Effective L-Tyrosine Hydroxylation by Native and Immobilized Tyrosinase (original) (raw)
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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).
Tyrosinase (EC 1.14.18.1) from edible mushroom was immobilized onto agar particles activated by various concentrations of sodium periodate solution. It was found that agar activated with 200 and 300 mM periodate solution could adsorb the highest amount of tyrosinase. The progress of the reactions of two forms of tyrosinases (soluble tyrosinase and tyrosinase covalently bound to activated agar particles) were linear up to 50 and 120 min, respectively. The Michaelis-Menten constant (K m) values of free and immobilized tyrosinases were determined as 0.8 and 0.9 mM, respectively by Lineweaver-Burk plots. The optimum temperature of the immobilized tyrosinase increased from 25 to 35°C while the optimum pH of the immobilized form remained unchanged relative to free tyrosinase. The immobilized tyrosinase on agar was recycled 6 times maintaining 50% of its original activity at the end of the last cycle.
Journal of Biotechnology, 2007
Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, d-sorbitol, d-manitol, 1,2-O-isopropylidene-␣-d-glucofuranose, d-glucuronic acid, d-gulonic acid, sucrose, d-glucosone, d-arabinose, d-fructose, d-glucose, ethyl-d-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-d-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-␣-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and dl-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K app m were lower than these obtained for tyrosinase in solution while, when dopamine and dl-dopa were used, the K app m were higher. The order of the substrates as regards their ionizable groups, dl-dopa (two ionizable groups) > dopamine (one ionizable group) > 4-tert-butylcatechol (no ionizable group) coincided with the order of the K app m values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K app m values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.
Indirect inactivation of tyrosinase in its action on tyrosine
2014
Under aerobic conditions, tyrosinase is inactivated by dopa as a result of suicide inactivation, and, under anaerobic conditions, as a result of irreversible inactivation. However, tyrosine protects the enzyme from being inactivated by dopa under anaerobic conditions. This paper describes how under aerobic conditions the enzyme acting on tyrosine is not directly inactivated but undergoes a process of indirect suicide inactivation provoked by reaction with the o-diphenol originated from the evolution of o-dopaquinone and accumulated in the reaction medium.
Production of l-DOPA using Cu-alginate gel immobilized tyrosinase in a batch and packed bed reactor
Enzyme and Microbial Technology, 2007
In this study, production of l-DOPA (l-3, 4-dihydroxy phenylalanine) was investigated by using tyrosinase enzyme in batch and packed bed reactors. Tyrosinase has been immobilized with method of entrapment in copper-alginate gels. l-DOPA concentration obtained from batch reactor for free and immobilized enzyme was 9.5 and 4.5 mg/L, respectively. l-DOPA concentration was obtained as 1.2 mg/L in the packed bed reactor. When air was introduced l-DOPA production increased 6.4 times in packed bed reactor and the productivity was calculated as 110 mg/L h.
2007
We undertook a preliminary characterization of the tyrosinase produced by a strain of Lentinula boryana from Brazil, with a view to evaluate its potential for biotechnological applications. The enzyme was similar to other fungal tyrosinases in many respects. When the crude extract was characterized, the tyrosinase activity was optimal at pH=6 and was not particularly thermostable, with half-lives of about 10 min and 1 min at 50 and 60°C, respectively. We purified the enzyme with ammonium sulfate precipitation followed by ion exchange chromatography on a DEAE Sepharose column, obtaining a yield of 33 % and a 5.3-fold enrichment. The purified preparation gave three bands on SDS-PAGE, with molecular masses of 20, 27 and 47 kDa. This preparation showed substrate inhibition kinetics with L-DOPA (3,4-dihydroxy-L-phenylalanine), with a K M of 1.9 mM and a K I of 72 mM. Under the same reaction conditions, a commercial mushroom tyrosinase followed Michaelis-Menten kinetics, with a K M of 0.51 mM. Although the present study did not identify properties that would make the tyrosinase of L. boryana more suitable in biotechnological applications than tyrosinases from other mushrooms, it has made a contribution by showing that the enzyme suffers substrate inhibition by L-DOPA, something that has not previously been reported for mushroom tyrosinases.
Phenolic substrates and suicide inactivation of tyrosinase: kinetics and mechanism
Biochemical Journal, 2008
The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form Eox (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon...
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.
l-Dopa synthesis catalyzed by tyrosinase immobilized in poly(ethyleneoxide) conducting polymers
International Journal of Biological Macromolecules, 2013
a b s t r a c t 1-3,4-Dihydroxy phenylalanine called as l-Dopa is a precursor of dopamine and an important neural message transmitter and it has been a preferred drug for the treatment of Parkinson's disease. In this study, with regards to the synthesis of l-Dopa two types of biosensors were designed by immobilizing tyrosinase on conducting polymers: thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinylbenzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and tyrosinase immobilized by entrapment during electropolymerization. l-Tyrosine was used as the substrate for l-Dopa synthesis. The kinetic parameters of the designed biosensors, maximum reaction rate of the enzyme (V max ) and Michaelis Menten constant (K m ) were determined. V max were found as 0.007 mol/(min electrode) for PEO-co-PPy matrix and 0.012 mol/(min electrode) for CP-co-PPy matrix. K m values were determined as 3.4 and 9.2 mM for PEO-co-PPy and CP-co-PPy matrices, respectively. Optimum temperature and pH, operational and shelf life stabilities of immobilized enzyme were also examined.