6th World Congress on Oxidation Catalysis
REF
Laccase-Catalyzed
Microreactor Scale
L-DOPA
Oxidation
on
Macro
and
Marina 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, Croatia
b: Faculty of Chemical Engineering and Technology, University of Zagreb, Zagreb, Croatia
c: 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 LDOPA conversions for both systems revealed good agreement with models predictions. Up to 87 % conversions of LDOPA 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 enzymecatalysed 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. Laccasecatalyzed 3,4-dihydroxy-L-phenylalanine (L-DOPA)
oxidation was studied as a model process.
Experimental
Batch 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 yshaped 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 oxygensaturated laccase solution in the same buffer was
fed from the other (Fig. 1). Concentrations of LDOPA 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 LDOPA 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).
Results
For the laccases-catalysed reaction kinetics, a
double substrate Michaelis-Menten model was
suggested:
r
K
Vm  cL-DOPA  cO2  claccase
L-DOPA
m

 cL-DOPA   K m O2  cO2

Kinetic parameters, estimated by non-linear
regression analysis of data obtained in batch
1
reactor using Simplex and Least Squares method
implemented in the Scientist software, were:
100
Vm =6.90 ± 0.24 U/mg, K m L-DOPA = 0.47 ± 0.10
O2
experiment, ci, L-DOPA=5 mmol / L
80
= 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.)
0.6
experiment, ci,, L-DOPA=0.5 mmol / L
model, ci, L-DOPA=0.5 mmol / L
model, ci, L-DOPA=5 mmol / L
60
X [%]
mmol/L and Km
40
20
6
0
0
50
100
150
200
c0, L-DOPA = 5 mmol/L
0.5
5
c0, L-DOPA = 0.5 mmol/L
 [L / min]
0.4
4
0.3
3
0.2
2
0.1
1
0.0
0
0
10
20
30
40
cL-DOPA [mmol/L]
cL-DOPA [ mmol/L]
model
50
t [min]
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 LDOPA 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 LDOPA 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.
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 LDOPA concentration at the outlet of the microchannel at different
total fluid flow velocities through the main channel.
Conclusions
The 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.
Acknowledgements
This 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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