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The mixture was withdrawn from each reaction vessel, centrifuged at 9,000 rpm (8,332 × g) for 15 min and the clear supernatant was kept in the dark at ambient temperature (~20°C).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
Tyrosinase activity was determined following a previously described method [29]. Briefly, potassium phosphate buffer (2.60 ml, 50 mM), 0.10 ml L-catechol, 0.10 ml L-ascorbic acid and 0.10 ml EDTA were mixed by inversion and equilibrated to 25°C. The ΔA265 nm was monitored until constant, followed by the addition of 100 μl of reaction broth. The decrease in ΔA265 nm was recorded for approximately 5 min. The ΔA265 nm was obtained using the maximum linear rate for both the test and control. Enzyme activity was determined with the following formula,
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
L-dopa production and L-tyrosine consumption were determined following procedures previously described [10,30].
a) L-dopa
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.
b) L-tyrosine
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 [31] 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 [32]. 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 [33].
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