Lcc1 and Lcc5 are the main laccases secreted in li

Authors: Martin Rühl Andrzej Majcherczyk Ursula Kües

Publish Date: 2013/01/23

Volume: 103, Issue: 5, Pages: 1029-1039

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Abstract

The litterdegrading dung fungus Coprinopsis cinerea has the high number of seventeen different laccase genes In this work ten different monokaryons were compared in their ability to produce laccases in two different complete media at different temperatures Few strains showed laccase activity at the optimal growth temperature of 37 °C Nine of the strains gave laccase activities between 02 and 59 U mL−1 at the suboptimal temperature of 25 °C in mKjalke medium Laccase activities in YMG/T medium were detected for only three strains 05–45 U mL−1 Zymograms of supernatants from mKjalke medium resulted in total in 10 different laccase bands but strains differed in distribution LC–MS/MS analysis with Mascot searches of the annotated C cinerea genome identified isoenzymes from five different genes Lcc1 Lcc2 Lcc5 Lcc9 and Lcc10 and of Lcc1 three and of Lcc5 two distinct electrophoretical forms Lcc1 and Lcc5 were expressed in all laccase positive strains but not all forms were found in all of the strains Lcc2 Lcc9 and Lcc10 occurred only in three strains as minor laccases indicating that Lcc1 and Lcc5 are the main laccases of C cinerea secreted in liquid mKjalke mediumLaccases EC 11032 benzenedioloxygen oxidoreductase are four copperatoms containing oxidases capable of oxidising phenolic and other aromatic compounds Leonowicz et al 2001 In nature these multicopper oxidases occur widespread in the fungal kingdom particularly in basidiomycetes Hoegger et al 2006 In many higher fungi laccase genes exist in larger families that code for different isoenzymes with phenoloxidase activities Kilaru et al 2006a Courty et al 2009 Martinez et al 2009 Floudas et al 2012 Olson et al 2012 Pezzella et al 2012 Mainly laccases of straw and wooddegrading species have been studied with respect to production biochemical characteristics and potential biotechnological uses Baldrian 2006 In contrast biological functions of laccases usually remain speculative Kües and Rühl 2011Paralogous laccase genes can be differentially regulated Piscitelli et al 2011 Laccase production in higher basidiomycetes is frequently under nutritional control Dong et al 2005 Stajić et al 2006 Teerapatsakul et al 2007 or it is stressinduced Jaszek et al 2006 developmentally regulated Zhao and Kwan 1999 or part of a defence reaction Hiscox et al 2010 or it occurs in presence of specific inducers such as copper or potential phenolic substrates such as 24dimethylphenol ferulic acid syringic acid and vanillin Piscitelli et al 2011 or during growth on lignocellulosic substrates Stajić et al 2006 Rühl et al 2008 For routine fungal cultivation in laboratories laccases are usually produced in simple liquid media eg see Heinzkill et al 1998 Ikehata et al 2004 Han et al 2005 Tong et al 2007Often in liquid culture more than one enzyme is produced These may be isoenzymes coming from different genes or isoforms coming from a same gene and differing from each other due to differential splicing or by posttranslational modifications Palmieri et al 1997 2000 Dong et al 2005 D’SouzaTiclo et al 2006 Linke et al 2005 Lorenzo et al 2006 Giardina et al 2007 In some wooddegrading Trametes species 20 different isoenzymes and/or isoforms of specific laccases were detected Dong et al 2005 and strains of the edible whiterot mushroom Pleurotus ostreatus have been reported to produce isoenzymes from at least 4 distinct laccase genes summarised by Pezzella et al 2009The litterdegrading inkcap mushroom Coprinopsis cinerea has in total 17 different laccase genes which present one of the largest groups of laccase genes ever described for a fungus Kilaru et al 2006a Sixteen of the genes lcc1 to lcc14 lcc16 and lcc17 translate into fulllength proteins and 15 of these fulllength proteins all but Lcc8 have a typical secretion signal for release into the environment Hoegger et al 2004 Kilaru et al 2006a So far hardly anything is known about expression of these multiple laccase genes Schneider et al 1999 isolated laccase CcL synonymous to Lcc1 as the only enzyme from an unnamed C cinerea culture broth Nterminal sequencing allowed to assign CcL to a subcloned cDNA of gene lcc1 used for heterologous laccase production in Aspergillus oryzae Evidence for expression of two other genes lcc2 lcc3 is available from a cDNA library produced for subcloning laccase genes from mycelium of strain IFO 8371 grown at 26 °C in rich soybeanflourbased FG4 medium Yaver et al 1999Identification of different laccase isoenzymes and isoforms from culture supernatants is usually quite laborious Classically enzymes have individually to be purified and sequenced For this high amounts of laccase are required and in consequence such approaches are restricted to only a few species and for most species the number of expressed isoenzymes and forms remains unknown In contrast with available fungal genomes new proteomic techniques help in definition of enzymes even when present in minor amounts In this study we used LC–MS/MS with Mascot searches of the annotated C cinerea genome published by Stajich et al 2010 to identify laccases secreted in liquid culture by a collection of monokaryotic C cinerea strains of different genetic backgrounds Results show that media composition growth temperature and the strain used influenced the outcome of laccase production In modified Kjalke mKjalke medium at 25 °C under optimum yields of enzymatic activities 9 of 10 analysed strains produced laccases Lcc1 and Lcc5 whereas some of the strains in addition gave also different forms of Lcc2 Lcc9 and/or Lcc10

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