6th World Congress on Oxidation Catalysis

Laccase-Catalyzed L-DOPA Oxidation on Macro and Microreactor ScaleMarina Tišmaa,b, Bruno Zelićb, Đurđa Vasić-Račkib, Polona Žnidaršič-Plazlc and Igor Plazlc * a: Faculty of Food Technology, Josip Juraj Strossmayer University of Osijek, Osijek, Croatiab: Faculty of Chemical Engineering and Technology, University of Zagreb, Zagreb, Croatiac: Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia

______________________________________________________________Summary : Enzymatic oxidation of 3, 4-dihydroxyphenyl-L-alanine (L-DOPA) with laccase from Trametes versicolor was investigated in a batch laboratory-scale reactor and in a continuously operated microreactor. The reaction was described with double-substrate Michaelis-Menten kinetics. Kinetic parameters, evaluated in a 50 mL batch reactor, were used for the modelling of a microreactor, based on diffusion, convection and a non-linear reaction term, considering the velocity profile for laminar flow in a microchannel at steady-state conditions. Experimental data on L-DOPA conversions for both systems revealed good agreement with models predictions. Up to 87 % conversions of L-DOPA were achieved in a microreactor at residence times below 2 minutes.Keywords : laccase; L-DOPA; microreactor, modelling______________________________________________________________

Introduction Laccases (EC 1.10.3.2, p-diphenol: dioxygen

oxidoreductases) are currently seen as very interesting enzymes for industrial oxidation reactions, since they are capable of oxidizing a wide variety of substrates [1-4]. Because enzymatic reactions offer several advantages over traditional chemical processes, they represent great potential for optimisation of industrial processes, both economically and environmentally [5].

Microreaction technology is gaining importance in a broad range of areas. Due to the small amount of chemicals needed and high rate of heat and mass transfer, microreactors are an extremely efficient tool for the rapid screening of (bio)catalysts. Besides, the small length scale of reactors reduces transport limitations giving near gradientless conditions desirable for determination of reaction kinetics [6]. The integration of enzyme-catalysed processes with microreactor technology is of a great potential in this field [7].

The aim of this work was to theoretically and experimentally investigate enzymatic oxidation process on a macro and micro scale. Laccase-catalyzed 3,4-dihydroxy-L-phenylalanine (L-DOPA) oxidation was studied as a model process.

ExperimentalBatch experiments were carried out in a 250 mL

reactor (reaction volume of 50 mL) with different initial concentration of L-DOPA solutions (5 mmol/L and 0.5 mmol/L) continuously supplied by the air. Reactions were performed at previously defined optimal pH and temperature conditions (0.2 mol/L phosphate buffer, pH 5.4 and 25 ºC) with laccase concentration of 0.1 mg/mL [8].

Laccase-catalyzed L-DOPA oxidations were carried out also in a glass microreactor with the y-shaped inflow and outflow channels. The main channel was 220 μm wide, 50 μm high and 332 mm long. Oxygen-saturated (ci,O2= 1.15 mmol/L) L-

DOPA solution in 0.2 mol/L phosphate buffer (pH 5.4) was fed from one inflow, while oxygen-saturated laccase solution in the same buffer was fed from the other (Fig. 1). Concentrations of L-DOPA at the inlet were: ci,LD = 0.5 mmol/L and ci,LD = 5 mmol/L, while enzyme inlet concentration was 0.2 mg/mL. Both solutions were pumped in at equal and constant flow rates of 100, 10, 5 or 1 μL/min, so that the total flow rates in the microreactor were 200, 20, 10 or 2 μL/min. The concentration of L-DOPA in samples diluted with 0.1 mol/L HCl was evaluated by HPLC with UV detection at 280 nm [8].

Fig. 1. Scheme of the microchannel (2W = 220 µm, H = 50 µm, L= 332 mm).

ResultsFor the laccases-catalysed reaction kinetics, a

double substrate Michaelis-Menten model was suggested:

Kinetic parameters, estimated by non-linear regression analysis of data obtained in batch

1

REF

reactor using Simplex and Least Squares method implemented in the Scientist software, were:

=6.90 ± 0.24 U/mg, = 0.47 ± 0.10 mmol/L

and = 0.10 ± 0.02 mmol/L [8].Parameters were experimentally verified by a

set of experiments with or without constant supply of different oxygen and nitrogen mixtures and for different initial concentrations of L-DOPA and dissolved oxygen. The results of the experiments performed in batch reactor were in good correlation with the proposed model (Fig 2.)

t [min]

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c L -D

OP

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OP

A [

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c0, L-DOPA = 5 mmol/Lc0, L-DOPA = 0.5 mmol/Lmodel

Fig. 2. Laccase-catalyzed oxidation of L-DOPA in a batch reactor (25 ºC, 0.2 mol/L phosphate buffer, pH 5.4, claccase = 0.1 mg/mL, c0,O2= 0.24 mmol/L) with continuous oxygen supply for different initial L-DOPA concentrations.

For the description and prediction of microreactor performance, a two-dimensional mathematical model composed of convection, diffusion, and enzyme reaction terms, considering the velocity profile for laminar flow in a microchannel at steady-state conditions was developed. The results of the experiments of laccase-catalyzed L-DOPA oxidations in a microreactor, performed with two different inlet L-DOPA concentrations at different fluid flow rates were in good agreement with the model predictions for both inlet L-DOPA concentrations and for all applied flow rates (Fig 3.).

In a given microreactor geometry, up to 87 % conversion of L-DOPA was reached at inlet L-DOPA concentration of 0.5 mmol/L and at the residence time of 110.6 s (total fluid flow rate = 2 μL/min). For the same inlet substrate concentration, 6.7 % conversion was observed even at residence time of 11 s, which is much faster compared to the batch reactor, even at very efficient mixing assuring high oxygen transfer rates.

0 50 100 150 2000

20

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60

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experiment, ci, L-DOPA=5 mmol / Lexperiment, ci,, L-DOPA=0.5 mmol / Lmodel, ci, L-DOPA=0.5 mmol / Lmodel, ci, L-DOPA=5 mmol / L

[L / min]

X [%

]

Fig. 3. Enzymatic oxidation of L-DOPA in a microreactor – experimental data and the results of mathematical model calculations of conversion based on average dimensionless L-DOPA concentration at the outlet of the microchannel at different total fluid flow velocities through the main channel.

ConclusionsThe comparison of the results performed in

microreactor with the results from the batch reactor with continuous oxygen supply confirmed an advantage of microreactor technology over classical reactors. Based on the developed model simulations, which were in good agreement with experimental data, further microreactor design and process optimization are feasible.

AcknowledgementsThis work was supported by the Croatian

Ministry of Science, Education and Sports (Grant 125-1252086-2793) and by the Ministry of Higher Education, Science and Technology of the Republic of Slovenia (Grant P2-0191).

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[8] M. Tišma, P. Žnidaršič-Plazl, I. Plazl, Đ. Vasić-Rački, B. Zelić, Chem. Biochem. Eng. Q. 22 (2008) 307-313.

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