Inducive effect of L-methionine in transformation of L-tyrosine to L-Dopa and tyrosinase production by Streptomyces sp. VRS9

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1 Indian Journal of Biotechnology Vol 11, July 2012, pp Inducive effect of L-methionine in transformation of L-tyrosine to L-Dopa and tyrosinase production by Streptomyces sp. VRS9 Santosh Patil, Vandana Rathod* and E Ranganath Department of Microbiology, Gulbarga University, Gulbarga , India Received 16 March 2011; revised 21 July 2011; accepted; 23 September 2011 A total of 52 isolates of actinomycetes were isolated from dry land soils of Gulbarga region, Karnataka, India. The actinomycete designated as isolate VRS9 was selected for its high tyrosinase (monophenolase) activity (67 U/mL) in standard medium. The taxonomical properties of the isolate were examined according to the International Streptomyces Project (ISP) scheme and the isolate VRS9 was assigned to the Genus Streptomyces. The inducive effect of L-methionine on transformation of L-tyrosine to L-Dopa (3,4-dihydroxyphenyl-L-alanine) under submerged conditions using Streptomyces sp. VRS9 was investigated. The fermentation was carried at 35 C and ph 8.0, which were optimum physical conditions applied to test the effect of methionine on tyrosinase and L-Dopa production. Studies showed 1.15 and 0.52 folds higher production of tyrosinase and L-Dopa, respectively as compared to bioreaction conducted without addition of methionine. VRS9 gave the maximum L-Dopa production (0.90 mg/ml) and tyrosinase activity (289 U/mL) at 10 mm concentration of methionine added during the beginning of log phase. Keywords: Inducers, L-Dopa, Parkinson`s disease, Streptomyces sp., tyrosinase Introduction L-Dopa (3,4-dihydroxyphenyl-L-alanine) is a well known sympathetic stimulant. It is an amino acid and a precursor of the catecholamine neurotransmitters, dopamine, norepinephrine and epinephrine. It occurs naturally in seedlings, pods and beans of Vicia faba, and in the roots and seeds of Stizolobium hassjoo and Mucana pruriens 1. L-Dopa is a useful drug in the treatment of Parkinson s and myocardial diseases following neurogenic injury 2. It also helps in pigmentation of human skin by facilitating the activation of tyrosinase in both cytosol and melanosomes of human epidermal melanocytes. L-Dopa acts as an important modulator of renal dopamine synthesis, the deficiency of which causes diabetes. The product of L-Dopa oxidation prevents H 2 O 2 -induced oxidative damage to cellular DNA by enhancing the cellular antioxidant defense mechanisms 3. The world market for L-Dopa is about 250 tons per year 4. Several biological sources produce L-Dopa in various quantities; however, it is also synthesized by chemical methods 5. Most of the L-DOPA sold *Author for correspondence: Mobile: ; Tel: (O) Fax: drvandanarathod@rediffmail.com commercially is synthesized from vanillin and hydantoin by a chemical process that involves eight reaction steps 6. Chemical synthesis of L-Dopa is a time-consuming process, which involves several chemicals that are extremely costly and requires catalysts that are not ecofriendly 7. Conventional production of L-Dopa involves the extraction of L-Dopa from M. pruiens seeds and it is marketed as tablets under various brand names, such as, Sinemet, Atamet, Parcopa and Stalevo 3. Enzymatic bioconversion can constitute an alternative to the chemical synthesis when natural compounds are required. It has also been employed for the biosynthesis of L-Dopa. For the first time, Sih et al 8 reported the production of L-Dopa from L-tyrosine by fungi. Later, several workers explored the biological routes for L-Dopa synthesis 2,4,9. Conversion of L-tyrosine to L-Dopa is a one-step oxidation reaction, catalyzed by the enzyme tyrosinase, tyrosine hydroxylase 7 and tyrosine phenollyase 10. Tyrosinase (mono-phenol, o-diphenol: oxygen oxidoreductase EC: ) is a copper containing enzyme which catalyzes both orthohydroxylation of monophenols and oxidation of o-diphenol to o-quinones and then to complex polyphenolic heteropolymers called melanin 11. Many interesting determinants and regulations are found in the

2 321 PATIL et al: L-METHIONINE EFFECT ON PRODUCTION OF L-DOPA BY STREPTOMYCES SP. VRS9 synthesis and secretion of tyrosinase in genus Streptomyces spp 12,13. Methionine has been reported to serve as an inducer of tyrosinase production in S. glaucescens 12, S. antibioticus 13, and S. castaneoglobisporus 14. Since tyrosinase is a metallo-protein and contains copper in its active site, the presence and concentration of Cu 2+ during the tyrosinase synthesis has profound effect. A product from mel operon (responsible for the synthesis of tyrosinase) was suggested to function as a transactivator which facilitates the incorporation of copper into apotyrosinase 15. All these facts stimulated us to conduct studies on the effects of methionine on biotransformation of L-tyrosine to L-Dopa and tyrosinase production by Streptomyces sp. VRS9. Material and Methods Isolation of Actinomycetes A survey for isolating some of the untrapped wild strains of actinomycetes was carried out. Soil samples were collected from agricultural fields, graveyard and garden soils of Gulbarga region, Karnataka, India. This region comes under semi-arid tropical northern part of Karnataka, composed of Deccan trap with a general elevation of m from the mean sea level and is situated between and East longitude and and North latitude. The climate of the region is generally dry, with temperature ranging from 5 C in winter to 47 C in summer and annual rainfall about 750 mm. The soil samples were serially diluted and plated on starch casein agar and chitin agar. Cultures were obtained from selected colonies and checked for purity. The colonies of all rare actinomycetes were picked up and maintained on yeast extract malt extract agar slants at 4 C for further analysis. Screening and Fermentation Medium Peptone yeast extract iron agar (PYIA) and tyrosine agar were used to detect tyrosinase activity 16. Those isolates produced melanoid and diffused pigmentation on the above-mentioned screening medium were reinoculated into standard medium 16 to quantify the tyrosinase activity. The potential isolate was cultivated on a slat of starch yeast extract agar and incubated for 4 d at 30 C. Spore suspension was prepared from agar slants by adding 10 ml of sterile deionized water and carefully scraping the surface of the agar with an inoculation needle. The resulting suspension was adjusted approx. to spores per ml. The fermentation was carried out in enriched potato dextrose broth (EPB) of ph 7.5; potato, 200 g/l; dextrose, 20 g/l; and CuSO 4.5H 2 O, 40 µg/ml. Tyrosinase Assay The activity was determined with an aliquot of the supernatant fluid obtained from the culture broth by centrifugation at 4500 rpm/sec for 20 min. Monophenolase activity was assayed by dopachrome method 17. The standard mixture contained 0.5 ml of 2 mm L-tyrosine, 1.5 ml of 100 mm sodium phosphate buffer (ph 6.8) and the enzyme extract in total volume of 2.5 ml. The reaction took place in cuvette with a path length of 1 cm and the absorbance at 475 nm was monitored continuously for 5 min in the UV-VIS spectrophotometer (Thermo Spectronic Genesys 10 UV Spectrophotometer) at 30ºC by the development of reddish product. One cresolase unit is defined as the amount of enzyme required to oxidize 1 µmole of L-tyrosine per minute under above said conditions. To check the o-diphenolase activity of tyrosinase, 0.5 ml of 10 mm L-Dopa dissolved in 0.1 M sodium phosphate buffer (ph 6.2) was supplemented with 5 µm copper sulphate as substrate and 0.5 ml of crude enzyme extract, and the total volume was brought to 2.5 ml using above-said buffer. The absorbance was measured at 475 nm and monitored continuously for 5 min for determination of dopachrome formed within the reaction mixture. One unit of o-diphenolase activity is defined as amount of enzyme required to oxidize 1 µm of L-Dopa per min under the above conditions. Both the monophenolase and o-diphenolase activities were calculated using the molar extinction coefficient of dopachrome (3600 M -1 cm -1 ) 17. Generic Identification of Fermenting Organism Preliminary identification of the potential isolate among 52 screened isolates was done according to traditional morphological criteria (including characteristics of colonies on the plate, morphology of substrate and aerial hyphae, and pigment production) on various media as suggested by International Streptomyces Project (ISP), i.e., ISP2, ISP3, ISP4, ISP5, ISP7, Starch casein agar, Czapeks agar, Glucose asparagine agar and Nutrient agar 18. The ultra structure of mycelium and arrangement of spores and spore surface ornamentation were examined under light microscopy (Olympus) and scanning electron microscopy 19. Cell wall analysis of DAP

3 INDIAN J BIOTECHNOL, JULY (diaminopemilic acid) isomers in the cell wall composition was determined by thin layer chromatography 20. Fermentation and Reaction Procedure A submerged culture method in 250 ml Erlenmeyer flasks was employed with fermentation medium seeded with 1.0 ml of spore suspension of isolate VRS9. The flasks were incubated on a rotary incubator shaker (180 rpm) at 35ºC. At regular intervals, the contents of individual flasks were filtered under the laminar flow through Whatman filter paper no. 44 and centrifuged at 4500 rpm for 20 min. The obtained supernatant was used as crude enzyme source for biotransformation of L-tyrosine to L-Dopa. The reaction mixture containing L-tyrosine 2.5 mg/ml, L-ascorbic acid 5 mg/ml and 100 ml of supernatant (crude enzyme source) were taken in 250 ml Erlenmeyer flasks. The reaction was carried out aerobically at 35ºC for 25 min with magnetic stirrer. The samples were drawn, and kept under dark for further investigation. The specificity of tyrosinase induction was examined using various amino acids, including methionine and its analogues (ethionine & DLnorleucine). The profound effect of methionine on tyrosinase production initiated further work. The time of addition of methionine was investigated, using 4 different 250 ml sterile conical flasks supplemented with 100 ml of fermentation medium and 1 ml of spore suspension of the isolate VRS9. A concentration of 1 mm methionine was added in three conical flasks at different phases of growth, i.e., during the beginning of log phase (after 6 h of incubation), at mid log phase (after 12 h of incubation) and at end of log phase (after 24 h of incubation). One conical flask was kept devoid of methionine in the medium, which served as control. The effect of incubation period with methionine in the medium on tyrosinase and L-Dopa production was recorded at every 12 h of interval. The effect of methionine at different concentrations on biotransformation of L-tyrosine to L-Dopa and tyrosinase production was also evaluated. The time of addition of L-methionine along with varying concentrations of methionine (1, 10 & 20 mm) were investigated and evaluated. Estimation of L-Dopa L-Dopa production was determined as described by Arnow et al 21. The supernatant (1 ml) from the reaction mixture was added to 1.0 ml of 0.5 N HCL along with 1.0 ml of sodium nitrite molybdate reagent (10%, w/v sodium nitrite+10%, w/v sodium molybdate; a yellow colouration appeared), followed by the addition of 1.0 ml of 1.0 N NaOH (a red colouration appeared). Then total volume was brought to 5.0 ml with distilled water. The absorbance was recorded at 456 nm and the amount of L-Dopa produced was determined from the standard curve. Results and Discussion Isolation and Screening of Tyrosinase Producing Actinomycetes Among the 52 isolates, 9 have produced melanoid and/or diffused pigments when cultivated on PYIA and tyrosine agar, respectively, confirming that they are tyrosinase producer. The 9 positive isolates were inoculated into standard medium to quantify the amount of tyrosinase production. The isolate designated as VRS9 produced both melanin and diffused melanin on PYIA and tyrosine agar, respectively, with maximum tyrosinase activity (67 U/mL; Table 1) and, therefore, selected in further studies. Table 1 Tyrosinase activity and chromogenicity of positive isolates of Actinomycetes No. Isolates Sources of soil sampling Monophenolase activity (U/mL) Diphenolase activity (U/mL) Peptone yeast extract iron agar Tyrosine agar 1 VRS9 Soil near decaying barks of coconut tree KS32 Agricultural field (black soil) KS46 Graveyard soil US3 Agricultural field (red soil) KL50 Garden soil US7 Sediment soil KS46 Graveyard soil US23 Agricultural field (red soil) US11 Compost

4 323 PATIL et al: L-METHIONINE EFFECT ON PRODUCTION OF L-DOPA BY STREPTOMYCES SP. VRS9 Generic Identification of Isolate VRS9 Isolate VRS9 was Gram positive and showed good growth on several media, including ISP2, ISP3, ISP4, ISP5, ISP7, Starch casein agar, Czapeks agar, Glucose aspargine agar and Nutrient agar. Varying colours (brown, maroon, pink, red, white & yellow) of substrate and aerial mycelium were observed on these media. Diffusible pigments were produced on ISP7 medium (tyrosine agar). The cell wall peptidoglycan of isolate VRS9 contained L-diaminopemilic acid (L-DAP) and glycine, indicating that isolate VRS9 is a chemotype, cell wall type I 20. Comparing with the standard DAP isomers, ninhydrin-stained band appeared at the same position as a standard L-DAP on TLC plate (Fig. 1). The scanning electron microscopic observation showed that the mycelia were well branched and spores usually included in the chains of flexuous. Ornamentation of spores was confirmed as warty (Fig. 2). Thus, the identification of actinomycete isolate VRS9 was performed based on the morphological, physiological and biochemical characteristics and it was designated as Streptomyces sp. VRS9. leucine, tyrosine and DL-norleucine (Fig. 3). However, negligible or no increment in enzyme production was detected with other amino acids, such as, phenyl alanine, alanine and glutamic acid. In the present study, the increase in tyrosinase activity by Streptomyces sp. VRS9 with methionine, ethionine, leucine and norleucine were 1.04, 0.66, 0.65 and 0.5-fold, respectively. Ikeda et al 14 reported the enhancement of tyrosinase activity in S. castaneoglobisporus by leucine, phenyl alanine and tryptophan, while tyrosine and valine repressed the enzyme activity. However, in the present study, tyrosine was found to be a weak inducer by producing Inducive Effect of Amino Acids Induction of tyrosinase synthesis was the highest (274.2 U/mL) when fermentation medium was supplemented with L-methionine, which produced 0.86 mg/ml of L-Dopa, followed by ethionine, Fig. 2 Electron microscopy of isolate VRS9 Fig. 1 TLC illustrating cell wall amino acid of isolate VRS9 (M: Marker; 1: S. aureofaciencs (MTCC 325); & 2: Isolate VRS9) Fig. 3 Inducive effect of methionine and their analogues

5 INDIAN J BIOTECHNOL, JULY U/mL of tyrosinase activity and 0.49 mg/ml of L-Dopa. The results regarding methionine as an effective inducer are in agreement with the studies of Katz & Betancourt 21 and Ikeda et al 14. Geistlich et al 22 have proposed that a putative protein represses the tyrosinase expression in the absence of inducer and that repression can be removed by the presence of inducer or by deleting the repressor-binding site in mel operon. Initially, it is believed that methionine may serve as a regulatory signal at the level of transcription in the synthesis of inducible enzymes in Streptomyces spp. and the organization of the mel operon and the regulation of its expression in different species of Streptomyces are the same But Chen et al 15 in their study found that Cu 2+ induces tyrosinase formation in S. michiganensis and that the system is not regulated by methionine, indicating that there are different signals that control tyrosinase formation. Influence of Time and Concentration of Methionine The time course of methionine addition was found crucial on the tyrosinase activity and L-Dopa production (Figs 4 & 5). The tyrosinase induction and production of L-Dopa were significantly higher when methionine was added at 6 h (beginning of log phase) as compared to other periods of addition (mid log and log phase; 12 & 24 h). Further, the cultivation time of 36 h after addition of methionine was found optimum, producing 287 U/mL of tyrosinase activity and 0.91 mg/ml of L-Dopa, which latter gradually declined to 59 U/mL at 72 h in case of tyrosinase. This may be due to proteolytic activity of Streptomyces sp. VRS9 during fermentation and in bioreaction process. Kartz and Betancourt 13 have shown that de-nova synthesis of tyrosinase with S. antibioticus occurred as a function of time and methionine concentration. They hypothesized that methionine and some closely related analogues were specific in tyrosinase induction, while ethionine or norleucine would affect indirectly by utilization of these compounds in metabolism (protein synthesis) and endogenous methionine remained in reserve, later permitting its use for induction. Tyrosinase and L-Dopa were found gradually increased with the increase in methionine concentrations (1, 10 & 20 mm) in fermentation with Streptomyces sp. VRS9 (Fig. 6). The maximum production of L-Dopa was 0.91 mg/ml with 20 mm methionine added during the beginning of log phase, while it was 0.90 mg/ml with 10 mm of methionine with the same period of addition and incubation. Since there was no appreciable difference in L-Dopa production with both the concentrations, 10 mm concentration of methionine would make the production economical. Fig. 4 Effect of L-methionine (added at different time courses) on production of tyrosinase by Streptomyces sp. VRS9 Fig. 5 Influence of time of methionine addition on tyrosinase activity and L-Dopa production

6 325 PATIL et al: L-METHIONINE EFFECT ON PRODUCTION OF L-DOPA BY STREPTOMYCES SP. VRS9 Fig. 6 Influence of methionine concentration on tyrosinase and L-Dopa production Conclusion The studies with Streptomyces spp. with regards to L-Dopa and tyrosinase production are limited. The present work on the production of tyrosinase and L- Dopa by Streptomyces sp. VRS9 has used EPB medium. The medium used is an economical, low cost semi-synthetic medium, which provides natural inducers for tyrosinase and L-Dopa as potato contains L-Dopa, ρ-cresol, catechol and strong reducing agents, such as, ascorbic acid. Present results reveal 1.15 and 0.52-fold higher production of tyrosinase and L-Dopa, respectively, as compared to bioreaction conducted without addition of methionine. Thus, future work can focus on solid state fermentation using agro-based wastes rich in phenolic compounds for better yield of tyrosinase and L-Dopa. Genetic engineering approaches can also be adopted to achieve enhanced and stable tyrosinase production. Tyrosinase and L-Dopa have commercial applications, especially in pharmaceutical, food and cosmetic industry, so further research in this direction will be encouraging. Acknowledgement Authors are thankful to Department of Microbiology, Gulbarga University, Gulbarga, for providing the facilities to carry out the present investigation. References 1 Fling G & Paul J, Radiation induced production of 3,4-dihydroxyphenyl L-alanine by Aspergillus oryzae, J Anal Chem, 67 (2001) Raju B G S, Rao G H & Ayyanna C, Bioconversion of L-tyrosine to L-DOPA using Aspergillus oryzae, (CBS Publishers, Visakhapatnam, India) 1993, Ali S & Haq I, Innovative effect of illite on improved microbiological conversion of L-tyrosine to 3,4dihydroxy phenyl L-alanine (L-DOPA) by Aspergillus oryzae ME2 under acidic reaction conditions, Curr Microbiol, 53 (2006) Koyanagi T, Katayama T, Suzuki H, Nakazawa H, Yokozeki K et al, Effective production of 3,4-dihydroxyphenyl L-alanine (L-Dopa) with Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR, J Biotechnol, 115 (2005) Valdes R H, Puzer L, Gomes M, Marques C E S J, Aranda D A G et al, Production of L-DOPA under heterogeneous asymmetric catalysis, Catal Commun, 5 (2004) Reinhold D F, Utne T & Abramson N L, Process for L-dopa, U S Pat , 29 December, Lee S G, Hong S P & Sung M H, Development of an enzymatic system for the production of dopamine from catechol, pyruvate and ammonia, Enzyme Microb Technol, 25 (1999) Sih C J, Foss P, Rosazza J & Lembagar M, Microbial synthesis of L-3,4-dihydroxy phenylalanine, Am Chem Soc J, 91 (1969) Krishnaveni R, Rathod V, Thakur M S & Neelagund Y F, Transformation of L-tyrosine to L-Dopa by a novel fungus, Acremonium rutilum, under submerged fermentation, Curr Microbiol, 58 (2008) Para G M & Baratti J C, Effect of culture conditions on the production of tyrosine phenol-lyase by Erwinia herbicola, Appl Environ Microbiol, 48 (1984) Haghbeen K &Tan E W, Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates, Anal Biochem, 312 (2003) Crameri R, Ettlinger L, Hutter R, Lerch K, Suter M A et al, Secretion of tyrosinase in Streptomyces glaucescens, J Gen Microbiol, 128 (1982) Katz E & Betancourt A M, Induction of tyrosinase with L-methionine in Streptomyces antibioticus, Can J Microbiol, 34 (1988) Ikeda K, Tsutomu M & Masanori S, Effect of methionine and Cu 2+ on the expression of tyrosinase in Streptomyces castaneoglobisporus, J Biochem, 120 (1996) Chen LY, Leu W M, Wang K T & Wu Lee Y H, Copper transfer and activation of the Streptomyces apotyrosinase are mediated through a complex formation between apotyrosinase and its transactivator MelC1, J Biol Chem, 267 (1992) Aria T & Mikami Y, Chromogenicity of Streptomyces, Appl Microbiol, 23 (1972) Fling M, Horowitz N H & Heinemann S F, The isolation and properties of crystalline tyrosinase from Neurospora, J Biol Chem, 238 (1963) Goodfellow M & Cross T, Classification, in Biology of the actinomycetes, edited by M Goodfellow, M Mordarski & S T Williams, (Academic Press, London) 1984, Tresner H D, Davies M C & Backus E J, Electron microscopy of Streptomycetes spore morphology and its role in species differentiation, J Bacteriol, 81 (1961) Lechevalier M P & Lechevalier H A, The chemotaxonomy of actinomycetes, in Actinomycete taxonomy, spl publ 6 (Society for Industrial Microbiology, Arlington, VA, USA) 1980, Arnow L E, Colorimetric determination of the components of L-3,4-dihydroxyphenylalanine-tyrosine mixture, J Biochem, 118 (1937) Geistlich M, Irnigers S & Hutter R, Localization and functional analysis of the regulated promoter from the Streptomyces glaucescens me1 operon, Mol Microbiol, 3 (1989)