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Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon
Bioresource Technology 97 (2006) 786–789
Short Communication
Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon J.W. Wang a
a,b
, J.H. Wu a, W.Y. Huang a, R.X. Tan
a,*
Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210093, China b Department of Pharmaceutical Sciences, Soochow University, Suzhou 215006, China Received 12 January 2004; received in revised form 28 March 2005; accepted 28 March 2005 Available online 11 July 2005
Abstract The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by the endophytic fungus Monotospora sp. were evaluated. The optimal temperature and initial pH for laccase production by Monotospora sp. in submerged culture were found to be 30 °C and 8.5, respectively. Maltose (2 g l1) and ammonium tartrate (10 g l1) were the most suitable carbon and nitrogen source for laccase production. Under optimal culture medium, the maximum laccase activity was determined to be 13.55 U ml1, which was approximately four times higher than that in basal medium. This is the first report on laccase production by an endophytic fungus. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Monotospora sp.; Endophytic fungus; Laccase production; Submerged cultures
1. Introduction Laccases (benzenediol: oxygen oxidoreductases; EC1.10.3.2.) are glycosylated polyphenol oxidases (Thurston, 1994). These enzymes find important commercial applications in the pulp and paper industry, animal biotechnology, biotransformation and detoxification of phenolic pollutants (Brenna and Bianchi, 1994; Breen and Singleton, 1999). Laccase production is a common feature of many basidiomycetes, particularly those associated with wood decay or terminal stages of decomposition (Gianfreda et al., 1999). The genus Trametes, seems to be one of the most efficient laccase producers (Jang et al., 2002).
*
Corresponding author. Tel.: +86 25 83592945; fax: +86 25 83302728. E-mail address: [email protected] (R.X. Tan). 0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.03.025
As a less investigated microorganisms hidden within host plants, endophytes are obviously a rich and reliable source of bioactive metabolites with huge medical, agricultural and industrial potentials (Tan and Zou, 2001). Although the enzymes vary from isolate to isolate, the endophytic fungi tested all synthesize in vitro the enzymes necessary for penetrating and colonizing their plant hosts (Schulz et al., 2002). Such enzymes including pectinases, xylanase, cellulases and lipases, proteinase and phenol oxidase have been documented with some endophytes (Tan and Zou, 2001). However, little has been reported about laccase production for degrading lignin by endophytic fungi up to date. In continuation of our characterization of new metabolites from endophytic fungi (Lu et al., 2000; Liu et al., 2002, 2004), we herewith wish to explore the laccase production ability of Monotospora sp., an endophytic fungus in Cynodon dactylon. Various physicochemical parameters were examined to optimize laccase production.
J.W. Wang et al. / Bioresource Technology 97 (2006) 786–789
2.2. Inoculum preparation and flask cultures Monotospora sp. was initially grown on PDA medium in a Petri dish, and then transferred into the seed medium by punching out 5 mm of the agar plate culture with a self-designed cutter. Unless otherwise specified, a shake flask culture was carried out in a 250 ml flask containing 100 ml of the medium at 25 °C on a rotary shaker incubator at 150 rpm for 6 d. In order to optimize the cultural conditions, the initial pH in the MSB medium was adjusted to the desired value by addition of either 0.1 M HCl or 2.5 M NaOH. The flasks were incubated at various temperatures (20–40 °C). Sucrose, glucose, cellobiose, xylose, soluble starch, rhamnose, CMC-Na, lactose, mannitol, glycerol, maltose and arabinose were studied as carbon sources in relation to expression of laccase activity. Ammonium chloride, ammonium nitrate, ammonium sulfate, ammonium tartrate, meat peptone, yeast extract, acrylamide, urea, beaf extract and glycine were used in this study to select the best nitrogen source. For a time course study, the flasks were incubated at 30 °C on a rotary shaker at 150 rpm for 16 d. At 1 d interval, the contents of each flask were filtered through Whatman filter paper No. 1 (Whatman, Springfield Mill, UK), and the filtrate was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant thus obtained was treated as the enzyme extract. All experiments were performed at least in triplicate to ensure reproducibility. 2.3. Analytical determination The pH was measured with a digital pH meter (Oakton, Singapore, model 1000). The fungal biomass was harvested by filtration through filter paper and bio-
2.4. Statistical analyses Students t-test was performed using OriginTM version 6.0 (Microcal, Northampton, MA, USA). The values represented by their mean ± standard deviation: significant differences between groups were determined at q < 0.05.
3. Results and discussion The shake flask cultures using the basal medium (MSB) showed the ability of the endophyte Monotospora sp. in C. dactylon to produce appreciable amounts of laccases extracellularly. Maximum enzyme production (3.49 ± 0.92 U ml1) reached on day 8 in Monotospora sp. while the biomass maxima was achieved during 4–7 d (Fig. 1). The production maxima was also attained earlier as compared to some white rot fungi such as Phlebia floridensis (20th day) (Arora and Gill, 2000) and Pleurotus ostreatus (14th day) (Hou et al., 2004). To the best of our knowledge, this is the first report on laccase production by endophytic fungi. Our results revealed that an endophytic Monotospora sp. could synthesize in vitro laccase, being involved in various
33
60
30 27
50
24 40
21 18
30
15 12
20
9 6
10
Extracellular protein (µg ml-1)
Monotospora sp., strain number W823, is an endophytic fungus isolated from fresh stems of an apparently healthy C. dactylon collected in May 2002, in the suburb of Nanjing, China. Endophytic strains were isolated following the procedure described previously (Lu et al., 2000). The stock culture was maintained on a potato dextrose agar (PDA) (Sigma, St. Louis, USA) slant and subcultured every month. Slants were incubated at 25 °C for 5 d and subsequently stored at 4 °C. The seed culture was grown in 250 ml flask containing 100 ml of mineral salts broth (MSB) containing: sucrose, 5 g; yeast extract, 2.5 g; acetic acid, 0.6 ml; KH2PO4, 2 g; MgSO4 Æ 7H2O, 1 g; CaCl2 Æ 2H2O, 0.1 g, thiamine hydrochloride, 0.01 g; polysorbate 80, 1 g; trace element solution, 10 ml, in 1 l distilled water (initial pH 4.8), at 25 °C on a rotary shaker incubator at 150 rpm for 4 d.
Laccase activity (U ml-1)
2.1. Microorganism and media
mass yield was determined in terms of oven-dried (80 °C for 24 h) mycelium weight. The protein in the filtrate was estimated by method of Lowry et al. (1951). Laccase activity was determined with 2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as the substrate (Bourbonnais and Paice, 1990). One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 lmol of ABTS per min at 25 °C (Galhaup and Haltrich, 2001).
Mycelial dry weight (x10-1 gL-1)
2. Methods
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3 0
0 0
2
4
6
8
10
12
14
16
Inoculum age (d)
Fig. 1. The time course of mycelial dry weight ((n) under the basal medium (MSB) and (m) under optimized medium), extracellular protein ((h) under the basal medium (MSB) and (j) under optimized medium) and laccase production ((s) under the basal medium (MSB) and (d) under optimized medium) by Monotospora sp. in shake flask cultures at 30 °C on a rotary shaker at 150 rpm. Values represent the mean ± S.D. of triplicate samples.
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J.W. Wang et al. / Bioresource Technology 97 (2006) 786–789 5
5
Laccase activity (U ml-1)
(a)
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(b)
0 0
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40
Temperature (°C)
Initial pH
Fig. 2. Effect of pH (a) and temperature (b) on laccase production by Monotospora sp. in shake flask cultures. Values represent the mean ± S.D. of triplicate samples.
processes including the colonization and lignin biodegradation in plant hosts. Since enzyme production is also affected by some culture conditions, initial pH, temperature, the carbon and nitrogen source were chosen for optimization as these have been reported to influence laccase production by other microorganisms (Gianfreda et al., 1999; Meideieros et al., 1999). The effects of pH and temperature on laccase activity of Monotospora sp. are shown in Fig. 2. The optimum initial pH and incubation temperature were determined to 8.5 and 30 °C, respectively. The optimum concentrations of these components for the maximum laccase production by Monotospora sp. were: 2 g l1 maltose and 10 g l1 ammonium tartrate (that is C/N ratio was 0.2/1) (data not shown). Previous papers suggested that the expression of laccases by most white rot fungi required an acidic initial pH, N-limitation and higher C/N ratio in cultures (Swamy and Ramsay, 1999; Jang et al., 2002; Nyanhongo et al., 2002). In comparison to fungal plant pathogens and fungal soil isolates, endophytic fungi grow in different habitats and require distinct levels of nutrition (Schulz et al., 2002). The better enzyme production may be achieved as far as the environment in which these fungi grow is concerned. In this work, N-rich and C-limitation cultures at an alkaline initial pH resulted in a higher expression of laccase. Under the optimal conditions established, the maximum laccase activity of 13.55 ± 1.38 U ml1, which represents an approximately fourfold increase (q < 0.05) in laccase activity from 3.49 ± 0.92 U ml1 in basal medium was obtained in optimized medium containing (g/l): (2 maltose and 10 ammonium tartrate instead of 5 sucrose
and 2.5 yeast extract in MSB medium respectively, pH 8.5) (Fig. 1). As compared to laccase production determined by oxidation of ABTS in cultures of the much-studied white-rot fungi such as Tramets spp. (1.5–15.8 U ml1) (Jang et al., 2002; Rancan˜o et al., 2003), Monotospora sp. proved to be a high laccase producer. In our recent work, the endophytic fungi inside the healthy leaves of C. dactylon were versatile producers of new bioactive metabolites (Liu et al., 2004; Ma et al., 2004). Moreover, in the current study, based on higher laccase activity by the endophytic Monotospora sp., the potential for screening and optimizing more endophytic fungi for laccase production may be exploited in lignin degradation and other biotechnological applications.
Acknowledgements This work was co-financed by grants for RXT from NNSF (Nos. 30171104 and 30470191) and for JWW from MSDF (No. EE132514) of Soochow University.
References Arora, D.S., Gill, P.K., 2000. Laccase production by some white rot fungi under different nutritional conditions. Bioresource Technol. 73, 283–285. Bourbonnais, R., Paice, M.G., 1990. Oxidation of non-phenolic substrates. An expanded role of laccase in lignin biodegradation. FEBS Lett. 267, 99–102. Brenna, O., Bianchi, E., 1994. Immobilized laccase for phenolic removal in must and wine. Biotechnol. Lett. 24, 35–40.
J.W. Wang et al. / Bioresource Technology 97 (2006) 786–789 Breen, A., Singleton, F.L., 1999. Fungi in lignocellulose breakdown and biopulping. Curr. Opin. Biotechnol. 10, 252–258. Galhaup, C., Haltrich, D., 2001. Enhanced formation of laccase activity by the white-rot fungus Tremetes pubescens in the presence of copper. Appl. Microbiol. Biotechnol. 56, 225– 232. Gianfreda, L., Xu, F., Bollag, J.M., 1999. Laccases: a useful group of oxidoreductive enzymes. Bioremediation J. 3, 1–25. Hou, H., Zhou, J., Wang, J., Dua, C., Yan, B., 2004. Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye. Process Biochem. 39, 1415– 1419. Jang, M.Y., Ryu, W.R., Cho, M.H., 2002. Laccase production from repeated batch cultures using free mycelia of Trametes sp. Enzyme Microb. Technol. 30, 741–746. Liu, J.Y., Liu, C.H., Zou, W.X., Tian, X., Tan, R.X., 2002. Leptosphaerone, a metabolite with a novel skeleton from Leptosphaeria sp. IV403, an endophytic fungus in Artemisia annua. Helv. Chim. Acta 85, 2664–2667. Liu, J.Y., Song, Y.C., Zhang, Z., Wang, L., Guo, Z.J., Zou, W.X., Tan, R.X., 2004. Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites. J. Biotechnol. 114, 279–287. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
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Lu, H., Zou, W.X., Meng, J.C., Hu, J., Tan, R.X., 2000. New bioactive metabolites produced by Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant Sci. 151, 61–73. Ma, Y.M., Li, Y., Liu, J.Y., Song, Y.C., Tan, R.X., 2004. AntiHelicobacter pylori metabolites from Rhizoctonia sp. Cy064, an endophytic fungus in Cynodon dactylon. Fitoterapia 75, 451–456. Meideieros, M.B., Bento, A.V., Nunes, A.L.L., Oliveira, S.C., 1999. Optimization of some variables that affect the synthesis of laccase by Pleuratus ostreaties. Biopol. Eng. 71, 483–487. Nyanhongo, G.S., Gomes, J., Gubitz, G., Zvauya, R., Read, J.S., Steiner, W., 2002. Production of laccase by a newly isolated strain of Trametes modesta. Bioresource Technol. 84, 259–263. Rancan˜o, G., Lorenzo, M., Molares, N., Couto, S.R., Sanroma´n, ´ ., 2003. Production of laccase by Trametes versicolor in an M.A airlift fermentor. Process Biochem. 39, 467–473. Schulz, B., Boyle, C., Draeger, S., Ro¨mmert, A.-K., Krohn, K., 2002. Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol. Res. 106, 996–1004. Swamy, J., Ramsay, J.A., 1999. Effects of Mn2+ and NHþ 4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor. Appl. Microbiol. Biotechnol. 51, 391–395. Tan, R.X., Zou, W.X., 2001. Endophytes: a rich source of functional metabolites. Nat. Prod. Rep. 18, 448–459. Thurston, C.F., 1994. The structure and function of fungal laccases. Microbiology 140, 19–26.
Short Communication
Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon J.W. Wang a
a,b
, J.H. Wu a, W.Y. Huang a, R.X. Tan
a,*
Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210093, China b Department of Pharmaceutical Sciences, Soochow University, Suzhou 215006, China Received 12 January 2004; received in revised form 28 March 2005; accepted 28 March 2005 Available online 11 July 2005
Abstract The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by the endophytic fungus Monotospora sp. were evaluated. The optimal temperature and initial pH for laccase production by Monotospora sp. in submerged culture were found to be 30 °C and 8.5, respectively. Maltose (2 g l1) and ammonium tartrate (10 g l1) were the most suitable carbon and nitrogen source for laccase production. Under optimal culture medium, the maximum laccase activity was determined to be 13.55 U ml1, which was approximately four times higher than that in basal medium. This is the first report on laccase production by an endophytic fungus. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Monotospora sp.; Endophytic fungus; Laccase production; Submerged cultures
1. Introduction Laccases (benzenediol: oxygen oxidoreductases; EC1.10.3.2.) are glycosylated polyphenol oxidases (Thurston, 1994). These enzymes find important commercial applications in the pulp and paper industry, animal biotechnology, biotransformation and detoxification of phenolic pollutants (Brenna and Bianchi, 1994; Breen and Singleton, 1999). Laccase production is a common feature of many basidiomycetes, particularly those associated with wood decay or terminal stages of decomposition (Gianfreda et al., 1999). The genus Trametes, seems to be one of the most efficient laccase producers (Jang et al., 2002).
*
Corresponding author. Tel.: +86 25 83592945; fax: +86 25 83302728. E-mail address: [email protected] (R.X. Tan). 0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.03.025
As a less investigated microorganisms hidden within host plants, endophytes are obviously a rich and reliable source of bioactive metabolites with huge medical, agricultural and industrial potentials (Tan and Zou, 2001). Although the enzymes vary from isolate to isolate, the endophytic fungi tested all synthesize in vitro the enzymes necessary for penetrating and colonizing their plant hosts (Schulz et al., 2002). Such enzymes including pectinases, xylanase, cellulases and lipases, proteinase and phenol oxidase have been documented with some endophytes (Tan and Zou, 2001). However, little has been reported about laccase production for degrading lignin by endophytic fungi up to date. In continuation of our characterization of new metabolites from endophytic fungi (Lu et al., 2000; Liu et al., 2002, 2004), we herewith wish to explore the laccase production ability of Monotospora sp., an endophytic fungus in Cynodon dactylon. Various physicochemical parameters were examined to optimize laccase production.
J.W. Wang et al. / Bioresource Technology 97 (2006) 786–789
2.2. Inoculum preparation and flask cultures Monotospora sp. was initially grown on PDA medium in a Petri dish, and then transferred into the seed medium by punching out 5 mm of the agar plate culture with a self-designed cutter. Unless otherwise specified, a shake flask culture was carried out in a 250 ml flask containing 100 ml of the medium at 25 °C on a rotary shaker incubator at 150 rpm for 6 d. In order to optimize the cultural conditions, the initial pH in the MSB medium was adjusted to the desired value by addition of either 0.1 M HCl or 2.5 M NaOH. The flasks were incubated at various temperatures (20–40 °C). Sucrose, glucose, cellobiose, xylose, soluble starch, rhamnose, CMC-Na, lactose, mannitol, glycerol, maltose and arabinose were studied as carbon sources in relation to expression of laccase activity. Ammonium chloride, ammonium nitrate, ammonium sulfate, ammonium tartrate, meat peptone, yeast extract, acrylamide, urea, beaf extract and glycine were used in this study to select the best nitrogen source. For a time course study, the flasks were incubated at 30 °C on a rotary shaker at 150 rpm for 16 d. At 1 d interval, the contents of each flask were filtered through Whatman filter paper No. 1 (Whatman, Springfield Mill, UK), and the filtrate was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant thus obtained was treated as the enzyme extract. All experiments were performed at least in triplicate to ensure reproducibility. 2.3. Analytical determination The pH was measured with a digital pH meter (Oakton, Singapore, model 1000). The fungal biomass was harvested by filtration through filter paper and bio-
2.4. Statistical analyses Students t-test was performed using OriginTM version 6.0 (Microcal, Northampton, MA, USA). The values represented by their mean ± standard deviation: significant differences between groups were determined at q < 0.05.
3. Results and discussion The shake flask cultures using the basal medium (MSB) showed the ability of the endophyte Monotospora sp. in C. dactylon to produce appreciable amounts of laccases extracellularly. Maximum enzyme production (3.49 ± 0.92 U ml1) reached on day 8 in Monotospora sp. while the biomass maxima was achieved during 4–7 d (Fig. 1). The production maxima was also attained earlier as compared to some white rot fungi such as Phlebia floridensis (20th day) (Arora and Gill, 2000) and Pleurotus ostreatus (14th day) (Hou et al., 2004). To the best of our knowledge, this is the first report on laccase production by endophytic fungi. Our results revealed that an endophytic Monotospora sp. could synthesize in vitro laccase, being involved in various
33
60
30 27
50
24 40
21 18
30
15 12
20
9 6
10
Extracellular protein (µg ml-1)
Monotospora sp., strain number W823, is an endophytic fungus isolated from fresh stems of an apparently healthy C. dactylon collected in May 2002, in the suburb of Nanjing, China. Endophytic strains were isolated following the procedure described previously (Lu et al., 2000). The stock culture was maintained on a potato dextrose agar (PDA) (Sigma, St. Louis, USA) slant and subcultured every month. Slants were incubated at 25 °C for 5 d and subsequently stored at 4 °C. The seed culture was grown in 250 ml flask containing 100 ml of mineral salts broth (MSB) containing: sucrose, 5 g; yeast extract, 2.5 g; acetic acid, 0.6 ml; KH2PO4, 2 g; MgSO4 Æ 7H2O, 1 g; CaCl2 Æ 2H2O, 0.1 g, thiamine hydrochloride, 0.01 g; polysorbate 80, 1 g; trace element solution, 10 ml, in 1 l distilled water (initial pH 4.8), at 25 °C on a rotary shaker incubator at 150 rpm for 4 d.
Laccase activity (U ml-1)
2.1. Microorganism and media
mass yield was determined in terms of oven-dried (80 °C for 24 h) mycelium weight. The protein in the filtrate was estimated by method of Lowry et al. (1951). Laccase activity was determined with 2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as the substrate (Bourbonnais and Paice, 1990). One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 lmol of ABTS per min at 25 °C (Galhaup and Haltrich, 2001).
Mycelial dry weight (x10-1 gL-1)
2. Methods
787
3 0
0 0
2
4
6
8
10
12
14
16
Inoculum age (d)
Fig. 1. The time course of mycelial dry weight ((n) under the basal medium (MSB) and (m) under optimized medium), extracellular protein ((h) under the basal medium (MSB) and (j) under optimized medium) and laccase production ((s) under the basal medium (MSB) and (d) under optimized medium) by Monotospora sp. in shake flask cultures at 30 °C on a rotary shaker at 150 rpm. Values represent the mean ± S.D. of triplicate samples.
788
J.W. Wang et al. / Bioresource Technology 97 (2006) 786–789 5
5
Laccase activity (U ml-1)
(a)
4
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0
(b)
0 0
2
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40
Temperature (°C)
Initial pH
Fig. 2. Effect of pH (a) and temperature (b) on laccase production by Monotospora sp. in shake flask cultures. Values represent the mean ± S.D. of triplicate samples.
processes including the colonization and lignin biodegradation in plant hosts. Since enzyme production is also affected by some culture conditions, initial pH, temperature, the carbon and nitrogen source were chosen for optimization as these have been reported to influence laccase production by other microorganisms (Gianfreda et al., 1999; Meideieros et al., 1999). The effects of pH and temperature on laccase activity of Monotospora sp. are shown in Fig. 2. The optimum initial pH and incubation temperature were determined to 8.5 and 30 °C, respectively. The optimum concentrations of these components for the maximum laccase production by Monotospora sp. were: 2 g l1 maltose and 10 g l1 ammonium tartrate (that is C/N ratio was 0.2/1) (data not shown). Previous papers suggested that the expression of laccases by most white rot fungi required an acidic initial pH, N-limitation and higher C/N ratio in cultures (Swamy and Ramsay, 1999; Jang et al., 2002; Nyanhongo et al., 2002). In comparison to fungal plant pathogens and fungal soil isolates, endophytic fungi grow in different habitats and require distinct levels of nutrition (Schulz et al., 2002). The better enzyme production may be achieved as far as the environment in which these fungi grow is concerned. In this work, N-rich and C-limitation cultures at an alkaline initial pH resulted in a higher expression of laccase. Under the optimal conditions established, the maximum laccase activity of 13.55 ± 1.38 U ml1, which represents an approximately fourfold increase (q < 0.05) in laccase activity from 3.49 ± 0.92 U ml1 in basal medium was obtained in optimized medium containing (g/l): (2 maltose and 10 ammonium tartrate instead of 5 sucrose
and 2.5 yeast extract in MSB medium respectively, pH 8.5) (Fig. 1). As compared to laccase production determined by oxidation of ABTS in cultures of the much-studied white-rot fungi such as Tramets spp. (1.5–15.8 U ml1) (Jang et al., 2002; Rancan˜o et al., 2003), Monotospora sp. proved to be a high laccase producer. In our recent work, the endophytic fungi inside the healthy leaves of C. dactylon were versatile producers of new bioactive metabolites (Liu et al., 2004; Ma et al., 2004). Moreover, in the current study, based on higher laccase activity by the endophytic Monotospora sp., the potential for screening and optimizing more endophytic fungi for laccase production may be exploited in lignin degradation and other biotechnological applications.
Acknowledgements This work was co-financed by grants for RXT from NNSF (Nos. 30171104 and 30470191) and for JWW from MSDF (No. EE132514) of Soochow University.
References Arora, D.S., Gill, P.K., 2000. Laccase production by some white rot fungi under different nutritional conditions. Bioresource Technol. 73, 283–285. Bourbonnais, R., Paice, M.G., 1990. Oxidation of non-phenolic substrates. An expanded role of laccase in lignin biodegradation. FEBS Lett. 267, 99–102. Brenna, O., Bianchi, E., 1994. Immobilized laccase for phenolic removal in must and wine. Biotechnol. Lett. 24, 35–40.
J.W. Wang et al. / Bioresource Technology 97 (2006) 786–789 Breen, A., Singleton, F.L., 1999. Fungi in lignocellulose breakdown and biopulping. Curr. Opin. Biotechnol. 10, 252–258. Galhaup, C., Haltrich, D., 2001. Enhanced formation of laccase activity by the white-rot fungus Tremetes pubescens in the presence of copper. Appl. Microbiol. Biotechnol. 56, 225– 232. Gianfreda, L., Xu, F., Bollag, J.M., 1999. Laccases: a useful group of oxidoreductive enzymes. Bioremediation J. 3, 1–25. Hou, H., Zhou, J., Wang, J., Dua, C., Yan, B., 2004. Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye. Process Biochem. 39, 1415– 1419. Jang, M.Y., Ryu, W.R., Cho, M.H., 2002. Laccase production from repeated batch cultures using free mycelia of Trametes sp. Enzyme Microb. Technol. 30, 741–746. Liu, J.Y., Liu, C.H., Zou, W.X., Tian, X., Tan, R.X., 2002. Leptosphaerone, a metabolite with a novel skeleton from Leptosphaeria sp. IV403, an endophytic fungus in Artemisia annua. Helv. Chim. Acta 85, 2664–2667. Liu, J.Y., Song, Y.C., Zhang, Z., Wang, L., Guo, Z.J., Zou, W.X., Tan, R.X., 2004. Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites. J. Biotechnol. 114, 279–287. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
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