Production of 3, 4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus õ

Transcription

1 Agr. Biol. Chem., 37 (9), 2121 `2126, 1973 Production of 3, 4-Dihydroxyphenyl-L-alanine (L-DOPA) and Its Derivatives by Vibrio tyrosinaticus õ Hajime YOSHIDA, Yoshitake TANAKA and Kiyoshi NAKAYAMA Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd., Machida-shi, Tokyo, Japan Received April 10, 1973 Six strains of bacteria belonging to Vibrio and Pseudomonas were selected as good pro ducers of L-DOPA from L-tyrosine out of various bacteria. The condition for the forma tion of L-DOPA by Vibrio tyrosinaticus ATCC was examined and the following results were obtained. (1) Intermittent addition of L-tyrosine in small portions gave higher titer of L-DOPA than single addition of L-tyrosine. (2) Higher amount of L-DOPA was produced in stationary phase of growth than in logarithmic phase. (3) Addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, araboascorbic acid, hydrazine, citric acid and 5- ketofructose increased the amount of L-DOPA formed. (4) L-Tyrosine derivatives such as N-acetyl-L-tyrosine amide, N-acetyl-L-tyrosine, L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to the corresponding L-DOPA derivatives. In the selected condition about 4mg/ml of L-DOPA was produced from 4.3mg/ml of L-tyrosine. The development of the economical process for L-DOPA production was eagerly desired since the observation that L-DOPA remarkably relieves, or reduces, the symptoms of Parkin sonism when administered in relatively high doses, on an average of 3 `5g per day.1) Many groups of workers endeavoured to develop the economical process for the pro duction of L-DOPA, and many reports have already appeared. L-Tyrosine,2) 3, 4-dihydro xyphenyl pyruvate,3) pyrocatecohol4 `6) were used as the substrate in the fermentative pro cesses. Sih et al.7) have used N-substituted tyrosines as the substrates to block the further transformation of L-DOPA produced by some fungi. Added advantages are their increased bacteria were selected as good producers of L-DOPA from L-tyrosine out of various bac teria. The cultural conditions for the produc tion of L-DOPA and its derivatives by one of these bacteria, Vibrio tyrosinaticus were in vestigated in detail. MATERIALS AND METHODS Microorganisms. Various stock cultures of bacteria in our laboratories were screened for the ability to convert L-tyrosine into L-DOPA (Table I). One hundred and thirty seven cultures are the members of TABLE I. MICROORGANISMS TESTED FOR L-DOPA PRODUCTION solubility and their inertness to the action of racemases. Recently the authors' group has succeeded in developing a industrial fermentative process for L-tyrosine.8) Subsequently, interest was aroused in finding a process for the prepara tion of L-DOPA from L-tyrosine. In the present investigation, six cultures of õ Production of 3, 4-Dihydroxyphenyl-L-alanine (L-DOPA) by Fermentative Method. Part I.

2 2122 H. YOSHIDA, Y. TANAKA and K. NAKAYAMA TABLE IL COMPOSITION OF MEDIA to the aim of each experiment, an typical process is shown in Fig. 1. Namely, 10ml of a seed medium in a large test tube (25mm ~190mm) were inoculated with one loopful of an organism grown on a bouillon agar slant, and incubated on a test tube shaker. After incubation for 24hr at 30 Ž, 1ml of this seed culture was transferred into a 250ml Erlenmeyer flask contain ing 20ml of a fermentative medium. The flask was incubated at 26 Ž on a rotary shaker. After 48hr the reaction of the culture broth was adjusted to ph 5.5 with 1 N-HCl, and the culture broth was added with L-ascorbic acid dissolved in water. The flask was in cubated on a rotary shaker for 2hr at 26 Ž. At this time the substrate (L-tyrosine) and L-ascorbic acid were added to the flask and the cultivation was continued with repeated addition of the substrate and L-ascorbic acid at regular intervals. To solve the problem of solubility of L-tyrosine and to keep the ph-value of the a) 88mg Na2B4O7 E10H2O, 37mg (NH4)6Mo7O27 E 4H2O, 8.8mg ZnSO4 E4H2O, 270mg CuSO4 E 5H2O, 7.2mg MnCl2 E4H2O and 970mg FeCl3 E 6H2O, in a liter of distilled water. broth to near 5.5, equal amounts of acidic and basic solution were added. After the final addition of L- tyrosine, the incubation was continued for 15hr at 26 Ž. Growth experiment. Ten milliliters of a medium in a large test tube were inoculated with one drop of the cell suspension prepared from a bouillon agar slant (finally, ca. 107 `108cells/ml), and incubated on a large test tube shaker at 30 Ž for 72 `96hr. Growth was estimated turbidimetrically at 660mƒÊ, using Hitachi spectrophotometer "Model 125." Analysis. L-DOPA and L-tyrosine in culture broth was estimated colorimetrically by use of ninhydrin after paper-chromatographical separation. The paper - chromatography was performed on "Toyo Roshi No. 50" paper by the use of the following solvent system; n-butanol-acetic acid-water (4:1:2). The amount of L-DOPA was estimated also by Arnow's method,9) after appropriate dilution of culture broth. L-DOPA derivatives were identified from the identical Rf values with the authentic samples in the. paper-chromatogram located with ninhydrin or Ar now's reagent. FIG. 1. Test for L-DOPA Production. RESULTS 8 genera and 10 cultures were unidentified ones. One of the selected cultures, V. tyrosinaticus ATCC was used to investigate of the cultural conditions for L-DOPA production. Culture media. Seed medium, fermentative medium and minimal medium, the compositions of which were shown in Table II, were used. Cultural conditions. Though the cultural condi tion was partly modified in some experiments according I. Screening for L-DOPA producer One hundred and forty-seven strains of vari ous bacteria listed in Table I were screened for the ability to convert L-tyrosine into L-DOPA. Good L-DOPA producers were found in the genera, Vibrio and Pseudomonas as shown in Table III. The L-DOPA production by one of them, V tyrosinaticus was studied in the following experiments.

3 Production of L-DOPA by Vibrio tyrosinaticus 2123 TABLE III. MICROORGANISMS SELECTED AS GOOD L-DOPA PRODUCER a) Dry cell weight.b ) Ninhydrin method.c) Arnow's method. L-Tyrosine was added intermittently at a final concentration of 4.3mg/ml. TABLE IV. NUTRITIONAL REQUIREMENT OF V. tyrosinaticus (1) TABLE V. NUTRITIONAL REQUIREMENT OF V. tyrosinaticus (2) a) Dry cell weight. II. Nutritional requirement of V. tyrosinaticus V. tyrosinaticus ATCC was isolated from sewage water by Beijerinck10) in 1911 and had been called as Microspira tyrosinatica. Later, Stanier11) renamed it as Vibrio beijerinckii and now it has been revised as Vibrio tyrosi naticus.12) The production of L-DOPA by V. tyrosinaticus ATCC has already been described in 1962 by Larway and Evans.13) They grew it in tyrosine-salt medium. The growth of the bacterium in our minimal medium was rather poor. Consequently, some improvement of the growth was attempt ed. As shown in Table IV, the addition of amino acids mixture containing 21 amino acids each at the concentration of 1mg/ml stimulated the growth. The addition of vitamins mixture and bases mixture was not effective on it. Consequently, the effect of the addition of particular amino acid on the growth was examined. The growth was stimulated by the a) Dry cell weight. addition of L-asparatic acid, L-lysine, L-phenylalanine, L-tyrosine, L-histidine, L-glutamic acid, or L-asparagine, and inhibited by L- arginine, L-tryptophan, L-threonine or L- homoserine (Table V). III. Effect of the amount of substrate and the method of its addition on the production of L-DOPA The L-DOPA titer reached to the level of 2.2mg/ml at 15hr after addition of 4.3mg of L-tyrosine per ml all at one time. The yield of L-DOPA was improved when L- tyrosine was added intermittently in small

4 a 2124 H. YOSHIDA, Y. TANAKA and K. NAKAYAMA TABLE VI. EFFECT OF THE AMOUNT OF SUBSTRATE AND THE METHOD OF ITS ADDITION ON THE PRODUCTION OF L-DOPA ) Interval, 1hr. b) Reaction time, starting from initial addition of L-tyrosine. FIG. 2. Effect of Culture Age on L-DOPA Produc tion. a) Dry cell weight. \ : L-Tyrosine was added at a concentration of 4.3mg/ml. œ \ œ : L-Tyrosine was added at a concentration of 8.6mg/ml. \ : Growth. FIG. 3. Effect of Reaction Time on L-DOPA Produc tion. L-Tyrosine was added at a concentration of 4.3mg/ml to the flask by the method described in the text. a) Reaction time, starting from the final addition of L-tyrosine. \ : L-Tyrosine was added intermittently 4 times. œ \ œ : L-Tyrosine was added intermittently 2 times. portions during the reaction time. When L-tyrosine, 4.3mg/ml in total, was added in 5 times, L-DOPA titer reached to the level of 3.7mg/ml at 20hr after the initial addition. The conversion rate was 79% (Table VI). The increase of the total amount of L-tyrosine added from 4.3mg/ml to 8.6mg/ml decreased the L-DOPA titer and the conversion ratio (Table VI). IV Tune of tyrosine addition As shown in Fig. 2, the growth of the bac terium reached to the maximum level in 24hr. On the other hand, the activity of the cells to transform L-tyrosine into L-DOPA reached its maximum level at 48 `72hr. Thus the pre ferential synthesis of the relevant enzyme was indicated. V. Effect of reaction time on L-DOPA forma tion The amount of L-DOPA reached to the almost maximum level in 15hr after the final addition of L-tyrosine (Fig. 3). It was noted in both methods of L-tyrosine addition, i.e. addition in 4 times and addition in 2 times. VI. Effect of various antioxidants on L-DOPA production The effect of L-ascorbic acid to improve the L-DOPA yield by preventing its further oxi dation is well known.

5 Production of L-DOPA by Vibrio tyrosinaticus 2125 TABLE VII. EFFECT OF ANTIOXIDANTS ON L-DOPA PRODUCTION TABLE VIII. SUBSTRATE SPECIFICITY a) Each substrate was added intermittently at a final concentration of 4mg/ml. L-tyrosine amide, L-tyrosine methyl ester and L-tyrosine benzyl ester were converted to N- acetyl-l-dopa, N-acetyl-L-DOPA amide, L- DOPA amide, L-DOPA methyl ester and L- DOPA benzyl ester at a ratio of 21 `74%. The effect of various antioxidants, chelat ing agents and reductants on the production of L-DOPA by V. tyrosinaticus was examined. Each compound to be tested was added into the broth in place of L-ascorbic acid in the fermentative process, namely at the time of the adjustment of the ph of the broth and the time of L-tyrosine addition. The yield of L-DOPA increased 1.4 `3.8 times the yield without addition by the addition of some compounds as shown in Table VII. Ara boascorbic acid was as effective as L-ascorbic acid. Citric acid, 5-ketofructose and notably hydrazine were significantly effective. Many reductants containing sulfur such as dithio threitol inhibited L-DOPA production. VII. Conversion of L-tyrosine derivatives to L-DOPA derivatives Conversion of L-tyrosine derivatives or isomers of L-tyrosine, N-acetyl-L-tyrosine, N- acetyl-l-tyrosine amide, L-tyrosine methyl ester, L-tyrosine benzyl ester, L-tyrosine amide, D-tyrosine and m-tyrosine, into the correspond ing L-DOPA derivatives or isomers of L-DOPA by V. tyrosinaticus were examined. Each substrate added at a concentration of 4mg/ml. N-Acetyl-L-tyrosine, N-acetyl-L-tyrosine amide, However, the conversion of D-tyrosine and m- tyrosine hardly occured (Table VIII). DISCUSSION The addition of antioxidant, chelating agent or reductant such as L-ascorbic acid, citric acid, araboascorbic acid, hydrazine or 5- ketofructose improved the L-DOPA production by V. tyrosinaticus (Table VII). The preven tion of DOPA quinone formation from L- DOPA by these compounds may be men tioned as one mechanism of this effect. The yield of L-DOPA was improved when L-tyrosine was added intermittently in small portions during the reaction time (Table VI). Two possible explanations may be offered for this finding. First, it may be that since L-tyro sine crystallizes in the culture broth, it hardly make contact with the cells under the condition. The second possibility would be that large amount of L-tyrosine causes the substrate in hibition against the L-DOPA forming enzyme. As the enzymes catalyzing hydroxylation of L-tyrosine, tyrosinase14) and L-tyrosine hy droxylase15) have been known. Tyrosinase catalyzes the hydroxylation of D-tyrosine as well as L-tyrosine and L-tyrosine hydroxylase

6 2126 H. YOSHIDA, Y. TANAKA and K. NAKAYAMA from Bacillus16) catalyzes the hydroxylation of m-tyrosine as well as L-tyrosine. In the pre sent investigation it was found that both D- tyrosine, m-tyrosine were hardly utilized as the substrate of L-DOPA formation by V. tyro sinaticus. It is not clear whether the enzyme catalyzing hydroxylation of L-tyrosine in V. tyrosinaticus is tyrosinase or L-tyrosine hy droxylase from the results of the present in vestigation. As shown in Table VIII, various L-tyrosine derivatives were also converted into the cor responding L-DOPA derivatives with sub stantial conversion ratio. Some of these derivatives may be used as a substrate without the problem of solubility. Some L-DOPA derivatives thus obtained may have potential to be tested for the treatment of Parkinsonism. REFERENCES 1) G. C. Cotzias, P. S. Papavasiliou and R. Gellene, New Engl. J. Med., 280, 337 (1969). 2) K. Haneda, S. Watanabe and I. Tanaka, Amino acids and Nucleic acids, 25, 113 (1972). 3) T. Nagasaki and M. Sugita, Abstracts of Papers, Meeting of Nippon Nogei Kagaku Kai, Sendai, Japan, April, 1972, p ) H. Enei, H. Nakazawa, H. Matsui, S. Okumura and H. Yamada, Abstracts of Papers, Meeting of Amino acids and Nucleic acids Symposium, Osaka, November, 1971, p ) H. Enei, H. Matsui and S. Okumura, Biochem. Biophys. Res. Commum., 43, 1345 (1971). 6) H. Enei, H. Nakazawa, H. Matsui and S. Okumu ra, FEBS Letters, 21, 39, (1972). 7) C. J. Sih, P. Foss, J. Rosazza and M. Lemberger, J. Amer. Chem. Soc., 91, 6204 (1969). 8) H. Hagino and K. Nakayama, Nippon Nogei kagaku Kaishi, 46, N 107, (1972). 9) L. E. Arnow, J. Biol. Chem., 118, 531 (1971). 10) M. S. Beijerinck, Proc. Acad. Sci. Amist., 13, 1066 (1911). 11) R. Y. Stanier, J. Bacteriol., 42, 527 (1941). 12) Intern. J. Syst. Bacteriol., 16, 126 (1966). 13) P. Larway and W. C. Evans, Biochem. J., 85, 22 (1962). 14) S. H. Pomerantz, J. Biol. Chem., 238, 2351 (1963). 15) T. Nagatsu, M. Levitt and S. Udenfriend, ibid., 239, 2910 (1964). 16) J. N. Aronson and R. S. Vickers, Biochem. Bio phys. Acta, 110, 624 (1965).