Evaluation of Various Factors Affecting Bioconversion of l-Tyrosine to l-DOPA by Yeast Yarrowia lipolytica-NCIM 3450 Using Response Surface Methodology (original) (raw)
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Indian Journal of Microbiology, 2013
L-DOPA (3,4-dihydroxyphenyl-L-alanine) is the most widely used drug for treatment of Parkinson's disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of L-tyrosine to L-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of L-DOPA were: pH 7.31, temperature 42.9°C, 2.31 g l-1 cell mass and 1.488 g l-1 L-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l-1. This optimization of process parameters using RSM resulted in 4.609-fold increase in the L-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R 2) was 0.9758, indicating a good agreement between the experimental and predicted values of L-DOPA production. The highest tyrosinase activity observed was 7,028 U mg-1 tyrosine. L-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce L-DOPA.
BMC Biotechnology, 2007
Background: 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 Ltyrosine to L-dopa) and L-ascorbate, which acts as a reducing agent. Results: 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 Y p/s , Q s and q s over the control were observed. Conclusion: 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.
Optimization ofl-DOPA production byBrevundimonassp. SGJ using response surface methodology
Microbial Biotechnology, 2012
L-DOPA (3,4-dihydroxyphenyl-L-alanine) is an extensively used drug for the treatment of Parkinson's disease. In the present study, optimization of nutritional parameters influencing L-DOPA production was attempted using the response surface methodology (RSM) from Brevundimonas sp. SGJ. A Plackett-Burman design was used for screening of critical components, while further optimization was carried out using the Box-Behnken design. The optimized levels of factors predicted by the model were pH 5.02, 1.549 g l-1 tryptone, 4.207 g l-1 L-tyrosine and 0.0369 g l-1 CuSO4, which resulted in highest L-DOPA yield of 3.359 g l-1. The optimization of medium using RSM resulted in a 8.355-fold increase in the yield of L-DOPA. The ANOVA showed a significant R 2 value (0.9667), model F-value (29.068) and probability (0.001), with insignificant lack of fit. The highest tyrosinase activity observed was 2471 U mg-1 at the 18th hour of the incubation period with dry cell weight of 0.711 g l-1. L-DOPA production was confirmed by HPTLC, HPLC and GC-MS analysis. Thus, Brevundimonas sp. SGJ has the potential to be a new source for the production of L-DOPA.
BMC Biotechnology, 2010
Background: 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. Results: 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. Conclusion: 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.
Biochemical Engineering Journal, 2013
Dihydroxyphenylalanine (l-DOPA) is the most potent drug used for treatment of Parkinsonism. Statistical optimization of nutritional parameters for the production of l-DOPA by Pseudomonas sp. SSA has been carried out using response surface methodology (RSM). Four most significant medium constituents identified by initial screening method of Plackett-Burman (PB) were glucose, peptone, l-tyrosine and CuSO 4. In order to investigate quantitative effects of the four variables selected from PB design on l-DOPA production, Box-Behnken design was subsequently employed for further optimization. The medium having glucose 1.69 g l −1 , peptone 1.61 g l −1 , l-tyrosine 4.11 g l −1 and CuSO 4 0.03 g l −1 was found to be optimum for maximum l-DOPA production. The optimization strategies used lead to a 6.27-fold increase in l-DOPA yield (3.251 ± 0.12 g l −1) compared to non-optimized medium (0.572 ± 0.1 g l −1). l-DOPA produced was further characterized by spectroscopic techniques, such as HPTLC, HPLC and GC-MS.
Brazilian Journal of Microbiology, 2006
Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-DOPA from L-tyrosine using chemical mutation. Different putative mutant strains of organism were tested for the production of L-DOPA in submerged fermentation. Among these putative mutant strains, mutant designated SI-12 gave maximum production of L-DOPA (300 mg L-DOPA.g-1 cells). The production of L-DOPA from different carbon source solutions (So= 30 g.l-1) by mutant culture was investigated at different nitrogen sources, initial pH and temperature values. At optimum pH (pHo= 5.0), and temperature (t=30ºC), 100% sugars were utilized for production and cell mass formation, corresponding to final L-DOPA product yield of 150 mg.g-1 substrate utilized, and maximum volumetric and specific productivities of 125 mg.l-1.h-1, and 150 mg.g-1 cells. h-1, respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is the highest reported in literature. To explain the kinetic mechanism of L-DOPA formation and thermal inactivation of tyrosinase, the thermodynamic parameters were determined with the application of Arrhenius model: activation enthalpy and entropy for product formation, in case of mutant derivative, were 40 k j/mol and 0.076 k j/mol. K for L-DOPA production and 116 k j/mol and 0.590 k j/mol. K for thermal inactivation, respectively. The respective values for product formation were lower while those for product deactivation were higher than the respective values for the parental culture. Therefore, the mutant strain was thermodynamically more resistant to thermal denaturation.
l DOPA production by immobilized tyrosinase
Applied Biochemistry and Biotechnology, 2000
The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U IMO). The optimal conditions were t=24 h, G=2% (v/v), and U C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).
SpringerPlus, 2013
L-DOPA (3,4-dihydroxyphenyl-L-alanine), a modified amino acid, is an expansively used drug for the Parkinson's disease treatment. In the present study, optimization of nutritional parameters influencing L-DOPA production was attempted using the response surface methodology (RSM) from Mucuna monosperma callus. Optimization of the four factors was carried out using the Box-Behnken design. The optimized levels of factors predicted by the model include tyrosine 0.894 g l -1 , pH 4.99, ascorbic acid 31.62 mg l -1 and copper sulphate 23.92 mg l -1 , which resulted in highest L-DOPA yield of 0.309 g l -1 . The optimization of medium using RSM resulted in a 3.45-fold increase in the yield of L-DOPA. The ANOVA analysis showed a significant R 2 value (0.9912), model F-value (112.465) and probability (0.0001), with insignificant lack of fit. Optimized medium was used in the laboratory scale column reactor for continuous production of L-DOPA. Uninterrupted flow column exhibited maximum L-DOPA production rate of 200 mg L -1 h -1 which is one of the highest values ever reported using plant as a biotransformation source. L-DOPA production was confirmed by HPTLC and HPLC analysis. This study demonstrates the synthesis of L-DOPA using Mucuna monosperma callus using a laboratory scale column reactor.
Biotechnology and Applied Biochemistry, 2005
Aspergillus oryzae mutant strain UV-7 was further improved for the production of L-dopa from L-tyrosine using chemical mutation. Different putative mutant strains of the organism were tested for the production of L-dopa in triplicate shake-flask cultures. Among these putative mutants, the strain designated SI-12 gave a maximal production of L-dopa (444 + − 14 mg of Ldopa/g of cells). The regulation of L-dopa from different carbon source solutions [initial substrate concentration (S 0 ) = 30 g/l] by the mutant culture was investigated. At an initial pH (pH 0 ) of 5.0 and a temperature (T) of 30 • C, 100 % of sugars were utilized for product and cell mass formation, corresponding to final L-dopa product yield of 189 + − 8 mg/g of substrate utilized and maximum volumetric and specific productivities of 145 + − 5 mg/h per litre and 155 + − 8 mg/h per g of cells respectively. There was up to 3-fold enhancement in product formation rate. This enhancement is, to our knowledge, the highest reported in the literature. To explain the kinetic mechanism of L-dopa formation and its thermal inactivation, the thermodynamic parameters were determined with the application of the Arrhenius model. Activation enthalpy and entropy for product formation, in the case of the mutant derivative, were 40 kJ/mol and 0.076 kJ · mol −1 · K −1 for its production and 116 kJ/mol and 0.590 kJ · mol −1 · K −1 for thermal inactivation respectively. The respective values for product formation and product de-activation were lower than the respective values for the parental culture. Therefore the mutant strain was thermodynamically more resistant to thermal denaturation during the product-formation process.