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Induction of laccase activity in the edible straw mushroom, Volvariella volvacea
FEMS Microbiology Letters 218 (2003) 143^148
www.fems-microbiology.org
Induction of laccase activity in the edible straw mushroom, Volvariella volvacea Shicheng Chen a , Dengbo Ma a , Wei Ge a , John A. Buswell b
a;b;
a Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, PR China Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, PR China
Received 19 September 2002; accepted 4 November 2002 First published online 29 November 2002
Abstract Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste ‘compost’ representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 WM CuSO4 ) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development. 5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Laccase ; Mushroom; Secondary metabolism; Enzyme induction ; Volvariella volvacea
1. Introduction The edible straw mushroom, Volvariella volvacea, is among the most extensively cultivated mushrooms in tropical and sub-tropical regions. Although the mushroom currently ranks ¢fth in terms of annual production worldwide [1], biological e⁄ciency (i.e. conversion of growth substrate into mushroom fruit bodies) is considerably lower compared with the other major cultivated species. It has been suggested that the relatively low growth yields may be linked, in part, to an apparent inability of V. volvacea to produce laccase (p-diphenol oxidase, EC 1.10.3.2) [2]. This enzyme is widely distributed among fungi where it has been assigned roles in lignin degradation [3^5], in rendering phenolic compounds less toxic via oxidative coupling and polymerisation [6], and in mushroom fruit body morphogenesis [3,7,8]. Since all these three functions are of fundamental importance for the colonisation of the various lignocellulosic substrates used in mushroom cultivation systems and for sporophore development, we have re-assessed earlier studies that failed to detect laccase pro-
* Corresponding author. Tel. : +852 (2609) 6298; Fax : +852 (2603) 5645. E-mail address : [email protected] (J.A. Buswell).
duction by V. volvacea [2]. We now show that this commercially important mushroom produces multiple forms of the enzyme, and have identi¢ed various factors a¡ecting laccase induction during growth of the fungus in submerged culture. We also report some novel features associated with laccase production in both liquid culture and in miniaturised solid-state systems representative of the conditions used for industrial cultivation.
2. Materials and methods 2.1. Organism and growth conditions V. volvacea, strain V14, was obtained from the culture collection of the Centre for International Services to Mushroom Biotechnology located at The Chinese University of Hong Kong (accession no. CMB 002) and maintained on potato dextrose agar at room temperature with periodic transfer. The fungus was cultivated in stationary 250-ml Erlenmeyer £asks containing 50 ml of a de¢ned medium, pH 6.0, as described previously [9]. This medium was supplemented with di¡erent concentrations of CuSO4 as shown, and nitrogen was added as NH4 NO3 and L-asparagine at concentrations of 2.6 mM-N and 26 mMN for low nitrogen (LN) and high nitrogen (HN) media,
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respectively [10]. In experiments to examine the e¡ect of Cu on laccase production, CuSO4 was omitted from the trace element solution used to prepare the basal medium described in [9]. The e¡ect of aromatic compounds on laccase production was determined using basal medium with HN as described above. Cultures were grown for 3 days before the addition of inducer to a ¢nal concentration of 2 mM (0.02 mM in the case of 2,5-xylidine), and laccase activity was determined after a further 11 days incubation. In order to study laccase production during the di¡erent stages of fungal development, we have developed a miniaturised solid-state growth system to mimic the cultivation conditions used in commercial operations. Cylindricalshaped (6 cm diameter, 6 cm long), miniaturised compost cultures were prepared as follows: compost mixture consisting of 30 g (dry weight) cotton waste, 0.3 g lime and 60 ml distilled water was placed into a plastic bag which was then ¢tted with a stainless-steel neck ring and plugged with a paper plug. Bags were autoclaved at 121‡C for 1 h and, after cooling to room temperature, the centre of the upper surface of the compost was inoculated with 2U2cm2 pieces of fungal mycelium taken from a 7-day-old culture grown on potato dextrose agar (Difco) at 32‡C. Compost cultures were incubated at 32‡C for 12 days and then transferred to an environmental chamber maintained at 30‡C, 85% humidity, and with alternate 12 h (approximately) illumination (400 lux) and darkness periods. On day 15, the bags were removed to facilitate fruiting. 2.2. Sampling procedures Liquid cultures were harvested at speci¢ed time intervals and laccase activity was assayed in the culture supernatants. Fungal biomass was determined by ¢ltering mycelia through tared Whatman No. 1 ¢lter paper, washing with distilled water and drying to constant weight at 95‡C. At periodic intervals throughout a 25-day experimental period, the compost cultures (three replicates) were cut longitudinally through the centre of the cylinder and one portion (half the total weight of the compost culture) suspended in 10 volumes (v/dry weight) 50 mM KHPO4 bu¡er, pH 6.5, and shaken (150 rpm) for 2 h at 30‡C. In older cultures, fruit bodies at various stages of development were removed beforehand. The mixture was squeezed through nylon mesh, the ¢ltrate further clari¢ed by centrifugation at 5000Ug for 30 min at 4‡C, and the supernatant retained. This extraction procedure appears to be highly e¡ective since laccase levels detected in reaction mixtures containing samples of treated compost in place of bu¡er extract were always less than 5% of values recorded for corresponding volumes of extract supernatant.
2.3. Enzyme assay Laccase activity was determined at 32‡C using 2,2P-azino-bis-ethylbenthiazoline (ABTS) in reaction mixtures (1 ml) containing 0.1 M sodium acetate bu¡er (pH 5), 0.03% (w/v) ABTS, and an appropriate amount of culture supernatant. One unit of enzyme activity is de¢ned as the amount of enzyme required to oxidise 1 Wmol ABTS per minute using an O420 value for oxidised ABTS of 3.6U104 mol31 cm31 [11]. 2.4. Polyacrylamide gel electrophoresis (PAGE) and activity staining of gels Native PAGE (12% w/v gel) was performed on aliquots of culture £uids using the Mini-Protean II system (BioRad). Protein bands exhibiting laccase activity stained green with ABTS (0.03% w/v) in 0.125 M acetate bu¡er, pH 5.0. 2.5. Protein determination Protein was determined by the method of Bradford [12] with bovine serum albumin as standard.
3. Results and discussion 3.1. E¡ect of copper on laccase induction in submerged culture In submerged cultures of V. volvacea, two laccase isoforms were induced, one more strongly than the other, in response to copper supplementation of the culture medium. The e¡ect of di¡erent Cu concentrations on laccase production in 14-day HN cultures of V. volvacea V14 is shown in Fig. 1. Readily detectable levels of laccase were observed in cultures supplemented with 50^300 WM CuSO4 with the highest enzyme titres recorded at concentrations of 200 WM CuSO4 . No laccase activity was detected in the absence of added Cu. Copper supplementation at concentrations over the range 0^300 WM only marginally a¡ected fungal growth in HN cultures and the in£uence of Cu on laccase production was not due to increased biomass production. Native PAGE analysis of culture £uids and activity staining of the gels revealed at least two protein bands (a strong staining band and a fainter second band) with laccase activity (Fig. 1). Positive regulation of laccase protein and laccase gene transcription levels by Cu was also reported in species of Trametes [13,14] and Pleurotus [15^17]. Experiments with puri¢ed laccase showed that Cu not only induces laccase by the expression of laccase genes, but it also positively a¡ects the activity and stability of the enzyme [17]. The e¡ect on enzyme stability may be due to inhibition by Cu of an
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50 40 30 20 10 0 300
Concentration of copper sulphate (µM) Fig. 1. E¡ect of copper on fungal growth and laccase induction. E Biomass, F laccase. Values represent the mean T S.D. of triplicate samples. Bands representing laccase proteins were separated by native PAGE and visualised by activity staining with ABTS.
extracellular protease shown to be responsible for the degradation of laccase [18]. 3.2. Laccase production over time courses under HN and LN conditions Time courses for laccase activity in the extracellular
40
5 0 4
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8 10 12 14 16
Time (days)
10 0
ro l
ru l Fe
LN
ic
nt Co
yd H p-
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8 8 Day 12 10 10 12 Fig. 2. Time courses of laccase production by V. volvacea under low and high nutrient nitrogen conditions. F Biomass (HN+copper), b biomass (LN+copper), E laccase, (HN+copper), a laccase (LN+copper). Values represent the mean T S.D. of triplicate samples. Cultures contained 2.6 mM (LN) or 26 mM (HN) nitrogen and 200 WM copper sulphate. Bands representing laccase proteins were separated by native PAGE and visualised by activity staining with ABTS.
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£uid of LN and HN V. volvacea cultures supplemented with 200 WM CuSO4 are shown in Fig. 2. In LN cultures, laccase was detectable after 6 days but enzyme levels were relatively low (3.4 U l31 ) and increased only slightly to a maximum of 7.2 U l31 over the 16-day growth period. In HN cultures, laccase activity was detected after 8 days incubation and reached peak levels (31.8 U l31 ) at day 14. Native PAGE analysis of culture £uids and activity staining of the gels (Fig. 2) revealed the same band patterns observed in Cu-induced cultures. Increased fungal biomass was not solely responsible for the higher laccase activities in HN cultures of V. volvacea since maximal mycelial growth yields were approximately twice those of LN cultures compared to a 4.4-fold increase in enzyme levels (Fig. 2). Enzyme levels were also higher in HN cultures of Lentinula edodes [19], and a direct correlation was observed between nutrient nitrogen and the expression level of a laccase gene in Trametes versicolor [14]. In Pleurotus sajor-caju, although peak levels of total laccase activity were considerably higher in HN compared to LN cultures, increased fungal biomass production accounted for this di¡erence and enzyme speci¢c activities (i.e. U mg31 biomass) were similar under both sets of growth conditions [20]. A major distinguishing feature of laccase production in liquid cultures of V. volvacea is that enzyme activity under both LN and HN conditions was detectable in culture £uids only in the later stages of primary growth when fungal biomass production had reached a maximum. This is in sharp contrast to the pattern of laccase production reported for other basidiomycetes where enzyme synthesis is associated with primary growth [14,16,20,21]. Lignin peroxidase and manganese peroxidase, two other enzymes generally considered to play key roles in lignin
Biomass (g/L) and Laccase activity (IU/L)
Laccase activity (IU/L) and biomass (g/L)
60
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Aromatic compound Fig. 3. E¡ect of aromatic compounds on laccase production and isozyme distribution in V. volvacea. Values represent the mean T S.D. of triplicate samples. E Biomass, F laccase. Bands representing laccase proteins were separated by native PAGE and visualised by activity staining with ABTS.
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degradation, are both normally expressed in ligninolytic fungi at the onset of idiophase [22,23]. 3.3. E¡ect of aromatic compounds on laccase induction in submerged culture The induction of laccase by di¡erent aromatic compounds in HN cultures of V. volvacea containing a reduced CuSO4 content (1.6 WM) is shown in Fig. 3. After 11 days incubation in the presence of inducer, highest titres of laccase were observed in cultures supplemented with ferulic acid (52.2 U l31 ), 2,5-xylidine (43.5 U l31 ), veratric acid (43.2 U l31 ) and 4-hydroxybenzoic acid (33.6 U l31 ). 4-Hydroxybenzaldehyde also induced lower levels of laccase (12.3 U l31 ) but no enzyme activity was detected in cultures supplemented with p-coumaric acid, syringic acid, vanillic acid, homovanillin or catechol. Native PAGE analysis of laccase-induced culture £uids revealed two protein bands staining with ABTS. However, in this case, the bands stained with equal intensity. Basidiomycetes exhibit widely varying responses in terms of the e¡ects elicited by aromatic compounds on laccase production. Thus, an individual aromatic compound may increase laccase titres without altering the isozyme pattern [21], induce new laccase isoforms [24,25], or have no induction e¡ect [26]. Induction is considered to be a protective response to toxic compounds produced either during the degradation of the lignin component of lignocellulosic residues which serve as the natural growth substrates of the fungi or as antimicrobial agents secreted by microbial competitors [14,21].
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Time (days) Fig. 4. Extracellular laccase production during the developmental cycle of V. volvacea. F Laccase, 8 total protein, Values represent the mean T S.D. of triplicate samples. Development stages: 0^16 days, substrate colonisation phase; 17 days, pinheads; 22 days, button stage; 23 days, egg stage; 24 days, elongation stage; 25 days, mature fruit body.
3.4. Laccase production in compost cultures Laccase activity in extracts of composts prepared at periodic intervals during mycelial growth and fruit body formation is shown in Fig. 4. Colonisation of these miniaturised compost cultures was normally complete after 10 days incubation at 32‡C. Sporophore development occurred over 7^9 days with the ¢rst pinheads detected after 17 days, and buttons observed after 20^22 days, respectively. Once the button stage was reached, the fruit bodies grew very rapidly: the egg stage was evident on day 23, elongation took place on day 24, and formation of the mature fruit body was usually complete after 25 days. Only very low laccase levels were detectable throughout the substrate colonisation phase but enzyme activity increased markedly during the period from fruit body initiation up to the maturation stage. The increase in laccase activity was observed only in those composts that produced fully developed sporophores. Total protein in extracts of the compost increased throughout the substrate colonisation phase (10 days) and declined after the onset of fruiting (Fig. 4). The pattern of laccase production by V. volvacea in the compost cultures is therefore completely di¡erent from that observed with other mushroom species such as Agaricus bisporus [7,27], Pleurotus cornucopiae var. citrinopileatus [28] and L. edodes [29,30]. High laccase activities were recorded during mycelial growth of A. bisporus, when a large proportion of the compost lignin was degraded [7]. Moreover, laccase gene expression measured as mRNA levels was maximal at the fully colonised stage prior to fruiting and then declined to very low levels during fruiting [31]. Similarly, the activity of laccase is strongly regulated during the development of L. edodes fruit bodies [29,30]. The level of laccase transcripts during growth of the fungus on a sawdust-based substrate was maximal during the mycelial growth stage and then declined rapidly at the fruiting stage. Laccase activity in straw cultures of P. cornucopiae var. citrinopileatus was also highest during the vegetative mycelial growth phase and declined sharply at the onset of fruiting [28]. The earlier fructi¢cation and increased growth yields achieved using cotton waste ‘composts’ compared with the traditional rice straw substrate [32] have led to suggestions that V. volvacea is poorly equipped to degrade the lignin component of lignocellulosic residues commonly used for mushroom cultivation [2]. Although we have now demonstrated that the fungus does produce laccase, the temporal correlation established here between laccase production and sporophore formation suggests that, rather than a lignin-degrading function, the enzyme instead plays an important role in the morphogenesis of the V. volvacea fruit body. The association of laccase activity with the onset of secondary metabolism in submerged cultures of V. volvacea (Fig. 3) lends further support for such a role.
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De Vries and coworkers [8] reported that laccase was required for fruit body formation in Schizophyllum commune, and Bu’lock [33] has proposed that phenoloxidases such as laccase may crosslink hyphal walls into coherent aggregates during primordium initiation. It has also been proposed that laccase continues to act on the hyphal surfaces throughout fruit body development [34]. We have recently isolated and cloned four di¡erent laccase genes from V. volvacea (Chen, Ge and Buswell, to be published) and are now examining the expression of these genes under di¡erent culture conditions in order to assign physiological functions more de¢nitively to the gene products. A fuller understanding of enzyme expression during substrate colonisation and sporophore formation will facilitate the development of strategies aimed at improving mushroom yields.
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgements
[18]
We thank Ce Mun Tang for critical reading of this manuscript. This work was supported by a Grant (CUHK 4063/01M) from the Hong Kong Research Grants Council, and a Strategic Research Grant from The Chinese University of Hong Kong.
[19]
[20]
References
[21]
[1] Chang, S.T. (1999) World production of cultivated edible and medicinal mushrooms in 1997 with particular emphasis on Lentinula edodes (Berk.) Sing. in China. Int. J. Med. Mushrooms 1, 291^300. [2] Cai, Y.J., Buswell, J.A. and Chang, S.T. (1994) Production of cellulases and hemicellulases by the straw mushroom, Volvariella volvacea. Mycol. Res. 98, 1019^1024. [3] Thurston, C.F. (1994) The structure and function of fungal laccases. Microbiology 140, 19^26. [4] Bourbonnais, R. and Paice, M.G. (1990) Oxidation of non-phenolic substrates. An expanded role for laccase in lignin degradation. FEBS Lett. 267, 99^102. [5] Archibald, F. and Roy, B. (1992) Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor. Appl. Environ. Microbiol. 58, 1496^1499. [6] Bollag, J.-M., Shuttleworth, K.L. and Anderson, D.H. (1988) Laccase-mediated detoxi¢cation of phenolic compounds. Appl. Environ. Microbiol. 54, 3086^3091. [7] Wood, D.A. (1980) Inactivation of extracellular laccase during fruiting of Agaricus bisporus. J. Gen. Microbiol. 117, 339^345. [8] De Vries, O.M.H., Koolstra, W.H.C.F. and Wessels, G.H. (1986) Formation of an extracellular laccase by Schizophyllum commune dikaryon. J. Gen. Microbiol. 132, 2817^2826. [9] Cai, Y.J., Chapman, S.J., Buswell, J.A. and Chang, S.T. (1999) Production and distribution of endoglucanase, cellobiohydrolase, and L-glucosidase components of the cellulolytic system of Volvariella volvacea, the edible straw mushroom. Appl. Environ. Microbiol. 65, 553^559. [10] Buswell, J.A., Mollet, B. and Odier, E. (1984) Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen su⁄ciency. FEMS Microbiol. Lett. 25, 295^299. [11] Bourbonnais, R. and Paice, M.G. (1988) Veratryl alcohol oxidases
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from the lignin-degrading basidiomycete Pleurotus sajor-caju. Biochem. J. 255, 445^450. Bradford, M.M. (1976) A rapid and sensitive method for detecting microgram amounts of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. Yaver, D.S., Xu, F., Golightly, E.J., Brown, K.M., Brown, S.H., Rey, M.W., Schneider, P., Halkier, T., Mondorf, K. and Dalboge, H. (1996) Puri¢cation, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa. Appl. Environ. Microbiol. 62, 834^841. Collins, P.J. and Dobson, A.D.W. (1997) Regulation of laccase gene transcription in Trametes versicolor. Appl. Environ. Microbiol. 63, 3444^3450. Palmieri, G., Giardina, P., Bianco, C., Fontanella, B. and Sannia, G. (2000) Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Appl. Environ. Microbiol. 66, 920^924. Soden, D.M. and Dobson, A.D.W. (2001) Di¡erential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147, 1755^1763. Baldrian, P. and Gabriel, J. (2002) Copper and cadmium increase laccase activity in Pleurotus ostreatus. FEMS Microbiol. Lett. 206, 69^74. Palmieri, G., Bianco, C., Cennamo, G., Giardina, P., Marino, G., Monti, M. and Sannia, G. (2001) Puri¢cation, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus. Appl. Environ. Microbiol. 67, 2574^2579. Buswell, J.A., Cai, Y.J. and Chang, S.T. (1995) E¡ect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula (Lentinus) edodes. FEMS Microbiol. Lett. 128, 81^88. Fu, S.Y., Yu, H.-S. and Buswell, J.A. (1997) E¡ect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Pleurotus sajor-caju. FEMS Microbiol. Lett. 147, 133^137. Eggert, C., Temp, U. and Eriksson, K.-E. (1996) The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: puri¢cation and characterization of the laccase. Appl. Environ. Microbiol. 62, 1151^1158. Faison, B.D. and Kirk, T.K. (1985) Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 49, 299^304. Gold, M.H., Kuwahara, M., Chiu, A.A. and Glenn, J.K. (1984) Puri¢cation and characterization of an extracellular H2 O2 -requiring diarylpropane oxygenase from the white-rot basidiomycete, Phanerochate chrysosporium. Arch. Biochem. Biophys. 234, 353^362. Bollag, J.-M. and Leonowicz, A. (1984) Comparative studies of extracellular fungal laccases. Appl. Environ. Microbiol. 48, 849^854. Lo, S.C., Ho, Y.S. and Buswell, J.A. (2001) E¡ect of phenolic monomers on the production of laccases by the edible mushroom Pleurotus sajor-caju, and partial characterization of a major laccase component. Mycologia 93, 413^421. Coll, P.M., Fernandez-Abalos, J.M., Villanueva, J.R., Santamaria, R. and Perez, P. (1993) Puri¢cation and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971). Appl. Environ. Microbiol. 59, 2607^2613. Bonnen, A.M., Anton, L.H. and Orth, A.B. (1994) Lignin-degrading enzymes of the commercial button mushroom, Agaricus bisporus. Appl. Environ. Microbiol. 60, 960^965. Kaviyarasan, V. and Natarajan, K. (1997) Changes in extracellular enzyme activities during growth and fruiting of Pleurotus cornucopiae var. citrinopileatus. In: Advances in Mushroom Biology and Production (Rai, R.D., Dhar, B.L. and Verma, R.N., Eds.), pp 309^320. Mushroom Society of India, Solan. Ohga, S., Cho, N.S., Thurston, C.F. and Wood, D.A. (2000) Transcriptional regulation of laccase and cellulase genes in Lentinula edodes on a sawdust-based substrate. Mycoscience 41, 149^153. Ohga, S. and Royse, D.J. (2001) Transcriptional regulation of laccase
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and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust. FEMS Microbiol. Lett. 210, 111^115. [31] Ohga, S., Smith, M., Thurston, C.F. and Wood, D.A. (1999) Transcriptional regulation of laccase and cellulase genes in the mycelium of Agaricus bisporus during fruit body development on a solid substrate. Mycol. Res. 103, 1557^1560. [32] Chang, S.T. (1974) Production of the straw mushroom (Volvariella volvacea) from cotton wastes. Mushroom J. 503, 348^354.
[33] Bu’lock, J.D. (1967) Fungal metabolites with structural function. In: Essays in Biosynthesis and Microbial Development: E.R. Squibb Lectures on Chemistry of Microbial Products, pp. 1^18. John Wiley, New York. [34] Leatham, G.F. and Stahmann, M.A. (1981) Studies on the laccase of Lentinus edodes: speci¢city, localization and association with the development of fruiting bodies. J. Gen. Microbiol. 125, 147^ 157.
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Induction of laccase activity in the edible straw mushroom, Volvariella volvacea Shicheng Chen a , Dengbo Ma a , Wei Ge a , John A. Buswell b
a;b;
a Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, PR China Centre for International Services to Mushroom Biotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, PR China
Received 19 September 2002; accepted 4 November 2002 First published online 29 November 2002
Abstract Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste ‘compost’ representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 WM CuSO4 ) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development. 5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Laccase ; Mushroom; Secondary metabolism; Enzyme induction ; Volvariella volvacea
1. Introduction The edible straw mushroom, Volvariella volvacea, is among the most extensively cultivated mushrooms in tropical and sub-tropical regions. Although the mushroom currently ranks ¢fth in terms of annual production worldwide [1], biological e⁄ciency (i.e. conversion of growth substrate into mushroom fruit bodies) is considerably lower compared with the other major cultivated species. It has been suggested that the relatively low growth yields may be linked, in part, to an apparent inability of V. volvacea to produce laccase (p-diphenol oxidase, EC 1.10.3.2) [2]. This enzyme is widely distributed among fungi where it has been assigned roles in lignin degradation [3^5], in rendering phenolic compounds less toxic via oxidative coupling and polymerisation [6], and in mushroom fruit body morphogenesis [3,7,8]. Since all these three functions are of fundamental importance for the colonisation of the various lignocellulosic substrates used in mushroom cultivation systems and for sporophore development, we have re-assessed earlier studies that failed to detect laccase pro-
* Corresponding author. Tel. : +852 (2609) 6298; Fax : +852 (2603) 5645. E-mail address : [email protected] (J.A. Buswell).
duction by V. volvacea [2]. We now show that this commercially important mushroom produces multiple forms of the enzyme, and have identi¢ed various factors a¡ecting laccase induction during growth of the fungus in submerged culture. We also report some novel features associated with laccase production in both liquid culture and in miniaturised solid-state systems representative of the conditions used for industrial cultivation.
2. Materials and methods 2.1. Organism and growth conditions V. volvacea, strain V14, was obtained from the culture collection of the Centre for International Services to Mushroom Biotechnology located at The Chinese University of Hong Kong (accession no. CMB 002) and maintained on potato dextrose agar at room temperature with periodic transfer. The fungus was cultivated in stationary 250-ml Erlenmeyer £asks containing 50 ml of a de¢ned medium, pH 6.0, as described previously [9]. This medium was supplemented with di¡erent concentrations of CuSO4 as shown, and nitrogen was added as NH4 NO3 and L-asparagine at concentrations of 2.6 mM-N and 26 mMN for low nitrogen (LN) and high nitrogen (HN) media,
0378-1097 / 02 / $22.00 5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 1 1 3 1 - X
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respectively [10]. In experiments to examine the e¡ect of Cu on laccase production, CuSO4 was omitted from the trace element solution used to prepare the basal medium described in [9]. The e¡ect of aromatic compounds on laccase production was determined using basal medium with HN as described above. Cultures were grown for 3 days before the addition of inducer to a ¢nal concentration of 2 mM (0.02 mM in the case of 2,5-xylidine), and laccase activity was determined after a further 11 days incubation. In order to study laccase production during the di¡erent stages of fungal development, we have developed a miniaturised solid-state growth system to mimic the cultivation conditions used in commercial operations. Cylindricalshaped (6 cm diameter, 6 cm long), miniaturised compost cultures were prepared as follows: compost mixture consisting of 30 g (dry weight) cotton waste, 0.3 g lime and 60 ml distilled water was placed into a plastic bag which was then ¢tted with a stainless-steel neck ring and plugged with a paper plug. Bags were autoclaved at 121‡C for 1 h and, after cooling to room temperature, the centre of the upper surface of the compost was inoculated with 2U2cm2 pieces of fungal mycelium taken from a 7-day-old culture grown on potato dextrose agar (Difco) at 32‡C. Compost cultures were incubated at 32‡C for 12 days and then transferred to an environmental chamber maintained at 30‡C, 85% humidity, and with alternate 12 h (approximately) illumination (400 lux) and darkness periods. On day 15, the bags were removed to facilitate fruiting. 2.2. Sampling procedures Liquid cultures were harvested at speci¢ed time intervals and laccase activity was assayed in the culture supernatants. Fungal biomass was determined by ¢ltering mycelia through tared Whatman No. 1 ¢lter paper, washing with distilled water and drying to constant weight at 95‡C. At periodic intervals throughout a 25-day experimental period, the compost cultures (three replicates) were cut longitudinally through the centre of the cylinder and one portion (half the total weight of the compost culture) suspended in 10 volumes (v/dry weight) 50 mM KHPO4 bu¡er, pH 6.5, and shaken (150 rpm) for 2 h at 30‡C. In older cultures, fruit bodies at various stages of development were removed beforehand. The mixture was squeezed through nylon mesh, the ¢ltrate further clari¢ed by centrifugation at 5000Ug for 30 min at 4‡C, and the supernatant retained. This extraction procedure appears to be highly e¡ective since laccase levels detected in reaction mixtures containing samples of treated compost in place of bu¡er extract were always less than 5% of values recorded for corresponding volumes of extract supernatant.
2.3. Enzyme assay Laccase activity was determined at 32‡C using 2,2P-azino-bis-ethylbenthiazoline (ABTS) in reaction mixtures (1 ml) containing 0.1 M sodium acetate bu¡er (pH 5), 0.03% (w/v) ABTS, and an appropriate amount of culture supernatant. One unit of enzyme activity is de¢ned as the amount of enzyme required to oxidise 1 Wmol ABTS per minute using an O420 value for oxidised ABTS of 3.6U104 mol31 cm31 [11]. 2.4. Polyacrylamide gel electrophoresis (PAGE) and activity staining of gels Native PAGE (12% w/v gel) was performed on aliquots of culture £uids using the Mini-Protean II system (BioRad). Protein bands exhibiting laccase activity stained green with ABTS (0.03% w/v) in 0.125 M acetate bu¡er, pH 5.0. 2.5. Protein determination Protein was determined by the method of Bradford [12] with bovine serum albumin as standard.
3. Results and discussion 3.1. E¡ect of copper on laccase induction in submerged culture In submerged cultures of V. volvacea, two laccase isoforms were induced, one more strongly than the other, in response to copper supplementation of the culture medium. The e¡ect of di¡erent Cu concentrations on laccase production in 14-day HN cultures of V. volvacea V14 is shown in Fig. 1. Readily detectable levels of laccase were observed in cultures supplemented with 50^300 WM CuSO4 with the highest enzyme titres recorded at concentrations of 200 WM CuSO4 . No laccase activity was detected in the absence of added Cu. Copper supplementation at concentrations over the range 0^300 WM only marginally a¡ected fungal growth in HN cultures and the in£uence of Cu on laccase production was not due to increased biomass production. Native PAGE analysis of culture £uids and activity staining of the gels revealed at least two protein bands (a strong staining band and a fainter second band) with laccase activity (Fig. 1). Positive regulation of laccase protein and laccase gene transcription levels by Cu was also reported in species of Trametes [13,14] and Pleurotus [15^17]. Experiments with puri¢ed laccase showed that Cu not only induces laccase by the expression of laccase genes, but it also positively a¡ects the activity and stability of the enzyme [17]. The e¡ect on enzyme stability may be due to inhibition by Cu of an
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50 40 30 20 10 0 300
Concentration of copper sulphate (µM) Fig. 1. E¡ect of copper on fungal growth and laccase induction. E Biomass, F laccase. Values represent the mean T S.D. of triplicate samples. Bands representing laccase proteins were separated by native PAGE and visualised by activity staining with ABTS.
extracellular protease shown to be responsible for the degradation of laccase [18]. 3.2. Laccase production over time courses under HN and LN conditions Time courses for laccase activity in the extracellular
40
5 0 4
6
8 10 12 14 16
Time (days)
10 0
ro l
ru l Fe
LN
ic
nt Co
yd H p-
4H
8 8 Day 12 10 10 12 Fig. 2. Time courses of laccase production by V. volvacea under low and high nutrient nitrogen conditions. F Biomass (HN+copper), b biomass (LN+copper), E laccase, (HN+copper), a laccase (LN+copper). Values represent the mean T S.D. of triplicate samples. Cultures contained 2.6 mM (LN) or 26 mM (HN) nitrogen and 200 WM copper sulphate. Bands representing laccase proteins were separated by native PAGE and visualised by activity staining with ABTS.
yd
ro
ro x
xy
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in e
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20
yl id
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X
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ac id
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50
e
1
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at ric
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er
20
yd
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V
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eh
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Laccase activity (IU/L)
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za ld
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en
250
oi ca ci d
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yb
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ac id
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£uid of LN and HN V. volvacea cultures supplemented with 200 WM CuSO4 are shown in Fig. 2. In LN cultures, laccase was detectable after 6 days but enzyme levels were relatively low (3.4 U l31 ) and increased only slightly to a maximum of 7.2 U l31 over the 16-day growth period. In HN cultures, laccase activity was detected after 8 days incubation and reached peak levels (31.8 U l31 ) at day 14. Native PAGE analysis of culture £uids and activity staining of the gels (Fig. 2) revealed the same band patterns observed in Cu-induced cultures. Increased fungal biomass was not solely responsible for the higher laccase activities in HN cultures of V. volvacea since maximal mycelial growth yields were approximately twice those of LN cultures compared to a 4.4-fold increase in enzyme levels (Fig. 2). Enzyme levels were also higher in HN cultures of Lentinula edodes [19], and a direct correlation was observed between nutrient nitrogen and the expression level of a laccase gene in Trametes versicolor [14]. In Pleurotus sajor-caju, although peak levels of total laccase activity were considerably higher in HN compared to LN cultures, increased fungal biomass production accounted for this di¡erence and enzyme speci¢c activities (i.e. U mg31 biomass) were similar under both sets of growth conditions [20]. A major distinguishing feature of laccase production in liquid cultures of V. volvacea is that enzyme activity under both LN and HN conditions was detectable in culture £uids only in the later stages of primary growth when fungal biomass production had reached a maximum. This is in sharp contrast to the pattern of laccase production reported for other basidiomycetes where enzyme synthesis is associated with primary growth [14,16,20,21]. Lignin peroxidase and manganese peroxidase, two other enzymes generally considered to play key roles in lignin
Biomass (g/L) and Laccase activity (IU/L)
Laccase activity (IU/L) and biomass (g/L)
60
145
Aromatic compound Fig. 3. E¡ect of aromatic compounds on laccase production and isozyme distribution in V. volvacea. Values represent the mean T S.D. of triplicate samples. E Biomass, F laccase. Bands representing laccase proteins were separated by native PAGE and visualised by activity staining with ABTS.
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degradation, are both normally expressed in ligninolytic fungi at the onset of idiophase [22,23]. 3.3. E¡ect of aromatic compounds on laccase induction in submerged culture The induction of laccase by di¡erent aromatic compounds in HN cultures of V. volvacea containing a reduced CuSO4 content (1.6 WM) is shown in Fig. 3. After 11 days incubation in the presence of inducer, highest titres of laccase were observed in cultures supplemented with ferulic acid (52.2 U l31 ), 2,5-xylidine (43.5 U l31 ), veratric acid (43.2 U l31 ) and 4-hydroxybenzoic acid (33.6 U l31 ). 4-Hydroxybenzaldehyde also induced lower levels of laccase (12.3 U l31 ) but no enzyme activity was detected in cultures supplemented with p-coumaric acid, syringic acid, vanillic acid, homovanillin or catechol. Native PAGE analysis of laccase-induced culture £uids revealed two protein bands staining with ABTS. However, in this case, the bands stained with equal intensity. Basidiomycetes exhibit widely varying responses in terms of the e¡ects elicited by aromatic compounds on laccase production. Thus, an individual aromatic compound may increase laccase titres without altering the isozyme pattern [21], induce new laccase isoforms [24,25], or have no induction e¡ect [26]. Induction is considered to be a protective response to toxic compounds produced either during the degradation of the lignin component of lignocellulosic residues which serve as the natural growth substrates of the fungi or as antimicrobial agents secreted by microbial competitors [14,21].
3
800
2.5
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200
1
0
Total protein (mg/g CW)
Laccase activity (mU/g CW)
1000
0.5 0
6
10
15
17
22
24
Time (days) Fig. 4. Extracellular laccase production during the developmental cycle of V. volvacea. F Laccase, 8 total protein, Values represent the mean T S.D. of triplicate samples. Development stages: 0^16 days, substrate colonisation phase; 17 days, pinheads; 22 days, button stage; 23 days, egg stage; 24 days, elongation stage; 25 days, mature fruit body.
3.4. Laccase production in compost cultures Laccase activity in extracts of composts prepared at periodic intervals during mycelial growth and fruit body formation is shown in Fig. 4. Colonisation of these miniaturised compost cultures was normally complete after 10 days incubation at 32‡C. Sporophore development occurred over 7^9 days with the ¢rst pinheads detected after 17 days, and buttons observed after 20^22 days, respectively. Once the button stage was reached, the fruit bodies grew very rapidly: the egg stage was evident on day 23, elongation took place on day 24, and formation of the mature fruit body was usually complete after 25 days. Only very low laccase levels were detectable throughout the substrate colonisation phase but enzyme activity increased markedly during the period from fruit body initiation up to the maturation stage. The increase in laccase activity was observed only in those composts that produced fully developed sporophores. Total protein in extracts of the compost increased throughout the substrate colonisation phase (10 days) and declined after the onset of fruiting (Fig. 4). The pattern of laccase production by V. volvacea in the compost cultures is therefore completely di¡erent from that observed with other mushroom species such as Agaricus bisporus [7,27], Pleurotus cornucopiae var. citrinopileatus [28] and L. edodes [29,30]. High laccase activities were recorded during mycelial growth of A. bisporus, when a large proportion of the compost lignin was degraded [7]. Moreover, laccase gene expression measured as mRNA levels was maximal at the fully colonised stage prior to fruiting and then declined to very low levels during fruiting [31]. Similarly, the activity of laccase is strongly regulated during the development of L. edodes fruit bodies [29,30]. The level of laccase transcripts during growth of the fungus on a sawdust-based substrate was maximal during the mycelial growth stage and then declined rapidly at the fruiting stage. Laccase activity in straw cultures of P. cornucopiae var. citrinopileatus was also highest during the vegetative mycelial growth phase and declined sharply at the onset of fruiting [28]. The earlier fructi¢cation and increased growth yields achieved using cotton waste ‘composts’ compared with the traditional rice straw substrate [32] have led to suggestions that V. volvacea is poorly equipped to degrade the lignin component of lignocellulosic residues commonly used for mushroom cultivation [2]. Although we have now demonstrated that the fungus does produce laccase, the temporal correlation established here between laccase production and sporophore formation suggests that, rather than a lignin-degrading function, the enzyme instead plays an important role in the morphogenesis of the V. volvacea fruit body. The association of laccase activity with the onset of secondary metabolism in submerged cultures of V. volvacea (Fig. 3) lends further support for such a role.
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De Vries and coworkers [8] reported that laccase was required for fruit body formation in Schizophyllum commune, and Bu’lock [33] has proposed that phenoloxidases such as laccase may crosslink hyphal walls into coherent aggregates during primordium initiation. It has also been proposed that laccase continues to act on the hyphal surfaces throughout fruit body development [34]. We have recently isolated and cloned four di¡erent laccase genes from V. volvacea (Chen, Ge and Buswell, to be published) and are now examining the expression of these genes under di¡erent culture conditions in order to assign physiological functions more de¢nitively to the gene products. A fuller understanding of enzyme expression during substrate colonisation and sporophore formation will facilitate the development of strategies aimed at improving mushroom yields.
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgements
[18]
We thank Ce Mun Tang for critical reading of this manuscript. This work was supported by a Grant (CUHK 4063/01M) from the Hong Kong Research Grants Council, and a Strategic Research Grant from The Chinese University of Hong Kong.
[19]
[20]
References
[21]
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