High performance microbiological transformation of L-tyrosine to L-dopa by Yarrowia lipolytica NRRL-143 (original) (raw)
Related papers
Natural Products and Bioprospecting, 2014
3,4-Dihydroxy L-phenylalanine (L-DOPA) is considered a potent drug for the treatment of Parkinson disease. Physical and nutritional parameters where optimized by using Yarrowia lipolytica-NCIM 3450 to accomplished the highest production of L-DOPA. Screenings of critical components were completed by using a Plackett-Burman design, while further optimization was carried out using the Box-Behnken design. The optimized factor levels predicted by the model were pH 6.1, 1.659 g L-1 yeast extract, 1.491 g L-1 L-tyrosine and 0.0290 g L-1 CuSO 4. The predicted yield of L-DOPA with these levels was 1.319 g L-1 , while actual yield obtained was 1.273 g L-1. The statistical analysis revealed that model is significant with F value 19.55 and R 2 value 0.9514. This process resulted in a 3.594-fold increase in the yield of L-DOPA. L-DOPA was confirmed by HPTLC and HPLC analysis. Thus, Yarrowia lipolytica-NCIM 3450 has potential to be a new source for the production of L-DOPA.
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.
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.
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.
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).
Biosynthesis of l-DOPA by Aspergillus oryzae
Bioresource Technology, 2002
The present investigation deals with the biosynthesis of L -DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae. There was a marked difference between the mycelial morphology and pellet type of parental and UV-irradiated mutant culture. The mutant strain of A. oryzae UV-6 exhibited pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L -tyrosine to L -DOPA because tyrosinase is an intracellular enzyme. The mutant was found to yield 3.72 fold higher production of L -DOPA than the parental strain. The mutant strain is stable and D -glc-resistant. The comparison of kinetic parameters was also done which showed the greater ability of the mutant to yield L -DOPA (i.e., Y p=x 40:00 AE 0:01d mg/mg with parent and 182:86 AE 0:02a mg/mg in case of mutant). When cultures grown for various incubation periods, were monitored for Q p , Q s and q p , there was significant enhancement (p < 0:0025-0.005) in these variables by the mutant strain of A. oryzae UV-7 over GCB-6 on all the rates. L -DOPA (3,4-dihydroxy phenyl L -alanine) is a drug of choice in the treatment of Parkinson's disease and myocardium following neurogenic injury. Ó
Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase
PLOS ONE, 2016
Hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine (L-DOPA) by immobilized tyrosinase in the presence of ascorbic acid (AH 2), which reduces DOPA-quinone to L-DOPA, is characterized by low reaction yields that are mainly caused by the suicide inactivation of tyrosinase by L-DOPA and AH 2. The main aim of this work was to compare processes with native and immobilized tyrosinase to identify the conditions that limit suicide inactivation and produce substrate conversions to L-DOPA of above 50% using HPLC analysis. It was shown that immobilized tyrosinase does not suffer from partitioning and diffusion effects, allowing a direct comparison of the reactions performed with both forms of the enzyme. In typical processes, additional aeration was applied and boron ions to produce the L-DOPA and AH 2 complex and hydroxylamine to close the cycle of enzyme active center transformations. It was shown that the commonly used pH 9 buffer increased enzyme stability, with concomitant reduced reactivity of 76%, and that under these conditions, the maximal substrate conversion was approximately 25 (native) to 30% (immobilized enzyme). To increase reaction yield, the pH of the reaction mixture was reduced to 8 and 7, producing L-DOPA yields of approximately 95% (native enzyme) and 70% (immobilized). A threefold increase in the bound enzyme load achieved 95% conversion in two successive runs, but in the third one, tyrosinase lost its activity due to strong suicide inactivation caused by L-DOPA processing. In this case, the cost of the immobilized enzyme preparation is not overcome by its reuse over time, and native tyrosinase may be more economically feasible for a single use in L-DOPA production. The practical importance of the obtained results is that highly efficient hydroxylation of monophenols by tyrosinase can be obtained by selecting the proper reaction pH and is a compromise between complexation and enzyme reactivity.
International journal of radiation applications and instrumentation. Part A, Applied radiation and isotopes, 1990
A fast, simple and inexpensive enzymatic preparation of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) from molecular oxygen and tyrosine using mushroom tyrosinase is described. The theoretical incubation time for production of [15O]L-DOPA with maximal specific activity from [15O]O2 can be calculated to be about 3 min. In practice, using a specially-designed glass reaction chamber to facilitate the incorporation of gaseous molecular oxygen into L-DOPA with zero lag-time, a 3 min reaction with 1% oxygen in nitrogen results in the formation of approx. 3.9 mumols of L-DOPA, representing conversion of about 14% of the tyrosine substrate. Given access to a supply of [15O]O2, the method should be applicable to the preparation of [15O]L-DOPA for use as a PET tracer.