Production of 3,4-dihydroxy L-phenylalanine by a newly isolated Aspergillus niger and parameter significance analysis by Plackett-Burman design
© Ali and Haq; licensee BioMed Central Ltd. 2010
Received: 19 June 2010
Accepted: 10 December 2010
Published: 10 December 2010
The amino acid derivative 3,4-dihydroxy L-phenylalanine (L-dopa) is gaining interest as a drug of choice for Parkinson's disease. Aspergillus oryzae is commonly used for L-dopa production; however, a slower growth rate and relatively lower tyrosinase activity of mycelia have led to an increasing interest in exploiting alternative fungal cultures. In the present investigation, we report on the microbiological transformation of L-tyrosine to L-dopa accomplished by a newly isolated filamentous fungus Aspergillus niger.
The culture A. niger (isolate GCBT-8) was propagated in 500 ml Erlenmeyer flasks and the pre-grown mycelia (48 h old) were used in the reaction mixture as a source of enzyme tyrosinase. Grinded mycelia gave 1.26 fold higher L-dopa production compared to the intact at 6% glucose (pH 5.5). The rate of L-tyrosine consumption was improved from 0.198 to 0.281 mg/ml. Among the various nitrogen sources, 1.5% peptone, 1% yeast extract and 0.2% ammonium chloride were optimized. The maximal L-dopa was produced (0.365 mg/ml) at 0.3% potassium dihydrogen phosphate with L-tyrosine consumption of 0.403 mg/ml.
Over ~73% yield was achieved (degree of freedom 3) when the process parameters were identified using 2k-Plackett-Burman experimental design. The results are highly significant (p ≤ 0.05) and mark the commercial utility (LSD 0.016) of the mould culture which is perhaps the first ever report on L-dopa production from A. niger.
The 3,4-dihydroxy L-phenylalanine (L-dopa) is known to be produced from L-tyrosine by a one-step oxidation reaction under submerged batch culture . The optimisation of cultural conditions is necessary for a successful cultivation 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.) or plant sources (Agaricus and Vicia spp.). However in microorganisms, enzyme activity is generally very weak; L-tyrosine along with L-dopa is rapidly decomposed to other metabolites. Thus, stoichiometric formation of L-dopa is difficult to achieve [3, 4]. In addition, L-dopa is an unstable product in the reaction mixture and is further converted into dopaquinone and the final product melanin . Because of the higher production cost and its greater commercial value, many researchers have investigated on the alternative production of L-dopa [6, 7]. Investigations have now centred upon microbiological L-dopa production from Erwinia herbicola and Escherichia coli [8, 9]. However, the production could be expensive due to the removal of proteins and hormones which would also be produced by the microbial cells. Another alternative method is the L-dopa production from L-tyrosine with an immobilized tyrosinase. The enzyme inhibits below pH 3.5 and thus decreases L-dopa production . The optimal production was obtained when glucose was used as a carbon source [11, 12]. The nitrogen sources as well as the concentration of nitrogen containing salts, sucrose and phosphate in the culture medium were found to greatly affect the biosynthetic pathway of L-dopa .
Aspergillus oryzae has largely been exploited as an organism of choice for L-dopa production; however its use has been limited drastically due to the strong tyrosinase inhibitors produced by the pre-grown mycelia . In addition, a much slower growth rate of this fungus has urged to find a better alternative microorganism [5, 15]. Therefore, in the present study, different strains of A. niger were isolated from bread wastes. Among them isolate GCBT-8 was found to be a faster growing culture and gave the highest product yield. An increase in biomass of this mould culture was attempted to further enhance L-dopa production under submerged cultivation. As tyrosinase is an intracellular enzyme, so mould mycelia were used for the biochemical conversion of L-tyrosine to L-dopa. The 2-factorial Plackett-Burman experimental design was used to further identify the significant variables influencing L-dopa production.
Maintenance of A. niger
Various strains of A. niger were isolated from bread wastes by pour plate method . The samples were collected in sterilized polythene bags from the local market of Lahore (Pakistan). The strains were maintained on potato dextrose agar (PDA) medium, pH 5.6 and incubated at 30°C for 4-6 days until maximal sporulation. Preliminary screening of fungal isolates was accomplished using PDA medium containing 0.1% L-tyrosine as an inducer and bromocresol green dye as an indicator .
The spore suspension was prepared by adding 10 ml of sterilized distilled water aseptically to a 4-6 day old slant culture having profuse growth on its surface. An inoculum needle was used to disrupt the clumps of spores. The tube was shaken gently to form a homogeneous suspension. The spore count was made on a haemocytometer.
Propagation, Harvesting and Ultrasonication of Fungal Mycelia
The propagation of mycelia was carried out by taking 100 ml of medium containing 4 g/l glucose, 2 g/l peptone, 0.6 g/l NH4Cl, 0.6 g/l KH2PO4, 0.04 g/l MgSO4.7H2O, 2 g/l yeast extract at pH 5 in 500 ml Erlenmeyer flasks. The flasks were cotton plugged and sterilized in an autoclave at 15 lbs/in2 pressure (121°C) for 20 min. The medium was inoculated with 5 ml of spore suspension (1.45×106 CFU/ml) of A. niger. The flasks were incubated at 30°C for 48 h in a rotary shaker (350 rpm). The mycelia were harvested by filtering through a funnel and washed free of adhering medium with ice cold water (4°C). These intact mycelia were dried in filter paper folds. The grinded form of mycelia was obtained after disrupting them by an ultrasonicator for 5 min . Both the intact and grinded mycelia were stored at 4°C in a cold cabinet. All the experiments were run parallel in triplicates.
Reaction Procedure and Critical Phases
The reaction for L-dopa production from L-tyrosine was carried out in a suspension of intact fungal mycelia . The reaction mixture was prepared by adding 0.0625 g/l L-tyrosine, 1.875 g/l mycelia (intact or grinded), 0.125 g/l L-ascorbic acid in 50 mM acetate buffer (pH 3.5). Twenty five millilitre of this reaction mixture was taken in 250 ml Erlenmeyer flasks. The reactions were performed aerobically at 50°C for 60 min in a shaking water bath (240 rpm).
The sample was withdrawn and centrifuged at 9000×g for 15 min. The supernatant was used for analysis of L-dopa produced and L-tyrosine consumed in the reaction mixture following the analytical methods of Arnow .
Estimation of L-Tyrosine
One millilitre of supernatant from the reaction mixture along with 1 ml of mercuric sulphate reagent was taken in a test tube. The tube was placed in a boiling water bath for 10 min. The assay mixture was cooled and 1 ml of nitrite reagent was added. The volume was raised upto 16 ml with distilled water. The diluted mixture was analyzed (A550 nm) by a spectrophotometer and the amount of residual L-tyrosine was determined after comparing with the tyrosine-standard.
Estimation of L-Dopa
Determination of Tyrosinase Activity
The kinetic parameters for L-dopa production and L-tyrosine consumption were studied according to the procedures of Pirt . The volumetric rates for substrate utilization (Qs mg/ml/min) and product formation (Qp mg/ml/min) were determined from the maximum slopes in plots of substrate utilized and L-dopa produced each vs. the time of reaction. The product yield coefficient Yp/s was determined using the relationship i.e., Yp/s = dP/dS (mg/ml). The specific rate constants for product formation (qp mg/mg cells/min) and substrate utilization (qs mg/mg cells/min) were determined by the equations i.e., qp = μ×Yp/x and qs = μ×Ys/x, respectively.
Statistical Analysis and Application of 2k-Factorial Design
In Eq. I, Eο is the effect of first parameter under study while M+ and M- are responses of L-dopa production by the filamentous fungus. N is the total number of optimizations. In Eq. II, E is the significant parameter, β1 is the linear coefficient, β2 the quadratic coefficient and β3 is the interaction coefficient for process parameters.
Results and Discussion
Screening of Fungal Cultures for L-Dopa Production
In the present study, several strains of A. niger were isolated from bread wastes by serial dilution method. Initially fungal isolates were screened on the basis of larger reddish zones of L-tyrosine hydrolysis in the growth medium, indicated by the presence of bromocresol green dye. Among them, isolate GCBT-8 was found to be a faster growing culture and gave the highest product yield. An increase in biomass of this mould culture was attempted to further enhance L-dopa production under submerged cultivation. Initially, the culture (strain GCBT-8) was used for the propagation of mycelia in 500 ml Erlenmeyer flasks for L-dopa production. The fungus A. niger GCBT-8 is capable of producing tyrosinase with activity 43.28 U/mg in an acidic reaction mixture which transformed tyrosine and its derivatives to L-dopa as reported by Krishnaveni et al. . The fungal strain was grown in a medium containing carbon sources, nitrogen sources, minerals, and other essential nutrients. To obtain optimal yield of L-dopa, it was imperative to add L-ascorbic acid in the reaction broth to prevent melanin formation. Since tyrosinase appears to be an inducible enzyme, its activity should be optimally increased to convert more L-tyrosine into L-dopa [13, 22].
Propagation of A. niger Mycelia
Effect of Initial pH
Role of Intact and Grinded Mycelia Developed at Various Glucose Concentrations
Evaluation of Nitrogen Sources
Evaluation of Potassium Sources
Comparison of kinetic variables for propagation of A. niger GCBT-8 for L-dopa production
L-dopa production (mg/ml)
Product formation parameters
0.0029 ± 0.001bc
0.0061 ± 0.003a
0.875 ± 0.03abc
0.905 ± 0.05ab
qp (mg/mg cells/min)
0.0012 ± 0.001b
0.0024 ± 0.0003a
Substrate consumption parameters
0.0033 ± 0.001bcd
0.0067 ± 0.003ab
qs (mg/mg cells/min)
0.0002 ± 0.0001bc
0.0003 ± 0.0001ab
Application of 2k-Plackett-Burman Design
Application of Plackett-Burman design at various process parameters (designated by different captions) for L-dopa production by A. niger GCBT-8
Process parameters at 2-factorial design
Tyrosinase activity (U/mg)
L-dopa production (mg/ml)
Time course (h)A
Glucose conc. (%, w/v)C1+C2
Ammonium chloride conc. (%, w/v)D
Potassium dihydrogen phosphate conc. (%, w/v)E
Statistical analysis of 2-factorial experimental design at various significant process parameters for L-dopa production by A. niger GCBT-8
Significant process parameters
Sum mean values
Degree of freedom
Tyrosinase is an intracellular enzyme so mould mycelia of a locally isolated A. niger GCBT-8 were used for the biochemical conversion of L-tyrosine to L-dopa in an ascorbate-acidulated reaction mixture. The maximal L-dopa (0.365 mg/ml) was found at L-tyrosine consumption of 0.403 mg/ml. The process parameters particularly nature of the carbon-limiting substrate, medium composition and nutritional supplements for L-dopa production were determined using Plackett-Burman design. The correlation (0.012E+0025), A, B, C2 and D for E values depicts that the model terms were highly significant (p ≤ 0.05). However, further work on the optimization of mycelial and L-tyrosine concentration is in progress prior to scale up studies.
revolutions per minute
mg L-dopa produced/ml/min
mg L-dopa produced/mg L-tyrosine consumed)
mg L-dopa produced/mg cells used/min
mg L-tyrosine consumed/ml/min)
mg L-tyrosine consumed/mg cells used/min
This work was completed by the collaborative support of Department of Botany and Institute of Industrial Biotechnology, GC University Lahore, Pakistan. We are extremely grateful to the honorable Vice Chancellor for his tremendous contributions to promote research culture in the University.
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