Appl Microbiol Biotechnol (2003) 60:708–712DOI 10.1007/s00253-002-1181-7

S H O R T C O N T R I B U T I O N

R. Pilz · E. Hammer · F. Schauer · U. Kragl

Laccase-catalysed synthesis of coupling productsof phenolic substrates in different reactors

Received: 10 July 2002 / Revised: 15 October 2002 / Accepted: 18 October 2002 / Published online: 18 December 2002� Springer-Verlag 2002

Abstract Substrate oxidation of aromatic substances bythe enzyme laccase followed by a heteromolecularcoupling with a co-substrate is a promising possibilityfor the synthesis of new compounds. To find a suitablereactor for the effective production of new compounds,the laccase-catalysed coupling of 3-(3,4-dihydrox-yphenyl)propionic acid with 4-aminobenzoic acid wasinvestigated as a model system. Based on the kineticparameters, a mathematical model was used to predict thereaction yield and oxygen demand in a discontinuouslystirred tank reactor and a continuously operated stirredtank reactor. Membrane processes were used for bubble-free aeration of the system and to recover the solubleenzyme.

Introduction

Laccases are enzymes of considerable biotechnologicalinterest, since they can be used for numerous applicationsincluding delignification or decolouration of paper pulp,detoxification of environmental pollutants, textile dye-bleaching or diagnostic assays (Xu 1999). All of theseapplications are based on the ability of the laccase tooxidise a wide range of aromatic substances includingphenolic substrates. During the laccase-catalysed reac-tion, O2 is reduced to water (Reinhammar and Malmstr�m1981).

The copper-containing enzyme laccase (E.C. 1.10.3.2)has been detected in various plant species (e.g. mango,mung bean, peach), some bacteria (e.g. Azospirillumlipoferum), various insects and in more than 40 different

fungi, which produce it as an extracellular enzyme(Gianfreda et al. 1999).

Laccase generally contains four copper atoms permonomer bound to three redox sites (type 1–3) and differfrom each other in their electronic properties. It isbelieved that the mechanism of laccase catalysis involvesthree steps: (1) the reduction of copper type 1 by reducinga substrate, (2) internal electron transfer between thedifferent copper types, and (3) the reduction of O2 towater at the type 2 and the type 3 copper site (Messer-schmidt 1994; Thurston 1994; Yaropolov et al. 1994).

The substrate oxidation catalysed by laccase is a one-electron reaction, in which a free (cation) radical isgenerated (Thurston 1994). The unstable radical mayundergo further non-enzymatic reactions, e.g. hydration,disproportionation or polymerisation. In addition, cross-coupling with different substances has been observed(Tatsumi et al. 1992, 1994). Using this reaction, it ispossible to produce novel compounds, e.g. with antibioticproperties (Schauer et al. 2001). For technical purposes,effective production of such new compounds by laccasecatalysis should be carried out in a bioreactor. To find asuitable reactor type we have determined the kineticparameters maximum reaction velocity (Vmax), Michaelis-Menten constant (Km), and the product inhibition (Ki) of amodel reaction. These parameters were then used for themathematical simulation of several reactor types. Themodel reaction used was the laccase-catalysed cross-coupling of 3-(3,4-dihydoxyphenyl)propionic acid and 4-aminobenzoic acid (Fig. 1).

Materials and methods

Chemicals

3-(3,4-Dihydroxyphenyl)propionic acid, 2,2-azino-bis(3-ethylben-zothiazoline-6-sulfonic acid)diammonium salt (ABTS) and phos-phoric acid (crystallised) were obtained from Fluka (Deisenhofen,Germany). p-Aminobenzoic acid, methanol, sodium acetate, andacetic acid were purchased from Baker (Deventer, The Nether-lands).

R. Pilz · U. Kragl ())Rostock University, Department of Chemistry, 18051 Rostock,Germanye-mail: udo.kragl@chemie.uni-rostock.deTel.: +49-381-4986450Fax: +49-381-4986452

E. Hammer · F. SchauerErnst Moritz Arndt University, Institute of Microbiology,17487 Greifswald, Germany

Preparation of laccase

Laccase was prepared from cultures of the white rot fungusPycnoporus cinnabarinus (DSM 15225) isolated from an oak treein northern Germany. Cultures were prepared by inoculating anitrogen-rich medium (Braun-L�llemann et al. 1997) according toJonas et al. (2000). After 6 days of incubation, the cultures wereharvested and the supernatant separated from mycelia by filtrationthrough glass fibre filters (GF6, Schleicher & Schuell, Dassel,Germany). The extracellular enzyme was bound on DEAE-Sephacel (Sigma, Steinheim, Germany) as described by Jonas etal. (2000), eluted by a high salt buffer (20 mM sodium acetate,700 mM sodium chloride, pH 6.0) and desalted over a HiPrep 26/10column (Pharmacia, Freiburg, Germany).

Discontinuously stirred tank reactor

To determine the mass balance and the kinetic parameters in adiscontinuously stirred tank reactor (STR), dark glass vials with avolume of 5 ml were used. The vials were filled with 1 ml reactionmixture and were vigorously shaken over the reaction period(400 min–1).

Continuously operated enzyme-membrane reactor

Continuous enzyme reactions were performed in a continuouslyoperated enzyme-membrane reactor (EMR). The reaction chamberconsisted of acrylic glass and had a volume of 44 ml. It hadconnections to substrate inlet and product outlet, temperaturecontrol fluid (inlet/outlet) and two sampling ports. The samplingports were located above the membrane, which retained theenzyme. The membrane used in the reactor was a NADIR C010F(NADIR Filtration, Wiesbaden, Germany) with a cut off of 10 kDa.

The acetate buffer (0.02 M, pH 5.0), which contained thereactants, was fed into the EMR by a membrane pump. The solutionin the reaction chamber was mixed by magnetic stirring. Theproduct and the residual reactants permeated through the membraneand left the reactor. Different residence times were achieved byvarying the flow rate of the inlet.

All reactions were carried out at 25�C. In the case ofexperiments with bubble-free aeration, the two sampling ports ofthe reaction chamber were connected to a silicon tube (2 m, i.d.4 mm, wall thickness 0.3 mm; BIW Isolierstoffe, Ennepetal,Germany) in the form of a closed loop. For this type of tube anoxygen transfer rate of 72 g day–1 m–2 was determined (Rissom1999). The reaction solution was pumped through the aeration tubeby a peristaltic pump (16 ml min–1). The volume of the tube(0.025 l) was added to the volume of the reaction chamber.

Analyses

The laccase activity was determined at pH 5.0 by monitoring theoxidation of ABTS at 420 nm (e420=36,000 M–1 cm–1, Bourbonnaisand Paice 1990). A 100-�l sample was added to 110 �l 5 mM ABTSsolution and to 790 �l 0.02 M sodium acetate buffer (pH 5.0) and thechange in absorbance monitored over 1 min (Jonas et al. 1998). Oneunit of enzyme was defined as 1 �mol of substrate oxidised per minute.

The concentrations of the reactants and the cross-couplingproduct were determined using an HPLC equipped with a UVdetector (Knauer, Berlin, Germany) operating at 280 nm. Thecolumn was a 125�2 mm reversed phase column (NucleosilC18100–3, Machery-Nagel, D�ren, Germany). The reaction mixturewas separated at a flow rate of 0.3 ml min–1 using an initial mobilephase of 20:80 (v/v) methanol:0.1% phosphoric acid, changing to80:20 (v/v) methanol:0.1% phosphoric acid run over a period of10 min and held for an additional 1 min.

To calibrate the HPLC system, the cross-coupling product wasisolated from the reaction mixture by solid phase extraction. TheC18-column type Bond elut (VARIAN, Harbor City, Calif.) filledwith 0.5 g material was activated with 2 ml each of methanol andwater and then charged with reaction mixture containing theproduct. Water containing 10–30% (v/v) methanol was used towash out the remaining reactants. After drying the column withargon, the cross-coupling product was eluted with acetonitrile anddried under vacuum.

The cross-coupling product was silylated with Silyl-991(BSTFA (N,O-bis-trimethylsilyl-trifluoracetamide)-TMCS (tri-methyl-chlorsilane) 99:1, Macherey-Nagel) and identified by gaschromatography/mass spectrometry (Mikolasch et al. 2002).

Results

The reaction

Incubation of 3-(3,4-dihydoxyphenyl)propionic acid and4-aminobenzoic acid (Fig. 1) with a laccase preparationfrom Pycnoporus cinnabarinus revealed one major prod-uct 4-[2-(2-carboxyethyl)-4,5-dihydoxyphenylamino]ben-zoic acid. The transformation was accompanied by achange in colour of the fluid to dark red.

Incubation of each substrate alone with laccase showedno enzyme activity in the case of 4-aminobenzoic acid,whereas 3-(3,4-dihydoxyphenyl)propionic acid was con-verted to several products. Therefore 3-(3,4-dihy-doxyphenyl)propionic acid must be the substrate that isbound to the enzyme.

To analyse the mass balance of the reaction (Fig. 1),the concentrations of the two reactants and of the cross-coupling product were measured with respect to time. Forthis experiment we used an STR with contact to theatmosphere; 81.1% of the 3-(3,4-dihydoxyphenyl)propi-onic acid was converted into the cross-coupling product(Fig. 2). After 120 min in the STR no further transfor-

Fig. 1 Cross-coupling reaction catalysed by laccase from Pycno-porus cinnabarinus

Fig. 2 Mass balance of the cross-coupling reaction, c0=0.001 M(reactants equimolar), cE=0.049 U ml–1

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mation could be detected although residues of 3-(3,4-dihydroxyphenyl)propionic acid as well as 4-aminoben-zoic acid were detected. HPLC analysis showed thepresence of small amounts (about 9%) of at least two by-products, which have not been identified so far. Theconcentration of the cross-coupling product decreasedafter 200 min, suggesting that the cross-coupling productitself might be transformed further by chemical orenzymatic reaction.

Determination of Vmax, Km and Ki

Experiments were carried out in the STR, to determine thekinetic parameters Vmax, Km and Ki from initial ratemeasurements. These parameters were required for themathematical simulation of the several reactor types. Therelationship between reaction velocity and substrateconcentration was obtained by varying the substrateconcentration at a constant concentration (0.01 M) of4-aminobenzoic acid and an enzyme activity of0.049 U ml–1 based on the standard assay. The resultingparameters were: Vmax =(50.1€1.58) 10–6 mol l–1 min–1

and Km =(10.4€1.38) 10–4 mol l–1. Substrate inhibitionwas not observed under these conditions.

Ki was determined by adding different amounts ofisolated product to an equimolar (0.004 M) reactionmixture before starting the coupling reaction, from whicha competitive Ki =(25.8€3.1) 10–5 mol l–1 was calculated.

Modelling

Computer aided modelling of the STR was carried outusing the software Scientist (Micromath Scientific Soft-ware, Salt Lake City, Utah) and the data of Vmax, Km andKi. The experimental data for the STR and the simulateddata for substrate consumption and product formation(Fig. 3A) show a good correlation, showing this simplemodel is sufficient to describe the reaction system. Thissuggests such simulations can provide an estimate of theyields of the reaction that could be reached in otherreactor types.

Comparatively stable enzymes can be used moreeffectively in a continuously operated reactor (CSTR)designed as an EMR than in a STR (Kragl et al. 1996). Inthe EMR, an ultrafiltration membrane is used to retain theenzyme whereas the reactants and the cross-couplingproduct pass through the membrane. In the followingexperiments an EMR was used with a membrane with acut-off of 10 kDa. In contrast to the results obtained forthe STR, the experimental and simulated data did notcorrelate well (Fig. 3B). A correlation was seen only forlow yields corresponding to 10–20 min residence time. Atlonger residence times synthesis of the coupling productslowed and yield was lower than expected from thesimulation. For instance, a residence time of 70 min gavea yield of only 40%, whereas the simulation predicted ayield greater than 70%. This difference in yield wasassumed to be caused by a limited oxygen supply in thereactor. The EMR is a sealed system, preventing access ofair to the reaction mixture. The acetate buffer (pH 5.0)used for the reaction contained 0.16 mmol l–1 dissolvedoxygen. However, total conversion of 1 mmol l–1

substrate consumes 0.25 mmol l–1 O2. Therefore, addi-tional oxygen is necessary for complete conversion. Thestandard method of feeding oxygen by bubbling air intothe reaction mixture might cause deactivation of theenzyme. For this reason, we tried an integrated bubble-free aeration system (Rissom et al. 1997) using a thin-walled silicon tube to supply oxygen. While the reactionmixture is pumped through the tube, the oxygen diffusesthrough the tube surface into the reaction mixture. Anaeration tube 2 m in length was sufficient to avoid oxygenlimitation. Under these conditions experimental andcalculated data were in good agreement (Fig. 3B).

Using integrated bubble-free aeration a yield of 80%was reached, similar to that in the STR. In order to reachtotal conversion of substrate at initial concentrationshigher than 0.001 M, a greater tube surface for thediffusion of oxygen might be necessary. This can beachieved by increasing the length of the tube. In total, thereactor was operated for 4 days. The average space-timeyield was 1.3 g l–1 day–1.

Fig. 3 Experimental and simu-lated data for the production of4-[2-(2-carboxyethyl)-4,5-dihy-droxyphenylamino]benzoic acidin a stirred tank reactor (STR),c0=0.001 M equimolar, cE=0.049 U ml–1 (A) and in thecontinuously operated stirredtank reactor (CSTR) c0=0.001 Mequimolar, cE= 0.170 U ml–1

(B)

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Discussion

The phenol-removing activity of laccase is going to beused not only for environmental applications but also inbleaching processes in the textile industry or in thetreatment of beverages (reviewed by Gianfreda et al.1999). In contrast, the synthesis potential of laccase-catalysed reactions was considered as moderate (Schmidet al. 2002). The coupling activity of the polyphenoloxidase laccase with a view to the synthesis of novelcompounds (Anyanwutaku et al. 1994; Bhalerao et al.1994; Lindequist and Schauer 2002; Mikolasch et al.2002) and materials (H�ttermann et al. 2001) has beenstudied in detail in only a few cases. Nevertheless, theconversion of phenolic substrates with laccase by cou-pling of small molecules is a promising possibility for thesynthesis of new compounds. It might be a way togenerate new therapeutic compounds for the treatment ofmicrobial infections or cancer, or to modify alreadyknown ones. To use such reactions effectively, it isnecessary to generate data to scale up the process andidentify possible bottlenecks. Immobilisation of thelaccase can reduce the amount of enzyme required foruse in the synthesis process. Several methods have beenshown to be applicable for this purpose (Palmieri et al.1994; Crecchio et al. 1995). However, in some cases theproducts synthesised may interact with the immobilisationmatrix, resulting in decreased yields. Therefore, wefavoured the use of a membrane reactor to retain theenzyme in a defined reaction volume.

On basis of the kinetic experiments we were able todetermine the basic kinetic parameters, Vmax, Km and Ki,necessary to carry out a mathematical simulation ofseveral reactor types. The estimation of the Km value with1 mmol l–1 point to a relatively low affinity of the enzymefor the substrate 3-(3,4-dihydroxyphenyl) propionic acid.The Km value for other laccase substrates like syringal-dazine, catechol or 2,6-dimethylphenol were described tobe in the range 0.001–0.26 mmol l–1 (Xu 1999).

In case of the STR, the simulated and the experimentaldata of the laccase-catalysed reaction were in goodagreement. Therefore, a mathematical calculation of theyield should be possible for any reactor type, thusavoiding scale up optimisation. However, the EMRexperiments showed that an optimal laccase-catalysedsynthesis strongly depends upon a sufficient supply ofoxygen. Without additional aeration the experimentalproduct yield was much lower than the calculated one.Because of this, some type of aeration device will be anecessary component of the reactor. Diffusion of oxygeninto the input is one possibility, air bubbling is another.We plan to investigate these processes in the future tooptimise the stability of the enzyme, further improve thereactor design and increase substrate concentrations inorder to scale up the process. This will also allow theproductivity of the process to be increased. Theseinvestigations will be of special importance if the reactionwill be used for synthetic purposes on a multi-gram scale.

Several examples of reaction systems of commercialinterest are described in Mikolasch et al. (2002).

Acknowledgements This work was financially supported by theEuropean Funds for Regional Development and by the Ministry forScience and Education of Mecklenburg-Vorpommern. We thank R.Jack (Greifswald, Germany) for revising the manuscript.

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