Laccase production by Phanerochaete chrysosporium ± an artefact caused by Mn(III)? H. Podgornik, M. Stegu, E. Zibert and A. Perdih University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia 2000/60: received 5 March 2001 and accepted 15 March 2001 H. PODGORNIK, M. STEGU, E. ZIBERT AND A. PERDIH. 2001. Aims: The possibility of laccase production by Phanerochaete chrysosporium was studied. Methods and Results: A relatively high initial Mn(II) concentration (1±4 mM) in the growth medium leads to the development of reddish-brown coloration and intensive oxidation of 2.2¢-azino-bis(3-etilbenz-tiazolin-6-sulfonate) (ABTS). The peak of ABTS oxidation was obtained approximately 1 day after the peak of MnP activity. Conclusions: ABTS oxidation was not caused by manganese peroxidase (MnP) nor by laccase but was the consequence of the action of Mn(III) which was stabilised in the growth medium. Decomposition of the complex took place after the biomass was removed from the growth medium and especially after the aeration of the culture was interrupted. Signi®cance and Impact of the Study: Mn(III) seems to be the cause of false positive laccase reactions. More reliable data on MnP activity can be obtained if the complex is decomposed by the fungus before MnP activity is measured in the medium. INTRODUCTION Phanerochaete chrysosporium has for a long time been known as a lignin peroxidase (LiP)±manganese peroxidase (MnP) producing white rot fungus (Hatakka 1994). LiPs and MnPs are a family of extracellular haemoproteins which are involved in lignin degradation and also in oxidation of different xenobiotics (Hammel 1995; Reddy 1995). Over the last few years, however, several publications dealing with laccase production by P. chrysosporium have been published (Srinivasan et al. 1995; Dittmer et al. 1997). The glycopro- tein laccase has been isolated from different plants and fungi (Thurston 1994). While in woody tissues it is a component of the lignin synthesizing system (Bao et al. 1993), the laccase produced by basidiomycete fungi is able to miner- alize lignin (Thurston 1994; Yaropolov et al. 1994). Laccase is a polyphenol oxidase (EC. 1.10.3.2) belonging to a group of blue copper oxidases (Yaropolov et al. 1994). During its catalytic action, reduction of oxygen to water is accompanied by oxidation of a phenolic substrate (Thurston 1994 2 ). Since laccases are remarkably non-speci®c, different substrates can be used to determine their activity. A substrate frequently used is 2,2¢-azino-bis(3-etilbenz-tiazo- lin-6-sulphonate) (ABTS), which can also act as a mediator in the oxidation of non-phenolic lignin model compounds (Bourbonnais and Paice 1990). Since ABTS can also be oxidized by Mn(III) produced by MnP, catalase is added to the reaction mixture to prevent direct oxidation of ABTS by MnP (Glenn and Gold 1985). Detection of laccase activity at a high manganese concen- tration, added as Mn(II) or Mn(IV) in the semi-solid growth medium of P. chrysosporium (Rodriguez Couto et al. 1998; Rodriguez et al. 1998; Couto et al. 1999), prompted this study of laccase production by P. chrysosporium during the submersed cultivation of the fungus. MATERIALS AND METHODS Organism The subcultures of P. chrysosporium MZKI B-223 (ATCC 24725) were prepared every 3±4 weeks on malt agar slants which were kept in a refrigerator until use. MnP production The fungus was grown in a nitrogen-limited medium (Podgornik et al. 1999), buffered by sodium tartrate (pH 4á5), in agitated 500 ml Erlenmeyer ¯asks containing Correspondence to: Dr H. Podgornik, University of Ljubljana, Faculty of Chemistry and Chemical Technology, As Ïkerceva 5, 1000 Ljubljana, Slovenia (e-mail: helena.podgornik@uni-lj.si). ã 2001 The Society for Applied Microbiology Letters in Applied Microbiology 2001, 32, 407±411