Biocontrol Science 2013 Vol 18 No 3 143-149 Original Phenol-Oxidizing Enzyme Expression in Lentinula edodes by the Addition of Sawdust Extract,Aromatic Compounds,or Copper in Liquid Culture Media ELJI TANESAKA*.HIRONORI TAKEDA AND MOTONOBU YOSHIDA Faculty of Agriculture.Kinki University.3327-204 Nakamachi.Nara 631-8505.Japan 'Present address:Yukiguni Maitake Co.,Ltd.,89 Yokawa,Minami-Uonuma City. Niigata 949-6695.Japan Received 31 January 2013/Accented 13 March 2013 This study examined how the addition of a sawdust extract from Castanopsis cuspidata several aro compou nds,and copper attected the expression of a phenc I-oxidizin enzyme n the te MYPG high (MYPG.S) concentrations of saw tex女 act hada marked on the motion of mycelial growth.No manganese peroxidase(MnP)production was observed in either MYPG MYPG media unti 35 days after inoculatic on.Howe er,MnP pro on v re in with a mark ase ot y at of MYPG-S3o0,increasing gradually thereafter until days 22-25.However,laccase (Lcc) produc tion was not observed ir any of the media until 35 days after inoculation.Additic on of 10 mM aroma 1,2-D nenol,hydroquinon and 4-an While the of 1 2 mM Cu"(CuSo.-5H-O)to MYPG-S. inhibited MnP production,this Cu"addition caused a marked increase in Lcc production at 17 and 6 days after the addition,respectively. Key words Copper/Laccase/Manganese peroxidase/Sawdust xtract/Shiitake INTRODUCTION reported to cretes the Lentinula (Berk.) 1032a idas valuable edible nushmoms cultured in the wodd (Cha (MOP EC 1 11 1 13)when cultiv ted on sawdust. and Miles,1989).This fungus has traditionally beer based media (Tokimoto et al., 1987:Makker et al cultivated on Fagace logs or.in 2001 Kamoto et al. 009:Nagai et a on sawdus s not typ 2009) et al 1 5.N including lignin and are frequently among the primar al.2002:Cavallazzi et al.2005:Saeki et al.2011) colonizers of decaying wood in nature (Shortle and Lcc production in white-rot basidiomycetes including saka et al.1993:Tanesaka L.edodes can als be promoted by the addition o 2000 ns e 002997 ath0Te+81-742.43.5135.Fax:+81 et al.2001.2003:Faraco al 2003)or 742-43-5271.E-mait:tanesaka (a)nara.kindai.ac.ip compounds (Fahraeus.1962:Scheel et al.2000)
Original ＊Corresponding author. Tel: +81-742-43-5135, Fax: +81- 742-43-5271, E-mail: tanesaka（a）nara.kindai.ac.jp Phenol-Oxidizing Enzyme Expression in Lentinula edodes by the Addition of Sawdust Extract, Aromatic Compounds, or Copper in Liquid Culture Media EIJI TANESAKA＊, HIRONORI TAKEDA1 AND MOTONOBU YOSHIDA Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan 1 Present address: Yukiguni Maitake Co., Ltd., 89 Yokawa, Minami-Uonuma City, Niigata 949-6695, Japan Received 31 January, 2013/Accepted 13 March, 2013 This study examined how the addition of a sawdust extract from Castanopsis cuspidata, several aromatic compounds, and copper affected the expression of a phenol-oxidizing enzyme in the white-rot basidiomycete, Lentinula edodes. Compared to liquid media that had not been supplemented with sawdust extract（MYPG）, MYPG containing low（MYPG-S100）or high（MYPG-S500）concentrations of sawdust extract had a marked effect on the promotion of mycelial growth. No manganese peroxidase（MnP）production was observed in either MYPG or MYPG-S100 media until 35 days after inoculation. However, MnP production was enhanced by culture in MYPG-S500, with a marked increase observed suddenly at 14 days after inoculation. Northern blot analysis revealed that the transcription of the lemnp2 gene coding extracellular MnP was initially observed at detectable levels at day 10 after the initial inoculation of MYPG-S500, increasing gradually thereafter until days 22-25. However, laccase（Lcc）production was not observed in any of the media until 35 days after inoculation. Addition of 10 mM aromatic compounds – 1,2-benzenediol, 2-methoxyphenol, hydroquinone, and 4-anisidine – into the MYPG-S500 medium completely inhibited MnP production and did not enhance any Lcc production. While the addition of 1 or 2 mM Cu2+ （CuSO4·5H2O）to MYPG-S500 medium completely inhibited MnP production, this Cu2+ addition caused a marked increase in Lcc production at 17 and 6 days after the addition, respectively. Key words：Copper / Laccase / Manganese peroxidase / Sawdust extract / Shiitake. Biocontrol Science, 2013, Vol. 18, No. 3, 143－149 reported to cause serious economic damage to timber （Dickinson, 1982）. Although L. edodes secretes the phenol-oxidizing enzymes involved in lignin-degradation, laccase（Lcc, EC 1.10.3.2）and manganese peroxidase （MnP, EC 1.11.1.13）, when cultivated on sawdustbased media（Tokimoto et al., 1987; Makker et al., 2001; Sakamoto et al., 2009; Nagai et al., 2009）, it does not typically secrete these enzymes in liquid media （Ikegaya et al., 1993; Buswell et al., 1995; Nagai et al., 2002; Cavallazzi et al., 2005; Saeki et al., 2011）. Lcc production in white-rot basidiomycetes including L. edodes can also be promoted by the addition of various metals, particularly Cu2+ （Collins et al., 1997; Palmieri et al., 2000; Galhaup et al., 2001, 2002; Soden et al., 2001, 2003; Faraco et al., 2003）, or by aromatic compounds（Fahraeus, 1962; Scheel et al., 2000）. INTRODUCTION The shiitake mushroom, Lentinula edodes（Berk.） Pegler, a white-rot basidiomycete, is one of the most valuable edible mushrooms cultured in the world（Chang and Miles, 1989）. This fungus has traditionally been cultivated on Fagaceae logs or, in recent decades, on sawdust-based media. White-rot basidiomycetes are capable of degrading whole wood components including lignin and are frequently among the primary colonizers of decaying wood in nature（Shortle and Cowling, 1978; Tanesaka et al., 1993; Tanesaka, 2012）. In addition, these mushrooms have been
144 E.TANESAKA ET AL Wood meal spread on the surface of aga edia has mvcelia were cuttured in MYPG liquid medium (2 5 been used for p ligninolytic culture conditions malt extract:1.0 g yeast ext for the white-and brown-rot basidiomycetes and the soft-rot deute sawdust extract (MYPG-S).The sawd al. :200 0). increase in Lc prepare l a mixture at 121 for 15 min before filtering it t ing 1996) filter paper (No.1.Advantec,Tokyo)and collecting the The substrates used for mushroom cultivation are extract Two concentrations of MYPG-S liquid media typically on were da t i MYPG-S Japanese chinguapin.Castanopsis cuspidata (Thunb containing extracts from 100 mg (MYPG-So ex Murray)Schottky.a member of the Fagaceae 500 mg (MYPG-Ss00)of sawdust in 30 ml media arm,temperate,broad-leave sub-cultured at nt and y U ia for ently reported that the produ the plates and used to inoculate 30 ml liguid media in of the primary MnP isozyme by L edodes cultured a 100 ml flask.which was then statically cultured at 200. nedia Sakan 25 C.All media t and enzyme sion of the lco isozyme,Lcc1 (Nagai et al.,2002) was strongly pro Dry weight of mycelia 2 mM Cu afte e colle ed by filtration through a me 6 extract made it easy to colle elia s washina the my elia twice with distilled wate the cuture liquid compared with cultures supplemented water was removed by blotting with a paper towel.The with solid sawdust or wood powder. However,whe mycelia were then dried overnight at 60C and then at edum、suppleme 105C for 1 h before being weighed. eith t that th En me expression of MnP and/or Lcc in L.edodes is controlle The phenol-oxidizing enzymes were assayed as Saeki et al.,2011:Tanesaka et production LCC pr fly.a 100ul ali quot of cult ure mediu mined the 2012 The e atant phenol-oxidiz enzymes in L.edodes under liquid the crude en yme solution and was as ved for lc culture conditions in response to the addition of vary peroxidases (Per.EC 1.11.1.x).and MnP in identical 5 ing com tions of the folld ng putative inducers c ml tes tube scontaining an Lcc nQyCtreconss tartrat (L ration o 1 mm)to ass sess Per activity.MnSO.5H.O of phenol-oxidizing enzyme expression employed by (final concentration 0.1 mM)was then added to the this fungus are also discussed. Per assay mixture to assess MnP activity.Aliquots we e ac MATERIALS AND METHODS after incubating these test tuhes at 37 for 10 mi Fungal stock and culture conditions the reactions were stopoed by the addition of 50ul of he L.edodes vanety. 'Hokken 600'(Hokken Co 40 mM NaN3 To inactivate the enzymes in the contro Tochigi,Japan) is we sulted to sawd sodium azide wa- to tubes that containe s in fiouid were spectro
144 E. TANESAKA ET AL. mycelia were cultured in MYPG liquid medium（2.5 g malt extract; 1.0 g yeast extract; 1.0 g peptone; 5.0 g glucose in 1,000 ml distilled water）supplemented with sawdust extract（MYPG-S）. The sawdust extract was prepared by adding 1 g of sawdust（C. cuspidata） to 30 ml of distilled water and then autoclaving the mixture at 121℃ for 15 min before filtering it through filter paper（No. 1, Advantec, Tokyo）and collecting the extract. Two concentrations of MYPG-S liquid media were then prepared by supplementing MYPG liquid medium with either a 1/10 or 1/2 volume of sawdust extract instead of distilled water, which gave MYPG-S containing extracts from 100 mg（MYPG-S100）or 500 mg（MYPG-S500）of sawdust in 30 ml media, respectively. Mycelia were then sub-cultured at 25℃ on MYPG 1% agar plates for 14 days. Three mycelial disks measuring 3 mm in diameter were then harvested from the plates and used to inoculate 30 ml liquid media in a 100 ml flask, which was then statically cultured at 25℃. All media treatments were performed in triplicate, and measurements of mycelial dry weight and enzyme activity were performed as described below. Dry weight of mycelia Mycelia samples were collected by filtration through a double nylon stocking mesh at 3-, 4- or 7-day intervals until sampling ended at day 35 of incubation. After washing the mycelia twice with distilled water, excess water was removed by blotting with a paper towel. The mycelia were then dried overnight at 60℃ and then at 105℃ for 1 h before being weighed. Enzyme assay The phenol-oxidizing enzymes were assayed as described previously（Saeki et al., 2011; Tanesaka et al., 2012）. Briefly, a 100μl aliquot of culture medium was collected during culture and centrifuged at 13,000 rpm for 10 min. The obtained supernatant was used as the crude enzyme solution, and was assayed for Lcc, peroxidases（Per, EC 1.11.1.x）, and MnP in identical 5 ml test tubes containing an Lcc assay mixture consisting of 0.1 mM o-dianisidine in 0.1 M sodium tartrate buffer（pH 5.0）and the addition of H2O2 （final concentration 0.1 mM）to assess Per activity. MnSO4·5H2O （final concentration 0.1 mM）was then added to the Per assay mixture to assess MnP activity. Aliquots （20μl）of crude enzyme solution were added to test tubes containing 980μl of each reaction mixture, and after incubating these test tubes at 37℃ for 10 min, the reactions were stopped by the addition of 50μl of 40 mM NaN3. To inactivate the enzymes in the control tubes, sodium azide was added to tubes that contained the Lcc assay mixture before incubation. Catalytic products of the reaction were spectroWood meal spread on the surface of agar media has been used for preparing ligninolytic culture conditions for the white- and brown-rot basidiomycetes and the soft-rot deuteromycetes（Enoki et al., 1989; Tanaka et al., 1999, 2000）. Similarly, a marked increase in Lcc production has been reported in Pleurotus ostreatus cultured together with cotton stalk extract（Ardon et al., 1996）. The substrates used for mushroom cultivation are typically dependent on the availability of agricultural wastes（Smith et al., 1988; Hadar et al., 1992）or the natural resources prevalent in a particular region. Japanese chinquapin, Castanopsis cuspidata（Thunb. ex Murray）Schottky, a member of the Fagaceae, is abundant in the warm, temperate, broad-leaved forests of Korea and Japan, and is commonly used as a component in sawdust-based media for L. edodes cultivation. We recently reported that the production of the primary MnP isozyme by L. edodes cultured on sawdust-based media, LeMnP2（Sakamoto et al., 2009）, was strongly promoted during liquid cultures supplemented with C. cuspidata sawdust extract（Saeki et al., 2011）. In addition, the expression of the Lcc isozyme, Lcc1（Nagai et al., 2002）, was strongly promoted by the addition of 2 mM Cu2+ seven days after the copper was added to the same media（Saeki et al., 2001）. Liquid cultures supplemented with sawdust extract made it easy to collect mycelia separately from the culture liquid compared with cultures supplemented with solid sawdust or wood powder. However, when cultured in liquid medium supplemented with the sawdust extract, L. edodes produced either MnP or Lcc, but not both. These observations suggest that the expression of MnP and/or Lcc in L. edodes is controlled by a negative feedback interaction, i.e., where MnP production inhibits Lcc production and MnP-inhibition enhances Lcc production（Tanesaka et al., 2012）. This study therefore examined the expression of phenol-oxidizing enzymes in L. edodes under liquid culture conditions in response to the addition of varying combinations of the following putative inducers of phenol-oxidizing enzymes: different concentrations of sawdust extract, 10 mM aromatic compounds, and 1 or 2 mM Cu2+ . Implications of the results in the regulation of phenol-oxidizing enzyme expression employed by this fungus are also discussed. MATERIALS AND METHODS Fungal stock and culture conditions The L. edodes variety, ʻHokken 600ʼ（Hokken Co., Ltd., Tochigi, Japan）, which is well suited to sawdust cultivation, was used in this study. To enhance the expression of phenol-oxidizing enzymes in liquid media
PHENOL-OXIDIZING ENZYME EXPRESSION IN LENTINULA EDODES 145 photometrically assaved using o-dianisidine as a using an established protocol (Sambrook et al.1989). substrate,and the activity of enzyme products was respective absorbance Data analysis 198 l.e.,Lcc acu swas performed ding the assay minus the Lcc assay:and MnP activity Mn OK)Analysis of va riance (ANOVA)and Tukey's mul assay minus the Per assay.One unit (U)of enzyme tiple comparison tests were performed to compare the activity was defined as the amount of enzyme require dry weight of mycelia and enzyme activities between 29,4 19 treatments dase (laninase RESULTS in the reaction mixture using veratryl alcohol as a substrate at a final concentration of 2 mM with 0.1 mM Promotion of growth and enzyme production by HO:in 0 sawdust ex the MYPG medium (with no sawdus tod (MYPG-S and MYPG-S had a marked effect on To examine the effects of aromatic compounds on promoting mycelial growth(FIG.1).Indeed,significant phenol-oxidizing enzyme production,the following aro 012). 4-anisidine fically.each substance was added MYPG-S liquid media was four-to-five-fold highe culture media to make 10 mM a than that observed in MYPG alone (P<0.001 at day noculation,and enzyme actMtie 35. lukey s test) M C (CUSO.5HO) on phe was not observed in any of the media until 35 day ined.All substance-addition treatments were conducted after inoculation.Although Per activity was detected meaisepwoepeme2pcaeraton after 25 days of culture in MYPG m edium and after RNA isolation and northern blot analysis two ty es of media.No obvious Mnp-p Total RNA was extracted from mycelia using TRlzol observed when mycelia were cultured in either MYPG Reagent (Invitrogen,CA)after varying incubation or MYPG-S0 media.Conversely,a marked increase in MnP activity was observed from day 14 on the LeMnP2En13r(5-GTCAGTGGTGAGATTTGGG AAGGGC),whic on ■MYPG-Ss0e et al mately 700 bp was then excised ● aldehyde gel,purified,and Prism 100-Avant Geneti Analyzer CA saccorcdngtoh 20 30 4 matchina le np2 were then labeled using a PCR-base Days after inoculation digoxigenin (DIG)-dUTP labeling kit (PCR DIG Probe Synthesis Kit;Roche Diagnostics,Germany) according FIG.1.Mycelial growth of L edodes in different liquid cultur manufacturer's ration sawdust extrac and in high hybridization after blotti 10 ug of total RNA onto Hybond-N'membrane (GE Healthcare,Switzerland) erors (vertical bars)for cultures grown in triplicate
PHENOL-OXIDIZING ENZYME EXPRESSION IN LENTINULA EDODES 145 using an established protoco（l Sambrook et al., 1989）. Data analysis Statistical analysis was performed using the STATISTICA statistical package（ver.6, StatSoft, Tulsa, OK）. Analysis of variance（ANOVA）and Tukeyʼs multiple comparison tests were performed to compare the dry weight of mycelia and enzyme activities between treatments. RESULTS Promotion of growth and enzyme production by sawdust extract Compared to the MYPG medium（with no sawdust extract）, the media supplemented with sawdust extract （MYPG-S100 and MYPG-S500）had a marked effect on promoting mycelial growth（FIG. 1）. Indeed, significant differences in mycelial growth were observed among the three media 7 days after the initial inoculation（F = 10.05, P = 0.012）. Mycelial growth in MYPG-S100 and MYPG-S500 liquid media was four-to-five-fold higher than that observed in MYPG alone（P < 0.001 at day 35, Tukeyʼs test）. Figure 2 shows the activities of the phenol-oxidizing enzyme in different culture media. Lcc production was not observed in any of the media until 35 days after inoculation. Although Per activity was detected after 25 days of culture in MYPG medium and after 14 days of culture in MYPG-S500 medium, the activities at day 35 were not significantly different between the two types of media. No obvious MnP-production was observed when mycelia were cultured in either MYPG or MYPG-S100 media. Conversely, a marked increase in MnP activity was observed from day 14 on the photometrically assayed using o-dianisidine as a substrate, and the activity of enzyme products was estimated by subtracting the respective absorbance values at 460 nm（Szklarz et al., 1989）: i.e., Lcc activity = Lcc assay minus the control assay; Per activity = Per assay minus the Lcc assay; and MnP activity = MnP assay minus the Per assay. One unit（U）of enzyme activity was defined as the amount of enzyme required to catalyze 1 μmol of o-dianisidine in 1 min（ε460 = 29,400 M-1 cm-1 ）（Paszczynski et al., 1988）. Lignin peroxidase（ligninase, LiP, EC 1.11.1.14）was assayed in the reaction mixture using veratryl alcohol as a substrate at a final concentration of 2 mM with 0.1 mM H2O2 in 0.1 M sodium tartrate buffer. This mixture was incubated at 37℃ for 10 min and the absorbance of veratryl aldehyde was measured at 310 nm（Tien and Kirk, 1984）. To examine the effects of aromatic compounds on phenol-oxidizing enzyme production, the following aromatic compounds were used: 1,2-benzenediol（catechol）, hydroquinone, 2-methoxyphenol（guaiacol）, and 4-anisidine. Specifically, each substance was added to MYPG or MYPG-S500 culture media to make 10 mM at day 10 after the initial inoculation, and enzyme activities were assayed daily, or at 2- to 3-day intervals thereafter. In addition, the effect of 1 or 2 mM Cu2+ （CuSO4·5H2O） on phenol-oxidizing enzyme production was also examined. All substance-addition treatments were conducted in triplicate, except for the 1 and 2 mM copper-addition treatments which were performed in duplicate. RNA isolation and Northern blot analysis Total RNA was extracted from mycelia using TRIzol Reagent（Invitrogen, CA）after varying incubation periods on MYPG-S500. cDNA was synthesized from total RNA using an RNA PCR Kit Ver.3.0（Takara Bio, Japan）, and amplified using the primer set LeMnP2En5f （5ʼ-TCCGACAGTGTCAATGACCTCGCTC）and LeMnP2En13r（5ʼ-GTCAGTGGTGAGATTTGGG AAGGGC）, which were designed based on the highly conserved lemnp2 region（DDBJ Acc. No. AB306944; Sakamoto et al., 2009）. A fragment measuring approximately 700 bp was then excised from the 1% agarose－formaldehyde gel, purified, and sequenced using an ABI Prism 3100-Avant Genetic Analyzer（Applied Biosystems, CA）according to the manufacturer’s instructions. Fragments with sequences matching lemnp2 were then labeled using a PCR-based digoxigenin（DIG）-dUTP labeling kit（PCR DIG Probe Synthesis Kit; Roche Diagnostics, Germany）according to the manufacturerʼs instructions. The resulting DIGlabeled probe, lemnp2, was then used for Northern hybridization after blotting 10 µg of total RNA onto a Hybond-N+ membrane（GE Healthcare, Switzerland） FIG. 1. Mycelial growth of L. edodes in different liquid culture media; without sawdust extract（MYPG）, in culture media supplemented with low-concentration sawdust extract （MYPG-S100）, and in high-concentration sawdust extract （MYPG-S500）, respectively. Values are means with standard errors（vertical bars）for cultures grown in triplicate
146 E.TANESAKA ET AL 20 MYPG 10 131619222528days 0 MYPG-S100 0 MYPG-S5o0 FIG.3.(top)Northern blot analysis of lemnp2 gene t20eshoanaboeechaneAipea8 coincided approximately with the changes observed in 30 MnP activities in the liquid culture medium. 10 20 40 Days after inoculation Effects of ard tion on enzy Symbols indicating the dif (1 and 2mM)on phenol-oxidizing the inmt TABLE1 on cultures grown in triplicate experiment.MnP activity in the control samples (n additives)exceeded 20 U/ml 20 davs after inoculatior MYPG-S medium,with the activity remaining above which was relatively lower than activities exceeding 50 60U/m until 35 MnP ac ties after days hicher than the M he production betw two media (P0.001).Lip activity was not detected in to slight differences in the preparation of the sawdust any of the culture media (data not shown). or the physiological condition of the mycelia in ure na se gene transc ar b f the CDNA which was prepa red from a mixed pool ot total RNA the addition of all substances-cate obtained from the mycelia of 10-.15-and 18-day-old guaiacol,anisidine,1 and 2 mM Cu completely in dentical to tha (TA known to code for intracellular MoP (data not shown) The finding indicated that sawdust extract induced the secretion of the same isozyme,LeMnP2 which is a U/ml by day 31.the ncrease nL major Mr prod blotting analysis with a probe 02 are sho ed after the addition of mMu no noticeable increase in lcc production was observed amount of lemnp2 mRNA was pree nt at days 10-19 during the of cu a not sho
146 E. TANESAKA ET AL. coincided approximately with the changes observed in MnP activities in the liquid culture medium. Effects of aromatic compound and copper addition on enzyme activities The effect of adding either 10 mM of aromatic compounds or Cu2+ （1 and 2 mM）on phenol-oxidizing enzyme activities 10 days after the initial inoculation in MYPG-S500 medium are summarized in TABLE 1. In this experiment, MnP activity in the control samples（no additives）exceeded 20 U/ml 20 days after inoculation, which was relatively lower than activities exceeding 50 U/ml that were observed after 14 days in a previous experiment（FIG. 2）. This disparity in the timing of MnP production between experiments may have been due to slight differences in the preparation of the sawdust extract or the physiological condition of the mycelia in the culture flasks. Even so, at approximately 60 U/m, MnP activity at 30 days after inoculation was similar between experiments. Compared to the control samples, the addition of all substances – catechol, hydroquinone, guaiacol, anisidine, 1 and 2 mM Cu2+ – completely inhibited MnP production（TABLE 1）. Instead of inhibiting MnP, the addition of 1 mM Cu2+ enhanced Lcc production after 17 days, with activities exceeding 45 U/ml by day 31. Similarly, the addition of 2 mM Cu2+ enhanced Lcc production after 6 days, with an activity exceeding 100 U/ml by day 31. Consequently, the increase in Lcc production observed after the addition of 2 mM Cu2+ was quicker and there was a higher activity than that observed after the addition of 1 mM Cu2+ . Conversely, no noticeable increase in Lcc production was observed in the MYPG liquid medium following the addition of any of the aromatic substances or Cu2+ （data not shown）. MYPG-S500 medium, with the activity remaining above 60 U/ml until day 35. MnP activities after 14 days of culture in the MYPG-S500 medium were significantly higher than the MnP activities obtained from the other two media（P < 0.001）. Lip activity was not detected in any of the culture media（data not shown）. Manganese peroxidase gene transcription The sequence of the fragment amplified from cDNA, which was prepared from a mixed pool of total RNA obtained from the mycelia of 10-, 15- and 18-day-old cultures cultured in MYPG-S500, was identical to that of lemnp2, which is known to code for extracellular MnP（LeMnP2）, but different from lemnp1, which is known to code for intracellular MnP（data not shown）. The finding indicated that sawdust extract induced the secretion of the same isozyme, LeMnP2, which is a major MnP isozyme that is secreted into sawdust media （Sakamoto et al., 2009）. The results of the Northern blotting analysis with a probe lemnp2 are shown in Figure 3. During culture on MYPG-S500, a detectable amount of lemnp2 mRNA was present at days 10-19 during the initial stage of culture, with the transcription increasing from day 22 to day 25 and then decreasing at day 28. These changes in lemnp2 transcription FIG. 2. Phenol-oxidizing activities of Lcc, Per and MnP from L. edodes cultured in liquid culture media containing different sawdust-extract concentrations. Symbols indicating the different enzymes are shown in the legend of the bottom graph. Values are means with standard errors（vertical bars）for cultures grown in triplicate. FIG. 3.（top）Northern blot analysis of lemnp2 gene transcript from L. edodes grown in MYPG-S500 liquid media. （bottom）10μg of total RNA used as a loading control. Days after inoculation are shown above each lane. Adapted from Tanesaka et al., 2012
PHENOL-OXIDIZING ENZYME EXPRESSION INLENTINULA EDODES TABLE 1.Addition of copper.phenolic and non-phenolic compounds and their effect on phenol oxidizing activities (U/ml)in L.edodes cultured on MYPG+ Days after substance was added' Substance Enzymes 0 d 6 10 17 20 31 Control 31 01a 022 0.3a a 326 Catechol 92 4.9 ne e Hydroquinone 0.3 0.3 1.0a 0.0a 88d e Guaiacol 8 a ne ne 0 42 1.9al ne ne Anisidine 0.3a 14a 怕伯 1 mM CuP 8 45.9b 89 4.6 .6a 1a 2 mM Cu 06 63.9b 98.0c 109 MnP 2.3 47a 3.4a Each substance was added 10 days after initial ino e reo the mean of thr r two replic ate flasks for Cu tters ar ne:not examined DISCUSSION ved in cultures aro wo in MYPG-s and not in MYPG-S. These observations suggested that MnP is he expression of lignin-degrading enzymes in only enhanced by specific,concentration-dependent wtoteutotobesicdiomceteshasbe attributed to the func the nig wth and nd Fc Cullen and Kersten 2004).Although L.edodes has only d due to the which was also observed in the low-concentration saw been reported dust extract (MYPG-.Transcription of the emnp sawa gene,which en odes t al 2009:Nag i et al 200g)the on MYPG-S. at d ings of the oresent study corroborated our onthese results.the addition of arpmatic compounds and copper 10 days after inoculation appeared to co ea produ 9 in hot pproximately,with the initiation of lemnp2 gene rar cnpt media was con components in the sawdust extract are unclear,the 2000;Piscitelli et al.,2011).However,in the presen compound(s) tor the ob study on L es,althougn the s sawdust extra 190 ned a var h was promoted in both MYPG- and MYPG-S. tested in this study enhanced I c media.a marked increase in MnP production was only production.In addition,all of the aromatic compounds
PHENOL-OXIDIZING ENZYME EXPRESSION IN LENTINULA EDODES 147 observed in cultures grown in MYPG-S500, and not in MYPG-S100. These observations suggested that MnP is only enhanced by specific, concentration-dependent, functional compounds in the high-concentration sawdust extract（MYPG-S500）. In other words, MnP enhancement was not only due to the vigorous growth, which was also observed in the low-concentration sawdust extract（MYPG-S100）. Transcription of the lemnp2 gene, which encodes LeMnP2, was initially observed at detectable levels by Northern blot analysis of cultures grown on MYPG-S500 at day 10 after inoculation. Based on these results, the addition of aromatic compounds and copper 10 days after inoculation appeared to coincide, approximately, with the initiation of lemnp2 gene transcription. In several white-rot fungi, Lcc production is promoted by aromatic substances（Fahraeus, 1962; Scheel et al., 2000; Piscitelli et al., 2011）. However, in the present study on L. edodes, although the sawdust extract likely contained a variety of phenolic and nonphenolic aromatic compounds（e.g. flavonoids）, none of the 10 mM aromatic compounds tested in this study enhanced Lcc production. In addition, all of the aromatic compounds DISCUSSION The expression of lignin-degrading enzymes in white-rot basidiomycetes has been attributed to the production of secondary metabolites under various growth and culture conditions（Kirk and Fenn, 1982; Cullen and Kersten 2004）. Although L. edodes has been reported to secrete the lignin-degrading enzymes Lcc and/or MnP when cultivated on sawdust-based media（Tokimoto et al., 1987; Makker et al., 2001; Sakamoto et al., 2009; Nagai et al., 2009）, the findings of the present study corroborated our previous studies（Saeki et al., 2011）and showed that extracts produced by autoclaving sawdust in hot water can also promote MnP activity. Compared to mycelial growth on MYPG medium, growth on MYPG-S100 and MYPG-S500 media was considerably more vigorous. Although the components in the sawdust extract are unclear, the compound（s）responsible for the observed vigorous growth may be flavonoids（Fahraeus, 1962; Ardon et al., 1996）. Interestingly, although mycelial growth was promoted in both MYPG-S100 and MYPG-S500 media, a marked increase in MnP production was only TABLE 1. Addition of copper, phenolic and non-phenolic compounds and their effect on phenol oxidizing activities（U/ml）in L. edodes cultured on MYPG+S500. Substance Enzymes Days after substance was added＊ 0 4 6 10 17 20 31 Control Lcc Per MnP 0.3† -0.1 0.3 0.0 ab‡ 0.7 2.5 0.1 a 2 a 5.7 0.2 a 7.2 b 20.1 c 0.2 a 16.4 c 45.8 b -0.3 a 22.2 c 57.0 b 0.3 a 27.9 65.9 b Catechol Lcc Per MnP 0.2 0.2 2.3 0.3 ab 3.2 5.0 0.5 a 3.2 a 4.9 1.5 a 2.0 a 4.1 a ne ne ne ne ne ne ne ne ne Hydroquinone Lcc Per MnP 0.3 0.1 0.7 -0.3 a 1.3 1.0 1.0 a 1.0 a 1.5 0.0 a 0.8 a 0.9 ab ne ne ne ne ne ne ne ne ne Guaiacol Lcc Per MnP 0.3 0.4 0.1 0.1 ab 0.7 1.2 -0.3 a 0.5 a 1.2 0.5 a 0.7 a 1.9 ab ne ne ne ne ne ne ne ne ne Anisidine Lcc Per MnP 0.4 0.6 1.7 0.3 ab 3.2 8.0 0.3 a 3.1 a 7.7 -0.3 a 1.4 a 3.7 ab ne ne ne ne ne ne ne ne ne 1 mM Cu2+ Lcc Per MnP 0.5 0.0 0.3 0.5 ab 0.4 1.4 0.3 a 0.2 a 0.8 0.2 a -0.1 a 0.4 ab 12.2 b -0.9 ab 0.6 a 27.0 b -0.1 a 0.7 a 45.9 b 4.6 -3.1 a 2 mM Cu2+ Lcc Per MnP 0.6 0.1 0.5 0.9 b 4.4 0.2 23.0 b 9.1 b 0.2 63.9 b 3.6 ab -2.3 a 98.0 c -4.0 a 4.7 a 95.1 c 2.7 ab -0.1 a 109 c 9.2 3.4 a ＊: Each substance was added 10 days after initial inoculation †: Each value represents the mean of three replicate flasks or two replicate flasks for Cu2+ ‡: Values with different lowercase letters are significantly different at the 5% level between treatments. Values with no lowercase letters are not significantly different between any pair of treatments on a given day ne: not examined
148 E.TANESAKA ET AL tested completely inhibited MnP production.These find However in this study Mnp inbibition by the additio inas those of a similar study on L.edode of ard matic compounds did not enhance Lcc produc which found that none of the 1 mM aromatic com tion.These enzyme expression dynamics are highly pounds tested (i.e.gallic acid,catechol,hydroxyben zolc acid, uction;rathe et al. 199 as well the findin vide sutficin Ikegaya et al.(1993)reported that the addition of 50 evidence for even the first part of the proposed nega ug/ml (0.33 mM)vanillin in a peptone-glucose liquid tive feedback interaction involving these two enzymes mediun may be attributable to differences in the concentrations ally independent. of substances added,nutritional conditions,or stocks mportantly.the and strans usd. in this study.ir rac nowr d to as Cu several fungi includino edodes (Nagai et al.,2002 wdust-based media) Cavallazzi et al. 2005:Saeki et al.2011).Indeed.Cu The liquid-culture media described here could therefore . be employed to the lignin-degradi van s an 4 s (Galhaun et al 2001 2002) ponent (s)in the sawdust extract and the correlation and T versicolor (Collins et al. 1997).For exar p between enzyme expression and regulation using this added) experimental system. ACKNOWLEDGMENTS et al 2002j Similarly,more than a 25-fold increase in Lcc produ This work was supported by the Academic Frontier' was achieved by the addition of 250uM or2 Project for Private Universities with a matching fund 20110. subsidy from e M coding the e (LAP2)of T.pu work was a fund from has copper-sensitive metal responsive elements(MRE Farmers Corporation, JA-Nakanoshi",Nakano. and 10 Nagano.Japan. M et al. 200 REFERENCES (Faraco et al,2003;Soden et al.,2003). Ardon,O.Kerem.Z and Hadar.Y.(1996)Enhancement o The main findings of this study were as follows a el. Cai,Y.,and Chang,S-T.(1995)Effect of only enhanced by the hiah Let.,T pletely inhibited M .M.and Sc ares,M.A (2005 C prod ion o odes in liquid medium.Brazn Microbio.36.383- anhanced le duction.Although the simultar ns and expression of MnP and Lcc was not observed in this study, e tr tion in eraction e nd MnP inhibitior biology ofignin degradat
148 E. TANESAKA ET AL. However, in this study, MnP inhibition by the addition of aromatic compounds did not enhance Lcc production. These enzyme expression dynamics are highly sensitive to nutrient nitrogen and manganese（Buswell et al., 1995）, as well as the fungal response to Cu2+ and aromatic compounds as discussed above. Indeed, the findings presented here do not provide sufficient evidence for even the first part of the proposed negative feedback interaction involving these two enzymes. However, the findings do show that MnP inhibition and Lcc production are affected by Cu2+ addition and that this inhibition-production relationship might be functionally independent. Importantly, the experimental conditions employed in this study, in which sawdust extract was shown to enhance mycelial growth and MnP production, were similar to the conditions employed to cultivate edible mushrooms（e.g., autoclaved sawdust-based media）. The liquid-culture media described here could therefore be employed to evaluate the lignin-degrading ability of cultivated mushroom varieties. Further analyses are therefore necessary to examine the functional componen（t s）in the sawdust extract and the correlation between enzyme expression and regulation using this experimental system. ACKNOWLEDGMENTS This work was supported by the“Academic Frontier” Project for Private Universities with a matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology（2004–2008）, Japan. This work was also partly supported by a fund from the Farmers Corporation,“JA-Nakanoshi”, Nakano, Nagano, Japan. REFERENCES Ardon, O., Kerem, Z., and Hadar, Y.（1996）Enhancement of laccase activity in liquid cultures of the ligninolytic fungus Pleurotus ostreatus by cotton stalk extract. J. Biotechnol., 51, 201-207. 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These findings corroborated those of a similar study on L. edodes which found that none of the 1 mM aromatic compounds tested（i.e. gallic acid, catechol, hydroxybenzoic acid, or vanillin）enhanced Lcc production; rather, the aromatic compounds either inhibited or repressed Lcc production（Cavallazzi et al., 2005）. In contrast, Ikegaya et al.（1993）reported that the addition of 50 μg/ml（0.33 mM）vanillin in a peptone-glucose liquid medium significantly promoted mycelial growth, laccase production and fruit-body formation by L. edodes. The differences observed in Lcc expression between studies may be attributable to differences in the concentrations of substances added, nutritional conditions, or stocks and strains used. The addition of metallic ions such as Cu2+ to media has also been used to induce Lcc production in several fungi, including L. edodes（Nagai et al., 2002; Cavallazzi et al., 2005; Saeki et al., 2011）. Indeed, Cu2+ is considered to be a strong Lcc inducer in the white-rot fungi, Pleurotus ostreatus（Palmieri et al., 2000; Faraco et al., 2003）, P. sajor-caju（Soden et al., 2001, 2003）, Trametes pubescens（Galhaup et al., 2001, 2002）, and T. versicolor（Collins et al., 1997）. For example, compared to a control treatment（no Cu2+ added）, a 1.5-fold increase in Lcc production was achieved in a liquid culture of L. edodes by the addition of 10 mM Cu2+ to the culture medium（Nagai et al., 2002）. Similarly, more than a 25-fold increase in Lcc production was achieved by the addition of 250μM or 2 mM Cu2+ to a liquid culture of L. edodes（Cavallazzi et al., 2005; Saeki et al., 2011）. The promoter region of the gene coding the major Lcc isozyme（LAP2）of T. pubescens has copper-sensitive metal responsive elements（MRE）, and Lcc gene transcription is induced within 10 h after the addition of 2 mM Cu2+ （Galhaup et al., 2001, 2002）. Copper regulation of Lcc by MRE consensus sequences has also been reported in Pleurotus spp （Faraco et al., 2003; Soden et al., 2003）. The main findings of this study were as follows: i） marked mycelial growth was observed in L. edodes cultured in low- and high-concentration sawdust extract produced from C. cuspidata. However, MnP production was only enhanced by the high-concentration sawdust extract; ii）all of the aromatic compounds tested completely inhibited MnP production and did not enhance Lcc production; iii）the addition of both 1 and 2 mM Cu2+ completely inhibited MnP production, but markedly enhanced Lcc production. Although the simultaneous expression of MnP and Lcc was not observed in this study, the hypothesis of whether a negative feedback interaction exists between MnP and Lcc is still unclear, i.e. whether MnP production initially inhibits Lcc production and MnP inhibition promotes Lcc production
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