High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolyticaNRRL-143
© Ali et al; licensee BioMed Central Ltd. 2007
Received: 14 March 2007
Accepted: 16 August 2007
Published: 16 August 2007
The 3,4-dihydroxy phenyl L-alanine (L-dopa) is a drug of choice for Parkinson's disease, controlling changes in energy metabolism enzymes of the myocardium following neurogenic injury. Aspergillus oryzae is commonly used for L-dopa production; however, potential improvements in ease of handling, growth rate and environmental impact have led to an interest in exploiting alternative yeasts. The two important elements required for L-dopa production are intracellular tyrosinases (thus pre-grown yeast cells are required for the transformation of L-tyrosine to L-dopa) and L-ascorbate, which acts as a reducing agent.
Pre-grown cells of Yarrowia lipolytica NRRL-143 were used for the microbiological transformation of L-tyrosine to L-dopa. Different diatomite concentrations (0.5–3.0 mg/ml) were added to the acidic (pH 3.5) reaction mixture. Maximum L-dopa biosynthesis (2.96 mg/ml L-dopa from 2.68 mg/ml L-tyrosine) was obtained when 2.0 mg/ml diatomite was added 15 min after the start of the reaction. After optimizing reaction time (30 min), and yeast cell concentration (2.5 mg/ml), an overall 12.5 fold higher L-dopa production rate was observed when compared to the control. Significant enhancements in Yp/s, Qs and qs over the control were observed.
Diatomite (2.0 mg/ml) addition 15 min after reaction commencement improved microbiological transformation of L-tyrosine to L-dopa (3.48 mg/ml; p ≤ 0.05) by Y. lipolytica NRRL-143. A 35% higher substrate conversion rate was achieved when compared to the control.
Tyrosinases (monophenol, o-diphenol:oxygen oxidoreductase, EC 184.108.40.206) belong to a larger group of type-3 copper proteins, which include catecholoxidases and oxygen-carrier haemocyanins . Tyrosinases are involved in the melanin pathway and are responsible for the first steps of melanin synthesis from L-tyrosine, leading to the formation of L-dopaquinone and L-dopachrome . Tyrosinases catalyse the o-hydroxylation of monophenols (cresolase activity or "monophenolase") and the ensuing oxidation of molecular oxygen. Subsequently, the o-quinones undergo non-enzymatic reactions with various nucleophiles, producing intermediates . The immobilization of tyrosinases on solid supports can increase enzyme stability [15–19], protect tyrosinase from inactivation by reaction with quinones, (preserving them from proteolysis) , improve thermal stability of fungal tyrosinases , and increase activity in comparison to soluble enzymes .
Diatomite (2:1 clay mineral) is a naturally occurring, soft, chalk-like sedimentary rock that is easily crumbled into a fine, off-white powder which has K+ in the interlayer. This powder has an abrasive feeling similar to pumice and is light-weight due to its porosity. By adding diatomite into the reaction, an increased substrate uptake and enzyme production rate with concomitant L-dopa production could result. We have previously reported the effect of cresoquinone and vermiculite on the microbial transformation of L-tyrosine to L-dopa by Aspergillus oryzae [23, 24]. In the present study, different concentrations of diatomite were added into the reaction mixture to achieve a high performance transformation of L-tyrosine to L-dopa.
A. oryzae is an organism typically used for L-dopa production. The easy handling, rapid growth rate and environmentally friendly nature of alternative yeasts such as Y. lipolytica have created an interest in their use for fermentation. Because tyrosinases are intracellular enzymes, pre-grown cells harvested from fermented broth were used for the microbiological transformation of L-tyrosine to L-dopa.
Results and discussion
The consumption of L-tyrosine, however, continued to increase despite the time of diatomite addition. The tyrosinase active center is comprised of dinuclear copper, coordinated with histidine residues, chelating substances or substances that are associated with this metal (as are quinones) which are irreversible inhibitors and/or inactivators of this enzyme . The addition of diatomaceous earth may remove these inhibitors and/or inactivators by active absorption. The absorption of inhibitors increased the enzyme activity of tyrosinases, β-carboxylases and tyrosine hydroxylases which was important for the catabolism of L-tyrosine to L-dopa under controlled conditions. Our data are both substantiated  and in contrast to previous reports  in which the production of L-dopa was achieved in minimal medium without additive supplementation (pH 7.0). Previous research efforts to produce L-dopa by the addition of 0.16 μg vermiculite during the reaction obtained 0.39–0.54 mg/ml of the desired product .
Comparison of parameters for L-dopa production by Y. lipolytica NRRL-143
Max. L-dopa production (mg/ml)
Level of significance <p>*$
In the present studies, Yarrowia lipolytica strain NRRL-143 was exploited for L-dopa production. The addition of 2.0 mg/ml diatomite (2:1 clay mineral) markedly improved the microbiological transformation of L-tyrosine to L-dopa. Diatomite addition 15 min after the start of reaction produced a 35% higher substrate conversion rate compared to the control (p ≤ 0.05). A biomass concentration of 2.5 mg/ml and reaction time of 30 min were also optimized. Because production of L-dopa is a high cost, low yield process, scaled up studies are a pre-requisite for commercial exploitation.
Microorganism and growth conditions
Yarrowia lipolytica strain NRRL-143 was grown on yeast extract agar slants (pH 5.4) and stored in a cold-cabinet (Model: 154P, Sanyo, Tokyo, Japan) at 4°C. Two hundred milliliters 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) (pH 5.5) were taken into individual 1.0 L Erlenmeyer flasks. The medium was autoclaved at 15 psi (121°C) for 20 min and seeded with 1.0 ml of yeast suspension (1.25 × 106 cells/ml). The flasks were incubated in a rotary shaking incubator (200 rpm) at 30°C for 48 h. A biomass ranging from 18–20 g/l was produced while 0.25% (w/v) glucose remained intact in the broth at 48 h of cultivation. Cells were harvested by centrifugation at 16,000 rpm (15,431 × g), washed free of adhering medium with ice-cold water (4°C), dried in filter paper folds (Whatman 44, Brazil) and stored at -35°C in an ultra-low freezer (Model: UF-12, Shimadzu, Tokyo, Japan).
Biochemical reaction and critical phases
The production of L-dopa from L-tyrosine was carried out in acetate buffer (pH 3.5, 50 mM) containing (mg/ml); L-tyrosine (3.5), L-ascorbic acid (5.0) and intact cells (3.0), dispensed to a 1.25 L capacity reaction vessel (Model: 2134-nmn, Perkin Elmer, NY, USA) with a working volume of 0.75 L. Different diatomite (Sigma, St. Louis, USA) concentrations (0.5–3.0 mg/ml) were added to the reaction mixture at different time intervals (5–25 min). Reactions were carried out aerobically (1.25 l/l/min air supply, 0.5% dissolved oxygen) on a digital hot plate with magnetic stirrers (Model: G542i, Inolab, Bonn, Germany) at 50°C for different time intervals (10–60 min). The level of dissolved oxygen (DO) was measured using a Rota meter equipped with a DO-sensor (Model: RM10, Inolab, Bonn, Germany).
The mixture was withdrawn from each reaction vessel, centrifuged at 9,000 rpm (8,332 × g) for 15 min and the clear supernatant was kept in the dark at ambient temperature (~20°C).
Determination of tyrosinase activity
One enzyme unit
One unit of tyrosinase activity is equal to a ΔA265 nm of 0.001 per min at pH 6.5 at 25°C in a 3.0 ml reaction mixture containing L-catechol and L-ascorbic acid.
Determination of L-dopa production and L-tyrosine consumption
One milliliter of supernatant from the reaction mixture was added to 1.0 ml of 0.5 N HCl along with 1.0 ml of nitrite molybdate reagent (10% w/v sodium nitrite + 10% w/v sodium molybdate) (a yellow coloration appeared) followed by the addition of 1.0 ml of 1.0 N NaOH (a red coloration appeared). Total volume was brought to 5.0 ml with distilled water. Transmittance (%) was compared using a double beam UV/VIS scanning spectrophotometer (Cecil-CE 7200-series, Aquarius, London, UK) at 456 nm wavelength and the amount of L-dopa produced was determined from the standard curve.
One millilitre of the supernatant from the same reaction mixture was added to 1.0 ml of mercuric sulphate reagent (15%, w/v mercuric sulphate prepared in 5.0 N H2SO4). The test tubes were placed in a boiling water bath for 10 min and then cooled to an ambient temperature. A total of 1.0 ml of nitrite reagent (0.2% w/v sodium nitrite) was added to each tube, followed by the addition of distilled water to a final volume of 5.0 ml. Transmittance was measured by spectrophotometer (546 nm wavelength) with the amount of residual L-tyrosine determined from the tyrosine-standard curve.
Determination of protein content
Protein in the reaction broth (with and without diatomite addition) was determined using Bradford reagent  with bovine serum albumin (BSA) as a standard.
Kinetic and statistical depiction
Kinetic parameters for L-dopa production and L-tyrosine consumption were previously studied . The product yield coefficient (Yp/s) was determined using the relationship Yp/s = dP/dS, while the volumetric rate for substrate utilization (Qs) was determined from the maximum slope in a plot of substrate utilized vs. time of biomass cultivation. Specific rate constants for substrate utilization (qs) were calculated by the equation i.e., qs = μ × Ys/x. The significance of results has been presented in the form of probability, using post-hoc multiple ranges under analysis of variance .
3,4-dihydroxy phenyl L-alanine
revolutions per minute, EDTA, ethylene diamine tetra acetic acid
bovine serum albumin.
The authors gratefully acknowledge Dr. CP Kurtzman, Microbial Genomics and Bioprocessing Research Unit, Peoria, Illinois, USA for providing the culture (Y. lipolytica NRRL-143).
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