Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson’s disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Designing Silver Nanoparticles for Detecting Levodopa (3,4-Dihydroxyphenylalanine, L-Dopa) Using Surface-Enhanced Raman Scattering (SERS)

Detection of the drug Levodopa (3,4-dihydroxyphenylalanine, L-Dopa) is essential for the medical treatment of several neural disorders, including Parkinson’s disease. In this paper, we employed surface-enhanced Raman scattering (SERS) with three shapes of silver nanoparticles (nanostars, AgNS; nanospheres, AgNP; and nanoplates, AgNPL) to detect L-Dopa in the nanoparticle dispersions. The sensitivity of the L-Dopa SERS signal depended on both nanoparticle shape and L-Dopa concentration. The adsorption mechanisms of L-Dopa on the nanoparticles inferred from a detailed analysis of the Raman spectra allowed us to determine the chemical groups involved. For instance, at concentrations below/equivalent to the limit found in human plasma (between 10−7–10−8 mol/L), L-Dopa adsorbs on AgNP through its ring, while at 10−5–10−6 mol/L adsorption is driven by the amino group. At even higher concentrations, above 10−4 mol/L, L-Dopa polymerization predominates. Therefore, our results show that adsorption depends on both the type of Ag nanoparticles (shape and chemical groups surrounding the Ag surface) and the L-Dopa concentration. The overall strategy based on SERS is a step forward to the design of nanostructures to detect analytes of clinical interest with high specificity and at varied concentration ranges.

LC-MS determination of L-DOPA concentration in the leaf and flower tissues of six faba bean (Vicia faba L.) lines with common and rare flower colors

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by the loss of muscle control, which causes trembling of the limbs and head as well as impaired balance. L-DOPA (L-3,4-dihydroxy phenylalanine) is the major ingredient of several prescription drugs used to treat PD. Faba bean (Vicia faba L.) is one of the few plant species that is known to produce L-DOPA and has the potential to be developed as a functional food crop for people suffering with PD.Objective: Aimed to provide needed information for people who want to use faba bean as a natural remedy or functional food to relieve PD symptoms, this study analyzed the variation of L-DOPA concentration in the leaf and flower tissues of six faba bean lines with common and rare flower colors.Methods: Leaf and flower samples were taken from field grown plants with different flower colors, namely, pink with purple lines and black dots, pure white, brown, and crimson. Samples were freeze-dried and L-DOPA was quantified by a LC-MS system consisting of an ACQUITY UPLC in line with a Synapt G2 HDMS quadrupole time-of-flight mass spectrometer. This experiment was carried out in two consecutive years (2012 and 2013) and the plants used in the second year were grown from the seeds harvested from the plants used in the first year.Results and Discussion: Our two-year study revealed a high level of variation in L-DOPA concentration for leaf and flower tissues among the six faba bean lines studied. The average L-DOPA concentration based on dry weight (DW) in flowers ranged from 27.8 to 63.5 mg/g and 18.2 to 48.7 mg/g for leaf tissues. There was no significant correlation between L-DOPA concentrations in flowers and leaves. The L-DOPA concentration in flowers and in leaves of the same line varied but were not statistically significant between the two years. Ideally, the genotype with the highest average L-DOPA concentration in both flowers and leaves would be grown as a natural source of this medicinally important molecule. When developing faba bean as a functional food crop for PD patients, a careful selection of genotype seems necessary for exploiting the full potential of this natural remedy.Conclusion: Consumption of young pods and leaves is the most practical means for direct intake or processing of L-DOPA. Lastly, favorable environmental conditions for growth will optimize L-DOPA yield (Landry, et al., 2015). Further analysis of the genetic control of L-DOPA synthesis and metabolism will be valuable, with the possibility of developing environmentally resilient cultivars that can produce desirable amounts of L-DOPA for pharmaceutical use. Key words: Faba bean, L-DOPA, Parkinson’s disease

Determination of Picogram Levels of Levodopa in Pharmaceutical Preparations and Biofluids by Flow-Injection Chemiluminescence

An ultrasensitive method for determination of L-dopa at picogram levels by flow-injection chemiluminescence (FI-CL) as presented for the first time, based on the quenching effect of levodopa (L-dopa) on the luminol-lysozyme reaction. It was found that the decrement of CL intensity was linearly proportional to the logarithm of L-dopa concentration ranging from 3.0 to 7.0 × 103pg mL-1(R= 0.9967), with the limit of detection (LOD) of 1.0 pg mL-1(3σ). The proposed procedure was successfully applied to the determination of L-dopa in pharmaceutical preparations, human saliva, serum, and urine samples with the recoveries ranging from 96.7% to 104.3% and RSDs less than 4.0% (n= 5).

Enhancement of L-DOPA Production in Micropropagated Plants of two Different Varieties of Mucuna pruriens L., Available in India

Reports on increment in L-DOPA content in micropropagated plants, regenerated through a simple technique following shoot bud multiplication in four strains of two different varieties of Mucuna pruriens are made. Nodal segments from in vitro grown seedlings were cultured on modified MS with various concentrations and combinations of BAP, 2iP, Kn either alone or with NAA. Highest shoot regeneration from the callus was achieved in modified MS fortified with BAP at 1.33 µM level. The regenerated shoots were rooted in vitro in half-strength of liquid MS supplemented with various levels of NAA (0.54 - 10.7 mM) or IBA (0.49 - 9.85 mM). However, roots of superior quality were obtained at 5.4 mM NAA level after two weeks in culture. The regenerants were acclimatized for 2 - 3 weeks and about 85% survival rate was observed after transferring to the field. Both chromosome number stability as well as stability in nuclear DNA contents of the regenerants of all these strains was recorded with complete absence of aneuploidy. The different strains have revealed two to three-folds increase in L-DOPA contents in the cultured plants. The L-DOPA concentration in leaves of the regenerated plants varied from11.85 - 15.42 mg/g dry weight in all these accessions. However, the highest amount was observed in the wild strain of M. pruriens. This is the first report on enhancement of L-DOPA content in differentiated tissue of cultured plants of both the varieties of M. pruriens. Key words: L-DOPA, Micropropagation, Mucuna pruriens, Nuclear DNA D. O. I. 10.3329/ptcb.v21i2.10224 Plant Tissue Cult. & Biotech. 21(2): 115-125, 2011 (December)

Human hypoxic ventilatory response with blood dopamine content under intermittent hypoxic training

Adaptation to intermittent hypoxia can enhance a hypoxic ventilatory response (HVR) in healthy humans. Naturally occurring oscillations in blood dopamine (DA) level may modulate these responses. We have measured ventilatory response to hypoxia relative to blood DA concentration and its precursor DOPA before and after a 2-week course of intermittent hypoxic training (IHT). Eighteen healthy male subjects (mean 22.8 ± 2.1 years old) participated in the study. HVRs to isocapnic, progressive, hypoxic rebreathing were recorded and analyzed using piecewise linear approximation. Rebreathing lasted for 5-6 min until inspired O2 reached 8 to 7%. IHT consisted of three identical daily rebreathing sessions separated by 5-min breaks for 14 consecutive days. Before and after the 2-week course of IHT, blood was sampled from the antecubital vein to measure DA and DOPA content. The investigation associated pretraining high blood DA and DOPA values with low HVR (r = -0.66 and -0.75, respectively), elevated tidal volume (r = 0.58 and 0.37) and vital capacity (r = 0.69 and 0.58), and reduced respiratory frequency (r = -0.89 and -0.82). IHT produced no significant change in ventilatory responses to mild hypoxic challenge (PetO2 from 110 to 70-80 mmHg; 1 mmHg = 133.3 Pa) but elicited a 96% increase in ventilatory response to severe hypoxia (from 70-80 to 45 mmHg). Changes in HVRs were not accompanied by statistically significant shifts in blood DA content (24% change), although a twofold increase in DOPA concentration was observed. Individual subject's changes in DA and DOPA content were not correlated with HVR changes when these two parameters were evaluated in relation to the IHT. We hypothesize that DA flowing to the carotid body through the blood may provoke DA autoreceptor-mediated inhibition of endogenous DA synthesis-release, as shown in our baseline data.Key words: hypoxic ventilatory response, dopamine, intermittent hypoxia.

Uptake and intracellular fate of l-DOPA in a human intestinal epithelial cell line: Caco-2

The aim of the present study was to examine the kinetic characteristics of thel-3,4-dihydroxyphenylalanine (l-DOPA) transporter and the fate of newly formed dopamine in Caco-2 cells. In the presence of 50 μM benserazide (an inhibitor of aromaticl-amino acid decarboxylase), l-DOPA was rapidly accumulated in Caco-2 cells. At equilibrium (30 min of incubation) the intracellular l-DOPA concentration was 10.2 ± 0.1 μM at a medium concentration of 0.5 μM. In saturation experiments the accumulation of l-DOPA was saturable with a Michaelis-Menten constant ( K m) of 60 ± 10 μM and a maximal reaction velocity ( V max) of 6.6 ± 0.3 nmol ⋅ mg protein−1 ⋅ 6 min−1; at 4°C the amount of l-DOPA accumulated in the cells was nonsaturable. When cells were incubated with increasing concentrations of l-DOPA (10–100 μM) in the absence of benserazide, a substantial amount of thel-DOPA that was taken up was decarboxylated to dopamine, with an apparent K m of 27.2 μM. In experiments performed in cells cultured in polycarbonate filters, the accumulation of l-DOPA in the presence of benserazide was greater when the substrate was applied from the basolateral cell border than when it was applied from the apical cell border. In the absence of benserazide, l-DOPA applied from the basolateral cell border resulted in a nonlinear formation of dopamine ( K m = 43 ± 7 μM, V max = 23.7 ± 1.2 nmol ⋅ mg protein−1 ⋅ 6 min−1). The amount of dopamine leaving the cell through the apical cell border was lower than the amount that escaped through the basolateral cell border, and the process was saturable ( K m = 623 ± 238 μM, V max = 0.19 ± 0.02 nmol ⋅ mg protein−1 ⋅ 6 min−1). In conclusion, the data presented here show that Caco-2 cells are endowed with an efficient l-DOPA uptake system, and intracellularl-DOPA was found to be rapidly converted to dopamine, some of which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way to look at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

The protein oxidation product 3,4-dihydroxyphenylalanine (DOPA) mediates oxidative DNA damage

A major product of hydroxy-radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA) which has reducing properties. Protein-bound DOPA (PB-DOPA) has been shown to be a major component of the stable reducing species formed during protein oxidation under several conditions. The aim of the present work was to investigate whether DOPA, and especially PB-DOPA, can mediate oxidative damage to DNA. We chose to generate PB-DOPA using mushroom tyrosinase, which catalyses the hydroxylation of tyrosine residues in protein. This permitted us to study the reactions of PB-DOPA in the virtual absence of other protein-bound oxidation products. The formation of two oxidation products of DNA, 8-oxo-7,8-dihydro-2ʹ-deoxyguanosine (8oxodG) and 5-hydroxy-2ʹ-deoxycytidine (5OHdC), were studied with a novel HPLC using gradient elution and an electrochemical detection method, which allowed the detection of both DNA modifications in a single experiment. We found that exposure of calf thymus DNA to DOPA or PB-DOPA resulted in the formation of 8oxodG and 5OHdC, with the former predominating. The formation of these DNA oxidation products by either DOPA or PB-DOPA depended on the presence of oxygen, and also on the presence and on the concentration of transition metal ions, with copper being more effective than iron. The yields of 8oxodG and 5OHdC increased with DOPA concentration in proteins. Thus PB-DOPA was able to promote further radical-generating events, which then transferred damage to other biomolecules such as DNA.