Financial support: Principal, Government College Lahore, Pakistan.

Keywords: amino acid, Aspergillus oryzae, fermentation, L-DOPA, L-tyrosine, Parkinson's disease.

L-DOPA (3,4-dihydroxy phenyl L-alanine) is a useful drug in the treatment of Parkinson's disease and Myocardium following neurogenic injury. It occurs naturally in beans of Vicia faba and seeds of Mucana pruriens. L-DOPA is produced from L-tyrosine by one-step oxidation reaction by submerged fermentation. The optimisation of cultural conditions is necessary for the successful fermentation process. The key enzyme responsible for biosynthesis of L-DOPA is tyrosinase. Tyrosinases are widely distributed and highly purified enzymes, derived from microbial (Aspergillus, Rhizopus and Neurospora spp) and plant sources (Agaricus and Vicia spp). However, in microorganisms tyrosinase activity is generally very weak and L-tyrosine and L-DOPA are rapidly decomposed to other metabolites. Thus, stoichiometric formation of L-DOPA is difficult to achieve. The mycelial activity of Aspergillus oryzae or Aspergillus flavus catalysing L-tyrosine to L-DOPA was observed in acidic range below pH 5.0. In present study, the authors have described the increase in biomass of Aspergillus oryzae and consequently the production of L-DOPA, using shake flasks. Tyrosinase is an intracellular enzyme. So, mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA.

The mutant strain of Aspergillus oryzae UV-3, a hyper producer of L-DOPA obtained after UV-irradiation was used for mycelial mutation. Hundred ml of vogel medium containing (g/l); trisodium citrate 2.5, NH₄NO₃ 2.0, KH₂PO₄ 5.0, (NH₄)₂SO₄ 4.0, MgSO₄.7H₂O 0.2, Peptone 2.0, Yeast extract 1.0 at pH 5.5 with 15-20 glass beads (2.0mm, diameter) in 1-L cotton wool plugged conical flask was sterilized at 15 lbs/inch² (121ºC) for 15 minutes. Small amounts of conidia from the slant (3-5 days old) were aseptically transferred with the help of an inoculating needle to the flask. The flask was incubated at 30ºC in an incubator shaker at 200 rpm for 24 hours. The inoculum was kept homogeneous and optical density was maintained at 1.0 with the help of a photoelectric colorimeter, using a 530 nm filter. Five ml of the vegetative inoculum was taken in a petriplate and then UV-treatment was given from 15-25 minutes, intervals, following the method of Pontecarvo et al. 1969. The mutant cultures were incubated at 30°C for 3-4 days for sporulation. The submerged culture method (Raju et al. 1993) was employed for cultivation of the mycelium. A conidial inoculum was prepared in 10 ml of Monoxal O.T. (Dioctyl ester of sodium sulpho succinic acid). Twenty-five ml of cultivation medium containing (% w/v); glucose 2.0, polypeptone 1.0, NH₄Cl 0.3, KH₂PO₄ 0.3, MgSO₄.7H₂O 0.02, yeast extract 1.0 at pH 5.0 was used in 250 ml shake flasks. The medium was autoclaved at 15-lb/inch² pressure (121ºC) for 15-minutes and seeded with 1.0 ml conidial suspension. The flasks were then incubated at a rotary incubator shaker (200 rpm) in 30ºC for 48 hours. The mycelium was harvested by filtering through a funnel and washed free of adhering medium with ice-cold water. The mycelium was dried in filter paper folds and it was stored at 5ºC in a refrigerator.

The reaction for L-DOPA production from L-tyrosine was carried out in a suspension of intact mycelia. The mycelia were suspended in reaction mixture (Haneda et al. 1973). Fifteen ml of acetate buffer (pH 3.5, 50mM) containing (mg/ml); L-tyrosine 2.5, L-ascorbic acid 5.0 and intact mycelia 75.0 were taken in 250 ml Erlenmeyer flasks. The reaction was carried out aerobically at 50ºC for 60 minutes in a hot plate with magnetic stirrer. The sample was withdrawn, centrifuged (6,000/g) and the supernatant was kept under dark for further investigation. L-DOPA: One ml of the supernatant was taken and in it 1.0 ml of 0.5N HCl along with 1.0 ml of nitrite molybdate reagent was added. A yellow color appeared. Then 1.0 ml of 1.0N NaOH was added, which gave red coloration and the total volume was made up to 5.0 ml. The color intensity was read by a photoelectric colorimeter (Model: AE-II, ERMA, Japan) using a green wratten filter of 530 nm and the amount of L-DOPA was determined from Arnow's standard curve of L-DOPA. L-tyrosine: One ml of supernatant from the reaction mixture was taken and in it 1.0 ml of mercuric sulfate reagent was added. It was placed in boiling water bath for 10 minutes. It was cooled and 1.0 ml of nitrite reagent was added. Total volume was made up to 5.0 with distilled water. It was compared in a colorimeter and the amount of L-tyrosine was determined from Arnow's standard curve of L-tyrosine.

Mutation can raise the status of microorganisms to hyper produce the actual product required. The mutant strain of Aspergillus oryzae UV-7 showed pellet-like mycelial morphology and improved tyrosinase activity due to the formation of thymidine-thymidine base pairs in its DNA structure. The product i.e., L-DOPA is a low cost and high yield product. By optimising the effect of phosphate sources on the mould growth, this mutant could be exploited for L-DOPA production.

HANEDA, K.; WATANABE, S. and TAKEDA, P. Production of L-DOPA from L- Tyrosine by microorganisms. Journal of Fermentation Technology, March 1973, vol. 51, no. 6, p. 398-406.

PONTECARVO, J.; ROPER, J.A.; HEMMOUS, L.M. and BUFTAN, W. Basic techniques of UV-irradiation. Advanced Genetics, September 1969, vol. 5, p. 141-183.

RAJU, B.G.S.; RAO, G.H. and AYYANNA, C. Bioconversion of L-tyrosine to L-DOPA using Aspergillus oryzae. CBS Publishers, Visakhapatnam, India, November 1993, p. 106-110.

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