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Comparison between the protease production ability of ligninolytic fungi cultivated in solid state media
Process Biochemistry 37 (2002) 1017– 1023 www.elsevier.com/locate/procbio
Comparison between the protease production ability of ligninolytic fungi cultivated in solid state media David R. Cabaleiro, Susana Rodrı´guez-Couto, Angeles Sanroma´n, Marı´a A. Longo * Department of Chemical Engineering, E.T.S.E.I., Uni6ersity of Vigo, Lagoas-Marcosende, E-36200 Vigo, Spain Received 14 June 2001; received in revised form 16 August 2001; accepted 16 October 2001
Abstract Solid state cultures of two white-rot fungi, Phanerochaete chrysosporium and Phlebia radiata, have been carried out, using an inert support (nylon sponge) and a support-substrate (corncob). The suitable medium and culture conditions have been chosen to favour the secretion of ligninolytic enzymes. The production of manganese peroxidase, lignin peroxidase, laccase and proteases has been monitored during the cultures, in an attempt to investigate the possible effect of the latter on the integrity of ligninolytic enzymes. The higher the protease concentration in the culture medium, the more irregular the profiles of ligninolytic enzyme activity. P. chrysosporium secretes proteolytic enzymes mainly during primary metabolism, while P. radiata produced these at the onset of secondary metabolism. Furthermore, different types of proteases produced were identified, P. chrysosporium secreted mainly thiol and acidic proteases, while P. radiata cultures contained thiol-, serin- and metalloproteases. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Protease; Ligninolytic enzymes; Phanerochaete chrysosporium; Phlebia radiata; Solid state cultures; Carrier
1. Introduction In recent years, much attention has been paid to the development of efficient processes for the treatment of industrial wastewater containing aromatic compounds, such as phenols or lignin [1 – 3]. These compounds are very persistent and can be highly toxic when found above certain concentrations [4]. White-rot fungi produce, when cultivated in the appropriate conditions, enzymic complexes (manganese peroxidase, lignin peroxidases and laccase), which catalyse the degradation of lignin and other aromatic compounds [5]. Among these, Phanerochaete chrysosporium and Phlebia radiata are outstanding. P. chrysosporium is currently the most widely studied lignin-degrading organism due to its good ligninolytic properties, fast growth and easy handling in culture [6,7]. P. radiata was first described as a ligninolytic fungus by Ander and Eriksson [8] and has many simi* Corresponding author. Tel.: + 34-986-81-2215; fax: +34-986-812201. E-mail address: [email protected] (M.A. Longo).
larities with P. chrysosporium in lignin degradation and production of lignin-modifying enzymes. Analogously to P. chrysosporium, lignin degradation by P. radiata requires a co-substrate and seems to be enhanced by an oxygen atmosphere and nitrogen limitation [9]. A number of recent studies have focused on the production, characterisation and potential applications of ligninolytic enzymes [10]. The development of industrial processes involving this kind of enzymes requires a large production of the biocatalysts at low cost. Nevertheless, one of the main problems encountered during the set-up of biotechnological processes for production of the above-mentioned biocatalysts is the fact that significant losses of activity occur during cultivation. The low stability of the produced peroxidases could be influenced by the simultaneous secretion of proteolytic enzymes by the microorganisms [11]. Solid state cultures are defined as the growth of microorganisms on solid materials in the presence of small quantities of free liquid [12]. The solid materials that can be employed in this type of cultivation are classified in two categories: inert supports (synthetic materials) and non-inert supports (agro-industrial
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wastes). The former act only as attachment places whereas the latter also provide some nutrients to the fungus, in account of which they are called supportsubstrates [13,14]. In the present work, solid state cultures of the ligninolytic fungi P. chrysosporium and P. radiata have been carried out, in conditions (i.e. media composition, temperature) which had been reported as adequate for production of ligninolytic enzymes [15,16]. Nylon sponge and corncob were selected as inert and non-inert supports, respectively, since both materials have been found to be very suitable for ligninolytic enzyme production by P. chrysosporium under solid-state conditions [17–19]. In all the cultures, the production of ligninolytic enzymes and proteases, which could greatly affect the longevity of the former, has been monitored. Furthermore, the different types of proteases produced have been identified. Knowledge about the amount and type of proteases produced, and the moment at which they are secreted, could be very useful in order to design strategies for minimising their deactivating action on ligninolytic biocatalysts. In addition, it was also of great interest to compare the behaviour of P. chrysosporium and P. radiata, two white-rot fungi that belong to the same family. 2. Materials and methods
2.1. Microorganisms and growth medium The microorganisms utilised were the ligninolytic fungi P. chrysosporium BKM-F 1767 (ATCC 24725) and P. radiata VTT-D-84236 (ATCC 64658). P. chrysosporium was maintained at 37 °C on 2% malt agar slants and plates. Spores were harvested, filtered through glass wool, and kept at − 20 °C until use [20]. The growth medium was prepared according to Tien and Kirk [21] with 10 g dm − 3 glucose as carbon source, and 20 mmol dm − 3 acetate buffer (pH 4.5) instead of dimethylsuccinate [22]. The fungus was grown in 100 cm3 of this medium at 37 °C in complete darkness for 48 h. After this, fungus and medium were homogenised for 1 min. This homogenate was used to inoculate (10% v/v) the production medium. P. radiata was grown for 10 days at 30 °C on Petri dishes containing 7.25% oatmeal agar (Difco) and 0.5% Kraft lignin (Indulin AT, Westvaco). For preparation of inoculum, pieces of agar with the fungus were cut (approximately 0.5 cm2) and grown in agitated (60 rpm) Erlenmeyer flasks with malt extract medium (2%) for 7 days at room temperature. Then, the insoluble materials were collected washed with sterile water, suspended into 100 cm3 of P. radiata production medium [23] and homogenised for about 20 s. This homogenate was used to inoculate (10% v/v) the production medium.
2.2. Carriers The selected fungi were cultivated under solid state conditions, using two different support materials: fibrous nylon sponge (Scotch Brite, 3M Company, Spain), as inert carrier, and chopped inside corncob as non-inert carrier. The latter could function both as physical support and source of nutrients. The nylon sponge (5 mm3-cubes) was pre-treated according to Linko [24] by boiling for 10 min and washing thoroughly three times with distilled water. Then, the cubes were dried at room temperature overnight, placed into the culture flasks (0.95 g nylon cubes/Erlenmeyer) and autoclaved before use. The chopped inside corncob (5 mm3-cubes) was placed into the Erlenmeyers (4 g corncob/Erlenmeyer), and autoclaved at 121 °C for 20 min.
2.3. Culture conditions 2.3.1. P. chrysosporium The production medium composition was that described by Tien and Krik [21], with 20 mmol dm − 3 acetate buffer (pH 4.5) instead of dimethylsuccinate [22]. Glucose was utilised as carbon source, at a concentration of 10 g dm − 3 for the nylon sponge cultures and 2 g dm − 3 for the corncob cultivations. The cultures were supplemented with sorbitan polyoxyethylene monooleate Tween 80 (0.5% v/v) and solid manganese (IV) oxide (1 g dm − 3) to stimulate ligninolytic enzyme production [15]. The former was added at the beginning of the cultivation, and the latter after the 1st day of incubation to prevent the inhibition of fungal growth. 2.3.2. P. radiata The cultivation medium was prepared as described by Kirk et al. [23]. Glucose was also used as carbon source (10 and 2 g dm − 3 in nylon sponge and corncob cultures, respectively). Sorbitan polyoxyethylene monooleate Tween 80 (0.05% v/v) and veratryl alcohol (1.5 mmol dm − 3) were added at the beginning of the cultivations to stimulate ligninolytic enzymes production [16]. For both fungi, experiments were carried out in 250 cm3 Erlenmeyer flasks containing the carrier (0.95 g nylon cubes or 4 g corncob) and 12 cm3 of production medium, which was previously inoculated with 10% (v/v) homogenised mycelium. The amounts of support and liquid were selected in order to obtain a monolayer of carrier, which was totally soaked after addition of the culture medium. The Erlenmeyer flasks were loosely capped with cellulose stoppers, which permitted passive aeration and incubated statically under an air atmosphere at 37 °C, and 80% relative humidity to avoid evaporation, in complete darkness.
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2.4. Analytical methods
3. Results and discussion
2.4.1. Reducing sugars Reducing sugars were measured by a dinitrosalicylic acid method using glucose as a standard, according to Ghose [25].
The white-rot fungi P. chrysosporium and P. radiata were cultivated under solid state conditions, using two different supports (nylon sponge and corncob). The culture media were selected in order to favour the production of ligninolytic enzymes [15,16], and the same carbon and nitrogen concentrations were utilised for both microorganisms. All cultures were carried out in buffered media (pH 4.5), and the pH remained between 4.5 and 5.5 throughout the cultivation time in all cases. Nutrient consumption, ligninolytic enzymes and protease production were analysed, in order to study the pattern of protease secretion by these fungi in solid state cultures and its influence in the activity of lignindegrading enzymes.
2.4.2. Nitrogen ammonium content Nitrogen ammonium content was determined by the phenol –hypochlorite method described by Weatherburn [26], using ammonium chloride as a standard. 2.4.3. Mn (II) -dependent peroxidase acti6ity Mn (II)-dependent peroxidase activity was assayed spectrophotometrically by the method of Kuwahara et al. [27], using 2,6-dimethyoxyphenol as substrate. One unit was defined as the amount of enzyme that oxidised 1 mmol of dimethoxyphenol per minute. The activities were expressed in U dm − 3. 2.4.4. Lignin peroxidase acti6ity Lignin peroxidase activity was analysed spectrophotometrically according to Tien and Kirk [28], using veratryl alcohol as substrate. One unit was defined as the amount of enzyme that oxidised 1 mmol of veratryl alcohol in 1 min, and the activities were reported as U dm − 3. 2.4.5. Laccase acti6ity Laccase activity was determined spectrophotometrically as described by Niku-Paavola et al. [29] with ABTS (2,2%-azino-di-[3-ethyl-benzothiazoline-(6)-sulphonic acid], Boehringer) as substrate. One unit was defined as the amount of enzyme that oxidised 1 mmol of ABTS per minute and the activities were expressed in U dm − 3.
3.1. P. chrysosporium cultures When P. chrysosporium was cultivated on nylon support, manganese dependent peroxidase (MnP) activity first appeared on the 4th day (300 U dm − 3), then increased to a maximum value of 510 U dm − 3 on the 6th day and from there onwards it progressively decreased. Protease production began on the 1st day with a value of 9 U cm − 3, and increased more or less continuously reaching values about 20 U cm − 3 (Fig. 1). The analysis of nutrient consumption (Fig. 2) showed that ammonium nitrogen was depleted in 24 h, while glucose (measured as reducing sugars) was gradually consumed at an approximate rate of 0.86 g dm − 3 per day.
2.4.6. Protease acti6ity Protease activity was determined according to Ginther [30], using a solution of azocasein (Sigma) at pH 4.5 as substrate and 37 °C during the whole reaction. One enzymic unit was defined as the amount of enzyme that produced an increase of 0.1 absorbance units for a reaction hour per cm3 of sample, and the activities were expressed in U cm − 3. 2.4.7. Protease characterisation Protease activity was measured in the presence of various protease inhibitors according to the method utilised by Bonnarme et al. [31]. Inhibitor concentrations were as follows: 0.36 mmol dm − 3 pepstatin A in methanol for acidic proteases; 4.8 mmol dm − 3 mercuric chloride (HgCl2) in distilled water for thiolproteases; 2.4 mmol dm − 3 ethylenediaminetetraacetic acid (EDTA) in distilled water for metalloproteases; 4.8 mmol dm − 3 phenylmethyl-sulphonyl fluoride (PMSF) in isopropanol for serine proteases.
Fig. 1. MnP and protease activities in solid state cultures of P. chrysosporium on different supports.
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Fig. 2. Nutrient consumption in solid state cultures of P. chrysosporium and P. radiata on different supports.
When P. chrysosporium was grown on corncob support, ammonium nitrogen was also depleted in 24 h, as in the nylon cultures. Glucose was consumed at a rate of 0.87 g dm − 3 per day during the two 1st days of cultivation, up to a value of around 0.25 g dm − 3. This concentration was maintained for 5 days, and from day 7 onwards it increased reaching values around 1 g l − 1. This was probably due to the occurrence of products of corncob degradation, indicating that the fungus was able to metabolise some of the nutritive substances contained in the support. MnP production began on 3rd day (209 U dm − 3), peaking on the 4th day (662 U dm − 3). Afterwards, it decreased, and from day 8 to the end of the cultivation it appeared again. In contrast, protease production started on the 1st day (5 U cm − 3), as in the nylon cultures, it then increased maintaining values around 35 U cm − 3 for the remaining culture time (Fig. 1). This value was about two-fold higher than that found in nylon cultures, which could contribute to the more irregular profile of MnP enzyme obtained in this case. Neither lignin peroxidase (LiP) nor laccase activities were detected in P. chrysosporium cultures, in the assayed conditions.
3.2. P. radiata cultures When P. radiata was cultivated on a nylon support, MnP activities were insignificant although, a peak of LiP activity (23 U dm − 3) was found (Fig. 3). Laccase was very low, reaching a maximum value of 13 U dm − 3. Protease production began on the 4th day (6 U cm − 3), and then abruptly increased showing maximum values above 50 U cm − 3 (Fig. 3). These values were three-fold higher than those encountered with P. chrysosporium.
In cultures on corncob support, MnP activity also approached zero. Nevertheless, higher LiP and laccase activities were detected compared to nylon cultivation , exhibiting maximum values of 83 and 44 U dm − 3, respectively (Fig. 3). This could be due to the lignin content of the corncob, which could act as both LiP and laccase inducer. This is in agreement with the reports of Vares et al. [32], who reported an increase of ligninolytic activities during wheat straw degradation by P. radiata. Proteases appeared on the 5th day (17 U cm − 3) and, then increased reaching values above 40 U cm − 3 (Fig. 3). These values were slightly higher than those detected in P. chrysosporium cultures. Nutrient consumption in both P. radiata cultures was quite slow (Fig. 2). Glucose (measured as reducing sugars) decreased quasi-linearly at an average rate of 0.6 g dm − 3 per day, while ammonium nitrogen diminished very slowly during the first 4 days, being totally depleted within 24 h. In summary, P. chrysosporium appeared to be a more adequate microorganism for MnP production in the assayed conditions, compared to P. radiata. On the other hand, the former did not secrete either LiP or laccase, while the latter showed the ability to produce both enzymes. In all cases, ligninolytic enzyme activities were higher in corncob cultures than in nylon cultures, although the difference was more significant in P. radiata cultures. Thus, nylon seems not to be an adequate support for the production of ligninolytic enzymes by P. radiata, although it has been demonstrated to be very suitable for P. chrysosporium [17,18,33]. Moreover, ligninolytic enzyme profiles were very irregular, mainly in P. radiata cultures. This fungus produced a higher amount of proteases than P. chrysosporium, especially in nylon cultures, which could
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be related to the lower production of ligninolytic enzymes detected (Fig. 3). Also, the ability of P. radiata to secrete proteases does not seem to be greatly influenced by the cultivation mode, since significant levels of these enzymes have been found both in solid state (this work) and submerged cultures [34,35]. As for the role of proteases in wood-rotting fungi, several hypotheses have been reported. Eriksson and Pettersson [36,37] indicated their possible implication in the release of ligninolytic enzymes from the fungal cell wall. On the other hand, Dosoretz et al. [11,38] postulated that one of the functions of the proteases produced by white-rot fungi is to recycle nitrogen by breaking down secreted proteins or proteins released into the medium on cell autolysis. Furthermore, several studies pointed out protease-mediated degradation as a major cause of the decay of extracellular enzyme activities (ligninases, amylases) in submerged cultures of white-rot fungi [38,39]. The results obtained in this work seem to support the second hypothesis, since the higher levels of proteases coincided with low levels of ligninolytic enzymes. Proteolytic action could be favoured in solid state cultures, compared to submerged ones, as a consequence of the poor mixing conditions encountered in the former. The pattern of protease production was different in both fungi. P. chrysosporium secreted proteases mainly during primary metabolism, whereas P. radiata produced them at the onset of the secondary metabolism (from day 4 onwards). In the case of P. chrysosporium, the results differed from those reported for submerged cultures, in which both primary and secondary metabolism proteases were detected [11,31]. This is not surprising, since solid state culture conditions may switch on enzyme systems that differ from their submerged culture counterparts [40].
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Moreover, the lower ligninase activities found in P. radiata cultures could indicate a predominant role of secondary proteases in extracellular enzyme degradation. This idea has been already reported in studies of the different proteases produced by P. chrysosporium in submerged cultures [11,38]. It could be interesting to investigate the regulation of secondary proteases in solid state cultures (i.e. by controlled addition of key nutrients) and its effect on ligninase production. Finally, it is noteworthy that no clear relationship could be found between the type of support utilised for solid state cultures and the pattern of protease production. It would seem that the amount of proteolytic enzymes and the stage of cultivation in which they are secreted are more related to the microorganism or the cultivation mode (solid state or submerged) than to the characteristics of the support. Subsequently, the types of proteases produced by both fungi in the operation conditions assayed were determined, utilising the suitable specific protease inhibitors as indicated.
3.3. Types of proteases Fig. 4 shows the different types of proteases as well as the total proteases existing in the extracellular liquid during P. chrysosporium cultures on nylon and corncob. The proteases have been characterised by means of specific inhibitors, and found to be acidic proteases (about 60%) and thiolproteases (around 80% after several culture days). This agrees with the investigations realised by Datta et al. [40] and Bonnarme et al. [31], in solid state and submerged cultures, respectively. In addition, the absence of serine proteases and metalloproteases has also been demonstrated, contrary to the
Fig. 3. Ligninolytic enzymes and protease activities in solid state cultures of P. radiata on different supports.
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Fig. 4. Total and different type of proteases produced in nylon and corncob cultures of P. chrysosporium.
data found by Dass et al. [41] in submerged cultures of P. chrysosporium. Moreover, some of the proteases secreted by P. chrysosporium were inactivated at the same time by inhibitors specific to thiol and acidic proteases. This indicated that both cystein and acidic aminoacid residues are directly involved in their catalytic ability, although the former seemed to be more important, especially for the proteases present in the extracellular liquid during secondary metabolism (from day 4 onwards). As for P. radiata, analysis of the proteases produced is shown in Fig. 5. When the fungus was cultivated on nylon sponge, the secreted proteases were mainly thiolproteases (around 80%), as occurred in P. chrysosporium cultures. In addition, similar amounts of serineproteases were also detected, which indicates that both cysteine and serine aminoacid residues are crucial for their catalytic activity. In P. chrysosporium cultures, acidic proteases production was not observed and only a low activity of metalloproteases was detected. The characterisation of the proteases produced by P. radiata cultivated on corncob also resulted in the identification of high amounts of thiol- and serineproteases, although in this case serine residues appeared to be less
Fig. 5. Total and different type of proteases produced in nylon and corncob cultures of P. radiata.
relevant for proteolytic activity (only 40% of the total proteases were affected by serine protease specific inhibitors). Also, a small amount of metalloproteases was detected. The results did not indicate a significant influence of the support (nylon sponge or corncob) in the type of proteases produced. It would seem that this is more related to the microorganism and the cultivation mode (solid state or submerged).
Acknowledgements This research was financed by Xunta de Galicia (Project PGIDT00PXI30118PR).
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Comparison between the protease production ability of ligninolytic fungi cultivated in solid state media David R. Cabaleiro, Susana Rodrı´guez-Couto, Angeles Sanroma´n, Marı´a A. Longo * Department of Chemical Engineering, E.T.S.E.I., Uni6ersity of Vigo, Lagoas-Marcosende, E-36200 Vigo, Spain Received 14 June 2001; received in revised form 16 August 2001; accepted 16 October 2001
Abstract Solid state cultures of two white-rot fungi, Phanerochaete chrysosporium and Phlebia radiata, have been carried out, using an inert support (nylon sponge) and a support-substrate (corncob). The suitable medium and culture conditions have been chosen to favour the secretion of ligninolytic enzymes. The production of manganese peroxidase, lignin peroxidase, laccase and proteases has been monitored during the cultures, in an attempt to investigate the possible effect of the latter on the integrity of ligninolytic enzymes. The higher the protease concentration in the culture medium, the more irregular the profiles of ligninolytic enzyme activity. P. chrysosporium secretes proteolytic enzymes mainly during primary metabolism, while P. radiata produced these at the onset of secondary metabolism. Furthermore, different types of proteases produced were identified, P. chrysosporium secreted mainly thiol and acidic proteases, while P. radiata cultures contained thiol-, serin- and metalloproteases. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Protease; Ligninolytic enzymes; Phanerochaete chrysosporium; Phlebia radiata; Solid state cultures; Carrier
1. Introduction In recent years, much attention has been paid to the development of efficient processes for the treatment of industrial wastewater containing aromatic compounds, such as phenols or lignin [1 – 3]. These compounds are very persistent and can be highly toxic when found above certain concentrations [4]. White-rot fungi produce, when cultivated in the appropriate conditions, enzymic complexes (manganese peroxidase, lignin peroxidases and laccase), which catalyse the degradation of lignin and other aromatic compounds [5]. Among these, Phanerochaete chrysosporium and Phlebia radiata are outstanding. P. chrysosporium is currently the most widely studied lignin-degrading organism due to its good ligninolytic properties, fast growth and easy handling in culture [6,7]. P. radiata was first described as a ligninolytic fungus by Ander and Eriksson [8] and has many simi* Corresponding author. Tel.: + 34-986-81-2215; fax: +34-986-812201. E-mail address: [email protected] (M.A. Longo).
larities with P. chrysosporium in lignin degradation and production of lignin-modifying enzymes. Analogously to P. chrysosporium, lignin degradation by P. radiata requires a co-substrate and seems to be enhanced by an oxygen atmosphere and nitrogen limitation [9]. A number of recent studies have focused on the production, characterisation and potential applications of ligninolytic enzymes [10]. The development of industrial processes involving this kind of enzymes requires a large production of the biocatalysts at low cost. Nevertheless, one of the main problems encountered during the set-up of biotechnological processes for production of the above-mentioned biocatalysts is the fact that significant losses of activity occur during cultivation. The low stability of the produced peroxidases could be influenced by the simultaneous secretion of proteolytic enzymes by the microorganisms [11]. Solid state cultures are defined as the growth of microorganisms on solid materials in the presence of small quantities of free liquid [12]. The solid materials that can be employed in this type of cultivation are classified in two categories: inert supports (synthetic materials) and non-inert supports (agro-industrial
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wastes). The former act only as attachment places whereas the latter also provide some nutrients to the fungus, in account of which they are called supportsubstrates [13,14]. In the present work, solid state cultures of the ligninolytic fungi P. chrysosporium and P. radiata have been carried out, in conditions (i.e. media composition, temperature) which had been reported as adequate for production of ligninolytic enzymes [15,16]. Nylon sponge and corncob were selected as inert and non-inert supports, respectively, since both materials have been found to be very suitable for ligninolytic enzyme production by P. chrysosporium under solid-state conditions [17–19]. In all the cultures, the production of ligninolytic enzymes and proteases, which could greatly affect the longevity of the former, has been monitored. Furthermore, the different types of proteases produced have been identified. Knowledge about the amount and type of proteases produced, and the moment at which they are secreted, could be very useful in order to design strategies for minimising their deactivating action on ligninolytic biocatalysts. In addition, it was also of great interest to compare the behaviour of P. chrysosporium and P. radiata, two white-rot fungi that belong to the same family. 2. Materials and methods
2.1. Microorganisms and growth medium The microorganisms utilised were the ligninolytic fungi P. chrysosporium BKM-F 1767 (ATCC 24725) and P. radiata VTT-D-84236 (ATCC 64658). P. chrysosporium was maintained at 37 °C on 2% malt agar slants and plates. Spores were harvested, filtered through glass wool, and kept at − 20 °C until use [20]. The growth medium was prepared according to Tien and Kirk [21] with 10 g dm − 3 glucose as carbon source, and 20 mmol dm − 3 acetate buffer (pH 4.5) instead of dimethylsuccinate [22]. The fungus was grown in 100 cm3 of this medium at 37 °C in complete darkness for 48 h. After this, fungus and medium were homogenised for 1 min. This homogenate was used to inoculate (10% v/v) the production medium. P. radiata was grown for 10 days at 30 °C on Petri dishes containing 7.25% oatmeal agar (Difco) and 0.5% Kraft lignin (Indulin AT, Westvaco). For preparation of inoculum, pieces of agar with the fungus were cut (approximately 0.5 cm2) and grown in agitated (60 rpm) Erlenmeyer flasks with malt extract medium (2%) for 7 days at room temperature. Then, the insoluble materials were collected washed with sterile water, suspended into 100 cm3 of P. radiata production medium [23] and homogenised for about 20 s. This homogenate was used to inoculate (10% v/v) the production medium.
2.2. Carriers The selected fungi were cultivated under solid state conditions, using two different support materials: fibrous nylon sponge (Scotch Brite, 3M Company, Spain), as inert carrier, and chopped inside corncob as non-inert carrier. The latter could function both as physical support and source of nutrients. The nylon sponge (5 mm3-cubes) was pre-treated according to Linko [24] by boiling for 10 min and washing thoroughly three times with distilled water. Then, the cubes were dried at room temperature overnight, placed into the culture flasks (0.95 g nylon cubes/Erlenmeyer) and autoclaved before use. The chopped inside corncob (5 mm3-cubes) was placed into the Erlenmeyers (4 g corncob/Erlenmeyer), and autoclaved at 121 °C for 20 min.
2.3. Culture conditions 2.3.1. P. chrysosporium The production medium composition was that described by Tien and Krik [21], with 20 mmol dm − 3 acetate buffer (pH 4.5) instead of dimethylsuccinate [22]. Glucose was utilised as carbon source, at a concentration of 10 g dm − 3 for the nylon sponge cultures and 2 g dm − 3 for the corncob cultivations. The cultures were supplemented with sorbitan polyoxyethylene monooleate Tween 80 (0.5% v/v) and solid manganese (IV) oxide (1 g dm − 3) to stimulate ligninolytic enzyme production [15]. The former was added at the beginning of the cultivation, and the latter after the 1st day of incubation to prevent the inhibition of fungal growth. 2.3.2. P. radiata The cultivation medium was prepared as described by Kirk et al. [23]. Glucose was also used as carbon source (10 and 2 g dm − 3 in nylon sponge and corncob cultures, respectively). Sorbitan polyoxyethylene monooleate Tween 80 (0.05% v/v) and veratryl alcohol (1.5 mmol dm − 3) were added at the beginning of the cultivations to stimulate ligninolytic enzymes production [16]. For both fungi, experiments were carried out in 250 cm3 Erlenmeyer flasks containing the carrier (0.95 g nylon cubes or 4 g corncob) and 12 cm3 of production medium, which was previously inoculated with 10% (v/v) homogenised mycelium. The amounts of support and liquid were selected in order to obtain a monolayer of carrier, which was totally soaked after addition of the culture medium. The Erlenmeyer flasks were loosely capped with cellulose stoppers, which permitted passive aeration and incubated statically under an air atmosphere at 37 °C, and 80% relative humidity to avoid evaporation, in complete darkness.
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2.4. Analytical methods
3. Results and discussion
2.4.1. Reducing sugars Reducing sugars were measured by a dinitrosalicylic acid method using glucose as a standard, according to Ghose [25].
The white-rot fungi P. chrysosporium and P. radiata were cultivated under solid state conditions, using two different supports (nylon sponge and corncob). The culture media were selected in order to favour the production of ligninolytic enzymes [15,16], and the same carbon and nitrogen concentrations were utilised for both microorganisms. All cultures were carried out in buffered media (pH 4.5), and the pH remained between 4.5 and 5.5 throughout the cultivation time in all cases. Nutrient consumption, ligninolytic enzymes and protease production were analysed, in order to study the pattern of protease secretion by these fungi in solid state cultures and its influence in the activity of lignindegrading enzymes.
2.4.2. Nitrogen ammonium content Nitrogen ammonium content was determined by the phenol –hypochlorite method described by Weatherburn [26], using ammonium chloride as a standard. 2.4.3. Mn (II) -dependent peroxidase acti6ity Mn (II)-dependent peroxidase activity was assayed spectrophotometrically by the method of Kuwahara et al. [27], using 2,6-dimethyoxyphenol as substrate. One unit was defined as the amount of enzyme that oxidised 1 mmol of dimethoxyphenol per minute. The activities were expressed in U dm − 3. 2.4.4. Lignin peroxidase acti6ity Lignin peroxidase activity was analysed spectrophotometrically according to Tien and Kirk [28], using veratryl alcohol as substrate. One unit was defined as the amount of enzyme that oxidised 1 mmol of veratryl alcohol in 1 min, and the activities were reported as U dm − 3. 2.4.5. Laccase acti6ity Laccase activity was determined spectrophotometrically as described by Niku-Paavola et al. [29] with ABTS (2,2%-azino-di-[3-ethyl-benzothiazoline-(6)-sulphonic acid], Boehringer) as substrate. One unit was defined as the amount of enzyme that oxidised 1 mmol of ABTS per minute and the activities were expressed in U dm − 3.
3.1. P. chrysosporium cultures When P. chrysosporium was cultivated on nylon support, manganese dependent peroxidase (MnP) activity first appeared on the 4th day (300 U dm − 3), then increased to a maximum value of 510 U dm − 3 on the 6th day and from there onwards it progressively decreased. Protease production began on the 1st day with a value of 9 U cm − 3, and increased more or less continuously reaching values about 20 U cm − 3 (Fig. 1). The analysis of nutrient consumption (Fig. 2) showed that ammonium nitrogen was depleted in 24 h, while glucose (measured as reducing sugars) was gradually consumed at an approximate rate of 0.86 g dm − 3 per day.
2.4.6. Protease acti6ity Protease activity was determined according to Ginther [30], using a solution of azocasein (Sigma) at pH 4.5 as substrate and 37 °C during the whole reaction. One enzymic unit was defined as the amount of enzyme that produced an increase of 0.1 absorbance units for a reaction hour per cm3 of sample, and the activities were expressed in U cm − 3. 2.4.7. Protease characterisation Protease activity was measured in the presence of various protease inhibitors according to the method utilised by Bonnarme et al. [31]. Inhibitor concentrations were as follows: 0.36 mmol dm − 3 pepstatin A in methanol for acidic proteases; 4.8 mmol dm − 3 mercuric chloride (HgCl2) in distilled water for thiolproteases; 2.4 mmol dm − 3 ethylenediaminetetraacetic acid (EDTA) in distilled water for metalloproteases; 4.8 mmol dm − 3 phenylmethyl-sulphonyl fluoride (PMSF) in isopropanol for serine proteases.
Fig. 1. MnP and protease activities in solid state cultures of P. chrysosporium on different supports.
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Fig. 2. Nutrient consumption in solid state cultures of P. chrysosporium and P. radiata on different supports.
When P. chrysosporium was grown on corncob support, ammonium nitrogen was also depleted in 24 h, as in the nylon cultures. Glucose was consumed at a rate of 0.87 g dm − 3 per day during the two 1st days of cultivation, up to a value of around 0.25 g dm − 3. This concentration was maintained for 5 days, and from day 7 onwards it increased reaching values around 1 g l − 1. This was probably due to the occurrence of products of corncob degradation, indicating that the fungus was able to metabolise some of the nutritive substances contained in the support. MnP production began on 3rd day (209 U dm − 3), peaking on the 4th day (662 U dm − 3). Afterwards, it decreased, and from day 8 to the end of the cultivation it appeared again. In contrast, protease production started on the 1st day (5 U cm − 3), as in the nylon cultures, it then increased maintaining values around 35 U cm − 3 for the remaining culture time (Fig. 1). This value was about two-fold higher than that found in nylon cultures, which could contribute to the more irregular profile of MnP enzyme obtained in this case. Neither lignin peroxidase (LiP) nor laccase activities were detected in P. chrysosporium cultures, in the assayed conditions.
3.2. P. radiata cultures When P. radiata was cultivated on a nylon support, MnP activities were insignificant although, a peak of LiP activity (23 U dm − 3) was found (Fig. 3). Laccase was very low, reaching a maximum value of 13 U dm − 3. Protease production began on the 4th day (6 U cm − 3), and then abruptly increased showing maximum values above 50 U cm − 3 (Fig. 3). These values were three-fold higher than those encountered with P. chrysosporium.
In cultures on corncob support, MnP activity also approached zero. Nevertheless, higher LiP and laccase activities were detected compared to nylon cultivation , exhibiting maximum values of 83 and 44 U dm − 3, respectively (Fig. 3). This could be due to the lignin content of the corncob, which could act as both LiP and laccase inducer. This is in agreement with the reports of Vares et al. [32], who reported an increase of ligninolytic activities during wheat straw degradation by P. radiata. Proteases appeared on the 5th day (17 U cm − 3) and, then increased reaching values above 40 U cm − 3 (Fig. 3). These values were slightly higher than those detected in P. chrysosporium cultures. Nutrient consumption in both P. radiata cultures was quite slow (Fig. 2). Glucose (measured as reducing sugars) decreased quasi-linearly at an average rate of 0.6 g dm − 3 per day, while ammonium nitrogen diminished very slowly during the first 4 days, being totally depleted within 24 h. In summary, P. chrysosporium appeared to be a more adequate microorganism for MnP production in the assayed conditions, compared to P. radiata. On the other hand, the former did not secrete either LiP or laccase, while the latter showed the ability to produce both enzymes. In all cases, ligninolytic enzyme activities were higher in corncob cultures than in nylon cultures, although the difference was more significant in P. radiata cultures. Thus, nylon seems not to be an adequate support for the production of ligninolytic enzymes by P. radiata, although it has been demonstrated to be very suitable for P. chrysosporium [17,18,33]. Moreover, ligninolytic enzyme profiles were very irregular, mainly in P. radiata cultures. This fungus produced a higher amount of proteases than P. chrysosporium, especially in nylon cultures, which could
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be related to the lower production of ligninolytic enzymes detected (Fig. 3). Also, the ability of P. radiata to secrete proteases does not seem to be greatly influenced by the cultivation mode, since significant levels of these enzymes have been found both in solid state (this work) and submerged cultures [34,35]. As for the role of proteases in wood-rotting fungi, several hypotheses have been reported. Eriksson and Pettersson [36,37] indicated their possible implication in the release of ligninolytic enzymes from the fungal cell wall. On the other hand, Dosoretz et al. [11,38] postulated that one of the functions of the proteases produced by white-rot fungi is to recycle nitrogen by breaking down secreted proteins or proteins released into the medium on cell autolysis. Furthermore, several studies pointed out protease-mediated degradation as a major cause of the decay of extracellular enzyme activities (ligninases, amylases) in submerged cultures of white-rot fungi [38,39]. The results obtained in this work seem to support the second hypothesis, since the higher levels of proteases coincided with low levels of ligninolytic enzymes. Proteolytic action could be favoured in solid state cultures, compared to submerged ones, as a consequence of the poor mixing conditions encountered in the former. The pattern of protease production was different in both fungi. P. chrysosporium secreted proteases mainly during primary metabolism, whereas P. radiata produced them at the onset of the secondary metabolism (from day 4 onwards). In the case of P. chrysosporium, the results differed from those reported for submerged cultures, in which both primary and secondary metabolism proteases were detected [11,31]. This is not surprising, since solid state culture conditions may switch on enzyme systems that differ from their submerged culture counterparts [40].
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Moreover, the lower ligninase activities found in P. radiata cultures could indicate a predominant role of secondary proteases in extracellular enzyme degradation. This idea has been already reported in studies of the different proteases produced by P. chrysosporium in submerged cultures [11,38]. It could be interesting to investigate the regulation of secondary proteases in solid state cultures (i.e. by controlled addition of key nutrients) and its effect on ligninase production. Finally, it is noteworthy that no clear relationship could be found between the type of support utilised for solid state cultures and the pattern of protease production. It would seem that the amount of proteolytic enzymes and the stage of cultivation in which they are secreted are more related to the microorganism or the cultivation mode (solid state or submerged) than to the characteristics of the support. Subsequently, the types of proteases produced by both fungi in the operation conditions assayed were determined, utilising the suitable specific protease inhibitors as indicated.
3.3. Types of proteases Fig. 4 shows the different types of proteases as well as the total proteases existing in the extracellular liquid during P. chrysosporium cultures on nylon and corncob. The proteases have been characterised by means of specific inhibitors, and found to be acidic proteases (about 60%) and thiolproteases (around 80% after several culture days). This agrees with the investigations realised by Datta et al. [40] and Bonnarme et al. [31], in solid state and submerged cultures, respectively. In addition, the absence of serine proteases and metalloproteases has also been demonstrated, contrary to the
Fig. 3. Ligninolytic enzymes and protease activities in solid state cultures of P. radiata on different supports.
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Fig. 4. Total and different type of proteases produced in nylon and corncob cultures of P. chrysosporium.
data found by Dass et al. [41] in submerged cultures of P. chrysosporium. Moreover, some of the proteases secreted by P. chrysosporium were inactivated at the same time by inhibitors specific to thiol and acidic proteases. This indicated that both cystein and acidic aminoacid residues are directly involved in their catalytic ability, although the former seemed to be more important, especially for the proteases present in the extracellular liquid during secondary metabolism (from day 4 onwards). As for P. radiata, analysis of the proteases produced is shown in Fig. 5. When the fungus was cultivated on nylon sponge, the secreted proteases were mainly thiolproteases (around 80%), as occurred in P. chrysosporium cultures. In addition, similar amounts of serineproteases were also detected, which indicates that both cysteine and serine aminoacid residues are crucial for their catalytic activity. In P. chrysosporium cultures, acidic proteases production was not observed and only a low activity of metalloproteases was detected. The characterisation of the proteases produced by P. radiata cultivated on corncob also resulted in the identification of high amounts of thiol- and serineproteases, although in this case serine residues appeared to be less
Fig. 5. Total and different type of proteases produced in nylon and corncob cultures of P. radiata.
relevant for proteolytic activity (only 40% of the total proteases were affected by serine protease specific inhibitors). Also, a small amount of metalloproteases was detected. The results did not indicate a significant influence of the support (nylon sponge or corncob) in the type of proteases produced. It would seem that this is more related to the microorganism and the cultivation mode (solid state or submerged).
Acknowledgements This research was financed by Xunta de Galicia (Project PGIDT00PXI30118PR).
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