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Two peaks of carbohydrase activities were observed, the ®rst relating to the early mycelial growth during the ®rst days after spawning and the second during ...
World Journal of Microbiology & Biotechnology 14, 513±519

Extracellular enzyme activities in six Lentinula edodes strains during cultivation in wheat straw G. Mata and J.-M. Savoie* Lentinula edodes (Berk.) Pegler is found in nature on dead broadleaf trees, but it is commercially produced on different substrates. The question of adaptation to different lignocellulosic substrates was addressed by measuring enzyme activities produced by six strains that were cultivated on wheat straw and that were able to produce sporophores. Despite quantitative variations, each strain of L. edodes had similar patterns of enzyme secretion into the wheat straw log matrix. Two peaks of carbohydrase activities were observed, the ®rst relating to the early mycelial growth during the ®rst days after spawning and the second during sporophore extension. Laccase activity in the early stage of colonization was related to the degradation of soluble phenolic compounds present in wheat straw. Manganese peroxidase activity was associated with mycelial growth. The strains with the earlier production and higher yield were able to hydrolyse and utilize straw cell wall components soon after inoculation, and developed high metabolic activities. Key words: Edible mushroom, hydrolases, Lentinula edodes, oxidases, shiitake, sporophore production.

Shiitake, Lentinula edodes (Berk.) Pegler is the second most important cultivated mushroom in the world (Chang 1996). The improvement of cultivation techniques tends to produce shiitake under conditions very different from its natural habitat. Traditionally, this white rot fungus is produced in Asia by inoculating wood logs in a climate favourable for outdoor cultivation. A synthetic-log system with indoor cultivation is now used in several European and American countries. It is based on plastic bags containing supplemented sawdust compacted in log form. The substrate is generally sterilized (Diehle & Royse 1986), but the feasibility of lower temperature treatments known as pasteurization has also been demonstrated (Delpech & Olivier 1991; Rinker 1991; Levanon et al. 1993). Delpech & Olivier (1991) proposed to cultivate shiitake by using cereal straw as substrate instead of wood products. This technique is now used on the industrial scale but the number of L. edodes strains able to produce sporophores on straw-based substrates is limited. Due to the differences between cultivation substrates,

G. Mata is with the Instituto de Ecologia, Apartado Postal 63, Xalapa, Veracruz 91000, Mexico. J.-M. Savoie is with the Station de Recherches sur les Champignons, INRA, BP 81, 33883 Villenave d'Ornon, France; fax: (+33) 05 56 84 31 78. *Corresponding author.

the selection of substrate-adapted genotypes is a way to improve shiitake production and to develop information about adaptation of saprotrophic fungi to speci®c lignocellulosic substrates. A difference in the control mechanism for inducible enzymes that degrade macromolecules was proposed as the major difference between a normal and a mutant strain of L. edodes with contrasting growth characteristics in a sawdust medium (ItaÈvaara et al. 1992). The types and levels of enzymes that can be produced for the depolymerization of some lignin constituents into absorbable forms are actually de®ned by the speci®c fungal genotype; it is also a component of the adaptation to a speci®c substrate (Shearer 1995). Changes in enzyme activities with the life cycle in saprotrophic Basidiomycetes is often observed (Wood & Goodenough 1977). Enzyme activities were studied during cultivation of L. edodes on wood derivatives but generally with only one strain in each study (Leatham 1985; Tokimoto et al. 1987; Matsumoto 1988). However, there is a lack of available information about cultivation on cereal- and straw-based substrates. The aim of the present work was to determine the spectrum of enzymes produced by six strains of L. edodes during cultivation on wheat straw and to determine if the levels of activities correlated with their ability to form sporophores.

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G. Mata and J.-M. Savoie

Materials and Methods Strains The six strains studied were maintained on malt extract agar (MEA). Three strains were cultivars obtained from spawn makers: VO84 (LeLion, France), 4055 (Sylvan, France), M115 (Lambert Spawn, USA). Three strains were hybrids of VO84 ´ 4055 (A9 A10 B19) obtained from the culture collection of INRA Mushroom Research Unit, France. Cultivation Rye grain was boiled in water and sterilized by autoclaving twice at 121 °C for 1 hour with incubation at room temperature for 48 hours between the two cycles. Bags containing 300 g of moist grains (65% H2O) were inoculated with 1 cm2 of MEA medium with L. edodes and placed at 25 °C in the dark for 28 days. Before use as inoculum for wheat straw, the grain precultures were stored at 4 °C for 7 days and then conditioned at 25 °C for 2 days. Wheat straw was shredded into pieces of 4± 6 cm in length and soaked in water for 24 hours at room temperature. After leaching, 10% (w/w) of gypsum was mixed with the straw and 1 kg of the mixture was placed in a microporous plastic bag for sterilization by autoclaving at 121 °C for 1 hour. Each block of substrate was inoculated under sterile conditions with 70 g of grain preculture by mixing the grains and the substrate in the bag in order to obtain a homogeneous mixture. For each strain, 15 blocks were prepared and inoculated simultaneously. The blocks were incubated at 25 ‹ 1 °C with a 12 hours light/12 hours dark cycle for 6 weeks. The plastic bags were then removed and the temperature decreased to 17 °C with the relative humidity maintained at 90%. This treatment was necessary to induce sporophore production after the spawnrunning period. The productivity of each strain was estimated by cumulating the yield of sporophores produced by ®ve blocks in one ¯ush and expressed as grams of mushroom (wet weight) per kg of substrate (wet weight). The sporophores were harvested at the beginning of cap opening. After the initial harvest no other sporophore production was observed. Sampling and Preparation of the Crude Enzyme Extract Samples were taken off just after spawning (sample 1). One block of each strain was then sampled every week during the spawn-running period (samples 2±7). The following samplings were taken when primordia were observable on the blocks (sample 8), at fruiting just prior to the harvest of sporophores (sample 9) and 1 week after harvesting (sample 10). The surface of the blocks (about 1.5 cm depth) was removed and the resting substrate was homogenized, lyophilized and ground in a domestic grinder. The powder was stored at 4 °C until used. Crude enzyme extracts were prepared in 100 ml ¯asks containing 0.7 g of powder and 10 ml of deionized water with 0.05% of Microo-Protect (Boehringer). The ¯asks were rotated end-over-end at 45 rev/min (for 30 min). The solids were removed by ®ltration through nylon mesh and centrifugation for 15 min at 12,000 ´ g, 4 °C. The crude enzyme extracts were used immediately for enzyme assays. Enzyme Assays All the enzyme activities were assayed by standard procedures using the pH known to be optimum for each activity. Endoglucanases (CMC), xylanases (XYL), mannanases (MAN) and laminarinases (LAM) were assayed in 0.1 M acetate pH 5.0 by

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release of reducing sugars measured with the DNS (3,5-dinitrosalicylic acid) reagent (Miller 1959), using the appropriate substrates which were respectively: carboxymethyl cellulose, xylan, mannan and laminarin (Mandels et al. 1976; Wood & Goodenough 1977). Standard curves were obtained with glucose for CMC and LAM, with xylose for XYL and mannose for MAN. b-Glucosidases (b-GLU), b-xylosidases (b-XYL), b-mannosidases (b-MAN), b-galactosidases (b-GAL), b-N-acetyl-glucosaminidases (NAG), and acid phosphatases (A-PH) were assayed in 0.1 M acetate pH 5.0 by monitoring the release of p-nitrophenol from p-nitrophenyl-b-D-glucopyranoside, p-nitrophenylb-D-xylopyranoside, p-nitrophenyl-b-D-mannopyranoside, p-nitrophenyl-b-D-galactopyranoside, p-nitrophenyl-N-acetyl-D-glucosamine, and p-nitrophenyl-phosphate respectively (Wood & Goodenough 1977). Basic phosphatase (B-PH) activity was assayed at pH 9.0 in 0.1 M NaOH±glycine. Standard curves were obtained with dilution of p-nitrophenol. Protease (PRO) was assayed with azocoll in 0.1 M acetate pH 5, by measuring the absorbency at 520 nm after removing the insoluble substrate (Jones & Grainger 1983). Laccase activity (LAC) was assayed by the oxidation of 3-dimethylaminobenzoic acid (DMAB) and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) in 50 mM succinate/lactate, pH 5.0. Changes in absorbance at 590 nm ( ˆ 3.29 104 M)1 cm)1) were monitored for 2 min (Lonergan & Baker 1995). MnSO4 and H2O2 were added to the mixture and the data obtained without MnSO4 and H2O2 (LAC) were subtracted to measure Mn-peroxidase activity (MnP). All the enzyme assays were performed at 30 °C. All enzyme activities except protease activity were expressed as U g)1 of cultivation substrate de®ned as the amount of enzyme producing 1 lmol of product min)1 g)1 of substrate extracted. The values given in the ®gures are the means of ®ve replications of measurements. Water Soluble Compounds The crude enzyme extracts were analysed for their reducing sugar and phenol contents. Water soluble reducing sugars (WSRS) were assayed with the DNS reagent (Miller 1959). Water-soluble phenols (WSP) were assayed with the Folin± Ciocalteu reagent (Box 1983). The values given in the ®gures are the means of ®ve replications of measurements. Metabolic Activity The fungal biomass-associated metabolic activity per weight of substrate was determined by measuring the ¯uorescein-diacetate-hydrolysing (FDA) activity (Lestan et al. 1996) in freshly collected samples without prior extraction of colonized wheat straw (Inbar et al. 1991; Libmond & Savoie 1993). One unit of FDA activity was equivalent to the hydrolysis of 1 lmol of FDA min)1 g)1 (dry weight) of substrate. FDA activity was not measured after the spawn-running period. For fruiting, the bags were removed and microorganisms other than L. edodes could hydrolyse FDA.

Results Productivity The productivity of VO84, M115 and A9 was 148 g kg)1 of culture medium (wet weight) 113 g kg)1 and 137 g kg)1 respectively. The three strains were classi®ed in the group of high yielding strains. The productivity of

Enzyme activities of shiitake in straw the low yielding strains was only 55 g kg)1, 39 g kg)1 and 32 kg)1 for A10, 4055 and B19 respectively. Primordia appeared 46 days after spawning for A9 and 48 days after spawning for VO84 and M115 whereas they appeared at day 49 for A10 and B19 and at day 52 for 4055.

(Figure 2) resulted from the high carbohydrase activities at fruiting. The level of enzyme activities at fruiting were related to the productivity of the strains. The means of activities of CMC, XYL, LAM, NAG were signi®cantly higher in the group with high yields than in the other group. At 42 days, no signi®cant difference between the two groups was observed and only CMC and XYL activities were signi®cantly and positively correlated with FDA activity. However A9 had the highest and 4055 the lowest activities for LAM and CMC. The oxidase activities (LAC, MnP) were signi®cantly lower during fruiting than during spawn-running and post-harvest periods (Figure 1). At 42 days no signi®cant correlation between LAC, MnP and FDA activity were observed but LAC and CMC were signi®cantly negatively correlated (T ˆ )0.97). During the spawn-running period, MnP activity increased to reach higher values between 28 and 42 days whereas LAC activity peaked signi®cantly at 7 days or 14 days, for most of the strains. In A10 and B19 however, MnP activity decreased signi®cantly between 35 days and 42 days. The pattern of WSP concentrations (Figure 2) appeared more related to LAC than to MnP activity. For each strain, LAC activity and WSP concentration were negatively related. This relationship was especially clear during the peak of LAC activity at the beginning of spawn-running. Low differences between 7 and 14 days were observed for both LAC activity and WSP concentration for V.O.84 and M115 whereas for other strains, LAC activity decreased and WSP concentration increased signi®cantly.

Metabolic Activity For each strain, a plateau of FDA activity was reached rapidly during the ®rst weeks of spawn-running (Table 1). After 28 days, the activity increased to reach values 2- to 4-fold higher at 42 days. No relationship between the metabolic activity at 42 days and the groups of productivity can be de®ned. However, A9, which showed the fastest primordia production, had the highest metabolic activity at the end of the spawning period whereas 4055, with the slowest primordia production, had the lowest metabolic activity. At spawning, the FDA activity due to the biological potential of the spawning material was signi®cantly greater in the high yielding group than in the low yielding group. Enzyme Activities and Changes in Water Soluble Compounds A-PH, B-PH, MAN, PRO, b-XYL, b-GAL, were not detected at signi®cant levels in this experiment. For most of the strains, CMC, XYL and LAM activities were signi®cant at spawning due to the activities present in the spawning material, and a peak of activities at 7 days was observed (Figure 1). The peak was associated with the increase in metabolic activity during the ®rst week (Table 1). A release of WSRS resulted from these activities (Figure 2). The high concentrations of WSRS decreased after 7 days and reached a plateau during the following part of the spawn-running period. The CMC, XYL, and LAM activities at the end of the spawn-running period were lower or equal to that at spawning and at 7 days whereas they were higher for b-GLU and b-MAN (Figure 1). For each strain, CMC, bGLU, XYL, b-MAN, LAM and NAG activities were signi®cantly higher at the stage of primordia than at 42 days and increased until fruiting. After harvesting they decreased to reach values close to those observed at 42 days. A second maximum of WSRS concentration

Discussion The results of this study show that each strain of L. edodes had similar patterns for secreting enzymes into the wheat straw log matrix. The same patterns were observed in other experiments with both sterilized and pasteurized wheat straw (unpublished data). Two cycles of carbohydrase activities were observed, with the ®rst related to the early mycelial growth during the ®rst days after spawning and the second during sporophore extension.

Table 1. Biological potential of six strains of Lentinula edodes during cultivation in wheat straw. Values are means of ®ve replicates of measurement of FDA activity (U g)1) and standard errors in brackets. Sample*

Strains VO84

1 2 3 5 7

1.3 11.6 10.4 14.0 32.6

(1.24) (0.62) (0.69) (1.75) (3.59)

M115 1.4 11.2 11.3 7.0 33.4

(0.16) (0.46) (0.19) (0.64) (4.66)

A9 1.4 15.1 22.4 21.9 50.7

(0.43) (0.79) (1.94) (2.46) (4.83)

4055 0.2 9.1 8.6 8.6 23.0

(0.40) (3.52) (0.33) (1.35) (2.86)

A10 0.8 17.9 11.9 14.4 45.5

(0.11) (4.32) (1.20) (0.99) (3.04)

B19 0.4 (0.19) 19.0 (1.33) 25.7 (1.24) 15.8 (2.25) 33.3(15.3)

* See Materials and Methods section for sample de®nitions.

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G. Mata and J.-M. Savoie Increases in carbohydrase activities in wood matrix at primordation and during sporophore production have been reported previously (Leatham 1985; Tokimoto et al.

1987; Matsumoto 1988; Okeke et al. 1994). In the present study, the level of carbohydrase activities was related to the yield of sporophores, this has also been reported for

Figure 1. Enzyme activities in straw-based substrates during cultivation of six strains of Lentinula edodes. See Materials and Methods for de®nition of sample numbers.

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Enzyme activities of shiitake in straw

Figure 2. Concentrations of water-soluble reducing sugars and phenols in straw-based substrates during cultivation of six strains of Lentinula edodes. See Materials and Methods for de®nition of sample numbers.

Agaricus bisporus (Wood & Goodenough 1977). Sporophore production was probably dependent on the ability to mobilize new carbon resources from polysaccharides. The maxima of carbohydrase activities at 7 days or 14 days were actually related with a signi®cant increase in the metabolic activity and maxima of concentrations of water-soluble sugars. In the present study, 4055, which was a low yielding strain, had the lowest metabolic activity throughout the spawn-running period, high carbohydrase activities at 7 days and the highest concentration of water soluble sugars until 21 days. This contrasts with the high yielding hybrid A9, which had the lowest carbohydrase activities and the lowest watersoluble sugar concentrations at 7 days whereas its metabolic activity was high. Early peaks of carbohydrase activities were previously observed by Leatham (1985) in laboratory scale experiments on oak wood chips. Cellulose and non-cellulose polysaccharides in straw cell walls are generally considered to be more available to hydrolytic enzymes than those in wood because of the lower lignin content. The observed release of excess watersoluble reducing sugars in the wheat straw log matrix is an important consequence of the maxima of carbohydrase activities. If they are not utilized rapidly by L. edodes they are putative substrates for other fungi or

bacteria. In complex substrates spores of some microorganisms are resistant to sterilization treatments or can contaminate the substrate afterwards. An alternative and less expensive cultivation technique, without sterilization, is also used to produce shiitake (Delpech & Olivier 1991; Levanon et al. 1993), but the colonization of the substrate by competitors utilizing available sugars released by the activity of L. edodes has been reported to be a problem. In the light of the present data, a high adaptation to sporophore production in wheat straw-based substrates by L. edodes is dependent on a high biological potential of the spawn and a high metabolic activity during spawnrunning for which FDA activity was found to be a suitable assay (Boyle & Kropp 1992; Lestan et al. 1996). However, the case of B19 shows that a high biological potential in the spawning materials and a high metabolic activity during the ®rst days after spawning were not suf®cient to obtain suitable yields. Other nutritional factors may be limiting during fruiting. Otherwise, the metabolic activity in the early mycelial growth of L. edodes in wheat straw was not directly related to secretions of enzyme but more probably linked with the ability to utilize soluble carbon resources. Differences in hyphal uptake rate could be one of the factors resulting in high-yielding and low-yielding strains. Carbohydrase activities were necessary to degrade carbohydrates but they were apparently not limiting for growth. At the end of the spawn-running period however, the metabolic activity was correlated with CMC and XYL activities. This could be the result of changes in substrate composition during the cultivation due to the action of carbohydrases and phenol oxidases. By cultivating L. edodes on wheat straw, in a laboratory scale experiment, Myoson & Verachter (1991) observed that lignin was degraded only after several weeks and then an improvement of enzymatic digestibility up to 43.5%, was obtained for 2 weeks without other changes afterward. Lignin is generally considered to be the major biodegradation obstacle in wood but several studies show that the ®ndings obtained with wood are rarely valid with cereal straw (Sharma et al. 1986; Miron & Ben-Ghedalia 1992; Savoie et al. 1994). The chemical nature of lignins and how they are linked with other cell wall polymers is as important as the total amount of lignin present (Cornu et al. 1994). The pattern of LAC activity in wood logs was previously described as complex (Leatham 1985). Okeke et al. (1994), observed one or two maxima during spawn-running and an increase of activity with the cold shock used to induce fruiting. In the present study in wheat straw, no cold shock was used but the incubation temperature was reduced to 17 °C for fruiting. As a consequence no increase of LAC activity was observed at the onset of primordiation and only one

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G. Mata and J.-M. Savoie peak at 7 days or 14 days was observed for each strain except 4055. Such differences in patterns could be related to differences in lignin and phenolic components in wheat straw and oak wood. LAC activity was related to WSP concentration for each strain. Laccases are known as inducible enzymes and a strain-dependent effect of the concentrations of water-soluble lignin derivatives on laccase induction has been reported for L. edodes (Savoie et al. 1995). Induction of LAC activity by the aromatic monomeric components of the water-soluble fraction of straw (Crestini et al. 1996) could be responsible for the high activities measured during the ®rst weeks of cultivation. MnP activity increased during spawn-running with a complex pattern and was clearly inactivated during primordiation. The pattern of Mn activity could be due to the fact that MnP is produced under nutrient nitrogen limitation (Buswell et al. 1996). Similar patterns have been reported for laccase and Mn-peroxidase in A. bisporus but the inactivation was effective only during sporophore extension (Bonnen et al. 1994). The changes in activities and WSP concentration during spawn-running observed here suggest that MnP activity could be involved in substrate depolymerization whereas LAC activity could be related to detoxi®cation of soluble phenolic compounds (Buswell et al. 1996). Comparisons of the pattern of enzyme activity secreted in the straw log matrix with reported patterns on wood logs are in agreement with the differences in time necessary to obtain fruiting bodies, which develop faster in wheat straw. The present results obtained by using the same enzymatic substrates agree with those of Leatham (1985) for the presence of CMC, XYL, LAM, b-GLU and NAG, also for absence of b-XYL. b-GAL found conspicously by Leatham (1985) was not found in this work. Mishra & Leatham (1990) and ItaÈvaara et al. (1992) also found CMC and b-GLU activities in shiitake mycelia grown on sawdust. The importance of carbohydrase activities and their changes during L. edodes development were in agreement with their ability to produce high yields of sporophores and to grow more rapidly in cellulose-rich wheat straw than in more ligni®ed substrates. L. edodes has been identi®ed as a ligninolytic fungus that is moderately cellulolytic (Leatham 1985). On the basis of enzyme activities detected, L. edodes appeared to have a preference for the hemicellulose component of the wood substrate (Leatham 1985; Buswell et al. 1996). In the present study, CMC and XYL activities in wheat straw were at the same level during the vegetative growth, although CMC was higher than XYL at fruiting. The strains giving higher yields and the earlier production on wheat straw were those able to hydrolyse straw cell wall components and utilize the soluble nutrients released quickly after spawning, in order to develop a high metabolic activity.

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A balance between carbohydrase production and velocity of utilization of soluble sugars has to be obtained to select L. edodes strains adapted to cultivation in wheat straw as well as being able to cope with the changes in the substrate during its degradation.

Acknowledgements The authors are grateful to colleagues Christophe Billette and Karen Stott for revising the manuscript. G. Mata wishes to thank CONACYT (Mexico) for funding his training period in France.

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(Received in revised form 17 September 1997; accepted 22 September 1997)

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