Gibberella fujikuroi                                                
                      IFO-5268 31    -29.5°                        
14    Torulopsis candida                                                  
                      IFO-0768 23    -29.8°                        
15    Hansenula anomala                                                   
                      IFO-0117 26    -30.5°                        
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     EXAMPLE 16 
     From the culture solution of Rhodotorula glutinis var. rebescens prepared in a manner as described in Example 4, the cells was collected by centrifugation and, then, washed twice with distilled water. 
     The washed cells were added to 100 ml of a 0.1M phosphate buffer solution having a pH of 7.0 and containing 2 g of DL-tryptophane amide and the mixture was incubated for 20 hours at a temperature of 30° C. 
     After the completion of the reaction, the cells were removed from the reaction mixture by centrifugation. The reaction mixture thus obtained was analyzed by high speed liquid chromatography. The resultant reaction mixture contained 983 mg of L-tryptophane (yield: 98%) and 1010 mg of D-tryptophane amide. From this reaction mixture the unreacted D-tryptophane amide was extracted by using ethyl acetate. 
     On the other hand, the pH of the water layer after the extraction was adjusted to 5.9 by using 2N sulfuric acid. Then, the resultant solution was vacuum concentrated until the total volume of the solution became about 20 ml. After ice-cooling, 330 mg of the crystallized L-tryptophane having a melting point of 280°-282° C. (decomposition) and [α] D   20  of -33.5° (C=0.5 H 2  O) was collected). 
     EXAMPLE 17 
     The washed cells of Rhodotolua glutinis var. rebescens prepared in a manner as described in Example 16 were washed with cold acetone. Thus, acetone dried cells were obtained. 
     On the other hand, DL-tryptophan amide was dissolved in distilled water and substrate solutions having various concentrations listed in Table 2 below and having a pH of 7.0 were prepared. 
     The above mentioned acetone dried cells were added to 10 ml of the substrate solution in such an amount that a weight ratio of the dried cells to the substrate were 0.2. Then, the reaction was carried out at a temperature of 30° C. for 20 hours. The resultant reaction mixture was analyzed to determine the yield of L-tryptophane by using high speed liquid chromatography. 
     The results are shown in Table 2 below. 
     
                       TABLE 2                                                     
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Concentration of Substrate                                                
                    Yield (%) of                                          
(i.e. DL-Tryptophane Amide)                                               
                    L-Tryptophane                                         
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 1                  100                                                   
 2                  100                                                   
 5                  100                                                   
10                  100                                                   
20                   92                                                   
30                   75                                                   
40                   55                                                   
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     EXAMPLE 18 
     The washed cells of Rhodotorula glutinis var. rebescens prepared in a manner as described in Example 16 were freeze dried. 
     The freeze dried cells were added to 10 ml of distilled water containing 10% by weight of DL-tryptophane amide (pH=7.5) in the weight ratio of the cells to the substrate listed in Table 3 below. Then, mixture was incubated at a temperature of 30° C. for 20 hours. 
     The reaction mixture was analyzed to determine the yield of L-tryptophane by high speed liquid chromatography. 
     The results are shown in Table 3 below. 
     
                       TABLE 3                                                     
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Freeze Dried Cells/Substrate                                              
                    Yield (%) of                                          
(weight ratio)      L-tryptophane                                         
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0.01                 73                                                   
0.05                 98                                                   
0.1                 100                                                   
0.5                 100                                                   
1.0                 100                                                   
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     EXAMPLE 19 
     50 mg of the freeze dried cells of Rhodotorula glutinis var. rebescens prepared in a manner as described in Example 18 were suspended in 5 ml of 0.2M phosphate acid buffer solution and, then, the cells were disrupted under cooling by using a French press (20,000 psi). The resultant mixture was centrifuged under 20,000×g for 30 mins. To 5 ml of the supernatant solution thus obtained, 250 mg of DL-tryptophane amides was added and the pH of the mixture was adjusted to 7.5. Thereafter, the mixture was incubated at a temperature of 30° C. for 20 hours. 
     The reaction mixture was analyzed by a high speed liquid chromatography. L-tryptophane was obtained at a yield of 95%. 
     EXAMPLE 20 
     To 5 ml of the cell-free extract of Rhodotorula glutinis var. rebescens prepared in a manner as described in Example 19, ammonium sulfate was added. The protein which was precipitated at a saturation of 25 to 75% was collected by centrifugation. Then, 5 ml of 0.2M phosphate buffer solution containing 250 mg of DL-tryptophane amide and having a pH of 7.5 was added thereto and the mixture was incubated at a temperature of 30° C. for 20 hours. 
     The reaction mixture was analyzed by high speed liquid chromatography. L-tryptophane was obtained at a yield of 63%. 
     EXAMPLE 21 
     10 ml of the cell-free extract of Rhodotorula glutinis var. rebescens prepared in a manner as described in Example 19 was passed through a column having a diameter of 1.5 cm and a length of 65 cm and packed with Sephadex G-75 and fractions having amidase activity were collected. These fractions were concentrated by using a semipermeable membrane method to a volume of 5 ml. Then, 250 mg of DL-tryptophane amide was added thereto and the mixture was incubated at a temperature of 30° C. for 20 hours. 
     The reaction mixture was analyzed by high speed liquid chromatography. L-tryptophane was obtained at a yield of 56%. 
     EXAMPLE 22 
     The washed cells (corresponding to 1.0 g of the freeze dried cells) of Rhodotorula glutinis var. rebescens prepared in a manner as described in Example 16 were suspended in 15 ml of 0.1M phosphate buffer solution having a pH of 7.0 and, then, 3.75 g of acrylamide monomer, 0.2 g of N,N&#39;-methylene bisacrylamide (i.e. crosslinking agent), 2.5 ml of a 5% aqueous 3-dimethylamino propionitrile solution (i.e. polymerization promotor) and 2.5 ml of 2.5% aqueous potassium persulfate solution (i.e. polymerization initiator) were added and mixed with one another. The mixture was allowed to stand at a temperature of 25° C. for 1 hour, whereby the gellation of the mixture was complete. 
     The gel thus obtained was crushed and washed with water. The resultant immobilized product, (i.e., gel particles, having a particle diameter of 0.2 to 0.5 mm) was packed into a column having a diameter of 2 cm and a length of 50 cm. Thereafter, distilled water containing 10% by weight of DL-tryptophane amide and having a pH of 7.5 was passed through the column at a temperature of 30° C. from the top of the column at a space velocity (SV) of 0.2. 
     In this continuous reaction, the yield of L-tryptophane was maintained at a yield of 80% or more until the reaction time became 200 hours. 
     EXAMPLE 23 
     Example 4 was repeated except that 2.0 g of DL-5-hydroxytryptophane amide was used as a substrate. As a result, L-5-hydroxytryptophane was obtained at a yield of 43%. 
     EXAMPLE 24 
     Example 15 was repeated except that 2.0 g of DL-6-methoxytryptophane amide was used as a substrate. As a result, L-6-methoxytryptophane was obtained at a yield of 27%. 
     EXAMPLE 25 
     Example 1 was repeated except that Candida kurusei (IFO-0013) was used as a microorganism and DL-5-methyltryptophane amide was used as a substrate. As a result, L-5-methyltryptophane was obtained at a yield of 23%. 
     EXAMPLE 26 
     Example 4 was repeated except that 2.0 g of DL-6-chlorotryptophane amide was used as a substrate. As a result, L-6-chlorotryptophane was obtained at a yield of 34%. 
     EXAMPLE 27 
     To 500 mg of D-tryptophane amide recovered in the manner as described in Example 16, 5 ml of concentrated hydrochloric acid was added and, then, the mixture was heated at a temperature of 90° C. for 3 hours, whereby the hydrolysis reaction was effected. After the completion of the reaction, concentrated hydrochloric acid was vacuum distilled off. 5 ml of water was added to the residue and the pH thereof was adjusted to 6.0. After ice cooling, 460 mg of the precipitated D-tryptophane having [α] D   20  of +33.3° (C=0.5 H 2  O) was collected by filtration. The yield of the isolated D-tryptophane was 92%.