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Production, purification and partial characterization of a novel endo-β-1,3-glucanase from Agaricus brasiliensis
Process Biochemistry 41 (2006) 1229–1233 www.elsevier.com/locate/procbio
Short communication
Production, purification and partial characterization of a novel endo-b-1,3-glucanase from Agaricus brasiliensis Chin-Hang Shu *, Chun-Jun Xu, En-Shu Lin Department of Chemical and Materials Engineering, National Central University, Taoyuan, Taiwan, ROC Received 7 September 2005; received in revised form 28 November 2005; accepted 1 December 2005
Abstract An endo-b-1,3-glucanase from Agaricus brasiliensis ATCC 76739 was first produced and purified in the submerged culture. Purified endo-b1,3-glucanase with a 13.1-fold purification and 6.7% yield was prepared by two steps: precipitation with 70% saturation ammonium sulfate and chromatography on hydrophobic interaction chromatography. The molecular mass of the enzyme was estimated to be 33 kDa by SDS-PAGE. The presence of endo-b-1,3-glucanase might explain the fall of the yield of bioactive exopolysaccharides in the late stage of the submerged cultures. The pH optimum for the enzyme was 4.5, and the temperature optimum was 45 8C. The enzyme showed high pH stability within the range of pH 3.5–6.0 and thermostability up to 50 8C, and exhibited a half-life of 30 min at 55 8C, which was better than that of a thermophilic fungus, Scytalidium thermophilum. The enzyme activity was strongly inhibited by HgCl2. # 2005 Elsevier Ltd. All rights reserved. Keywords: Agaricus brasiliensis; Endo-b-1,3-glucanase; Purification; Exopolysaccharides; Thermostable; Characterization
1. Introduction b-Glucans are not only the main structural components of the cell walls of most fungi but also responsible for the antitumor bioactivity [1–3] and the anti-diabetic activity [4] in several animal studies. The production of polysaccharides by the edible and medicinal mushroom Agaricus brasiliensis (previously named Agaricus blazei) has attracted considerable interest due to their strong antimutagenic effect [5,6], and information is available on the culture conditions which are known to affect their production [7,8]. Many studies indicated that the polysaccharides of A. brasiliensis are heteropolysaccharides [9,10]. The b(1 ! 3) backbone and the b(1 ! 6) branch of polysaccharides are probably responsible for their anti-tumor activity [11–13]. Edible mushrooms such as Agaricus bisporus, Lentinula edodes and Pleurotus sajor-caju produce endo-b-1,4-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), and b-glucosidase (EC 3.2.1.21) when cultivated on glucan and cellulose rich materials in either submerged culture or in solid-state systems [14,15]. However, most studies on the production and
regulation of fungal endoglucanases focused on Trichoderma reesei [16–18] and Phanerochaete chrysosporium [19,20], and studies of endoglucanases of edible mushrooms except A. bisporus are limited [14]. Fungal glucanases were commonly responsible for the degradation of exopolysaccharides such as pullulans in the late stage of the submerged cultures [21–23]. Enzymatically hydrolyzed oligosaccharides from A. brasiliensis by a bacterial glucanase have been shown to have twice the anti-diabetic activity in diabetic rats [4]. Thus, characterization of the glucanase of this economically important edible mushroom A. brasiliensis would be essential for a better understanding of its biological efficiency (i.e. conversion of growth substrate into mushroom fruit bodies) in the solid-state systems and the production of the bioactive polysaccharides and oligosaccharides. In this study, we first report the purification and characterization of an extracellular endo-b1,3-glucanase produced by A. brasiliensis when grown in an air-lift bioreactor.
2. Materials and methods 2.1. Microorganism and culture conditions
* Corresponding author at: #300 Jung-Da Road, Chung-Li, Taoyuan, Taiwan, ROC. Tel.: +886 3 4263749; fax: +886 3 4263749. E-mail address: [email protected] (C.-H. Shu). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.12.011
A. brasiliensis ATCC 76739 was grown in a culture medium containing (g/l): glucose 10; peptone 5; yeast extract 3; malt extract 3; KH2PO4 3; MgSO47H2O 0.3; Vitamin B1 0.01 [8]. The pH of the medium was adjusted with 1 M HCl and
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0.5 M NaOH to 5.2. A. brasiliensis was cultivated at 28 8C with 0.2 vvm aeration rate in a 2 l air-lift bioreactor for 25 day. Cell mass in the broth was removed by filtration through Whatman paper No. 1 in Millipore filtration apparatus, and the filtrate was extensively dialyzed against water at 4 8C.
2.2. Enzyme purification The purification procedure for endo-b-1,3-glucanase from A. brasiliensis involves two steps. 2.2.1. Step 1: ammonium sulphate precipitation Ammonium sulfate was added to the filtrate to give a concentration of 70% (w/v) saturation at 4 8C [14]. Precipitation was allowed for 10 h, and followed by centrifugation at 6000 rpm in a Hettich MIRO 22R refrigerated centrifuge for 20 min. The precipitate was dissolved in a minimal amount of 50 mM acetate buffer (pH 5) containing 1 mM EDTA, and dialyzed for 24 h with three changes in the same buffer. 2.2.2. Step 2: DEAE–Sepharose column chromatography Crude enzyme was applied to a phenyl–Sepharose (Amersham Bioscience, Piscataway, NJ, USA) column (1.6 cm 20 cm) previously equilibrated with the 50 mM acetate buffer (pH 5) containing 1.0 M ammonium sulfate. After washing with two bed volumes of the initial buffer, elution was performed with a linear gradient of 1.0–0.0 M ammonium sulfate at a flow rate of 60.0 ml/h. Fractions showing b-1,3-glucanase activity were pooled, concentrated by ultrafiltration (Centricon 10, Amicon Division, W.R. Grace & Co.) and stored at 20 8C.
2.3. b-1,3-Glucanase assay and protein determination b-1,3-Glucanase activity was routinely assayed by incubating 5 mg laminarin (b-1,3-glucan, Sigma), in 50 mM potassium acetate buffer, pH 5.0, with 1 ml of enzyme solution appropriately diluted in the same buffer [24]. After 30 min of incubation at 50 8C, the reaction was stopped by heating at 100 8C for 10 min. Then, the reducing sugars contents were determined by using the 3,5dinitrosalicylic acid (DNS) [25]. One unit of b-1,3-glucanase was defined as the amount of enzyme that catalyzes the release of 1 mmol of glucose equivalent per min. Protein concentration was determined by the method of Bradford [26], with bovine serum albumin as the standard.
2.4. Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in Laemmli’s system [27], using 4% acrylamide in the stacking gel and 12.5% acrylamide in the separating gel. Protein bands were visualized by staining with Coomassie R 250 brilliant blue. Low-molecular-mass standard proteins (Amersham Biosciences) were used for molecular mass determination as follows: phosphorylase b (94 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and alactalbumin (14.4 kDa).
component within the pH range of 3.0–8.0: sodium acetate (pH 3.0–5.0), phosphate (pH 5.0–7.0), and Tris–HCl (pH 7.0–8.0). The temperature optimum of the b-1,3-glucanase was determined by performing the standard assay within the temperature range of 25–65 8C. The thermal stability of the purified glucanase was examined by measuring the residual activity after incubation the enzyme mixture at each desired temperature for 30 min.
2.7. Determination of the effect of metal ions The effects of several metal ions and compounds on b-1,3-glucanase activity were investigated. The purified enzyme (12.5 U/mg) was preincubated with each 1 mM metals or organic compounds for 15 min at 50 8C. The b-1,3glucanase activity was determined by the standard assay as described above using laminarin as substrate. 100% activity corresponds to no reagents added.
2.8. Pattern of enzymatic hydrolysis Products released following time-course incubation (0–2 h) of laminarin solution with the purified enzyme, at 50 8C and pH 5.0, were separated and analyzed using NH2-HPLC (Nucleosil 10 NH2, 4.6 250 mm, GL Science, Japan) with RI detector (SFD, RI2000) [29]. The mobile phase was water– acetonitrile mixture (2:8) with a flow rate of 1.0 ml/min.
3. Results and discussion 3.1. Enzyme production Enzyme production was accomplished by using a glucan containing complex medium in an air-lift bioreactor. The activity of the endo-b-1,3-glucanase in the broth increased with mycelial biomass, but the activity reached its maximum, 2.5 U/ml, after 18 days of cultivation in the stationary phase as shown in Fig. 1. The presence of endo-b-1,3-glucanase activity might partially contribute to the fall of the yield of bioactive exopolysaccharides in the late stage of the submerged cultures [8]. 3.2. Purification of glucanase and size determination Purification of an endo-b-1,3-glucanase produced by A. brasiliensis was accomplished by a two-step procedure: precipitation with 70% saturation of ammonium sulfate and hydrophobic interaction chromatography on phenyl–Sepharose. Chromatography on phenyl–Sepharose resulted in the
2.5. Substrate specificity The activity of the purified b-1,3-glucanase was tested on various polymers with a- or b-glycosidic bonds at a final concentration of 5 mg/ml. In each case, degradation was assayed by the production of reducing sugars and measured as described above. Substrate blanks were included in parallel. Besides, a typical substrate for exo-b-glucanases p-nitrophenyl-b-D-glucopyranoside (pNPG) was included, and the enzyme activity toward pNPG was measured by the amount of pnitrophenol liberated from pNPG at 410 nm as described elsewhere [28].
2.6. Determination of pH and temperature optimum and their stability The pH optimum of the b-1,3-glucanase was determined by performing the standard enzyme assay except the appropriate buffers containing 100 mM each
Fig. 1. Extracellular production of endo-b-1,3-glucanase from A. brasiliensis in air-lift bioreactor during growth in 1.0% glucose supplemented culture: (*) extracellular protein (mg/ml); (~) biomass (g/l); (&) b-1,3-glucanase activity (U/ml).
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Table 1 Summary of purification scheme for the b-1,3-glucanase of A. brasiliensis Purification steps
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification factor
Culture filtrate 30–70%(NH4)2SO4 Phenyl–Sepharose
39.50 32.60 0.20
145.70 136.40 9.70
3.69 4.18 48.50
100 93.6 6.66
1 1.13 13.1
separation of one peak with b-1,3-glucanase activity at a low ammonium sulfate eluant. After the phenyl–Sepharose chromatography, the specific activity of b-1,3-glucanase was about 48.5 U/mg and 13.1-fold purification was achieved with a recovery of 6.7% (Table 1). A single protein band was observed with no trace of contaminants as it was electrophoresed on SDS-PAGE (Fig. 2). The molecular mass of the purified b-1,3glucanase from A. brasiliensis was estimated to be 33 kDa by SDS-PAGE (Fig. 2). The molecular masses of fungal b-1,3glucanases are species dependent [14], and values of 29, 32, and 35.5 kDa have been obtained for T. harzianum, A. bisporus, and Schizophyllum commune [14,30,31], respectively.
Table 2 Substrate specificity of the purified b-1,3-glucanase from A. brasiliensis on a variety of b-glucana Substrate
Main linkage b-1,3 b-1,3 b-1,3 b-1,3 b-1,3 b-1,3 b-1,4 b-1,4 a-1,4
3.3. Substrate specificity
Laminarin ABPSGc Oat glucan Barley glucan Lichenan Pustulan Chitosan Carboxymethylcellulose Amylose p-Nitrophenyl b-D-glucopyranoside
The results of substrate specificity of the purified b-1,3glucanase on various glucan substrates are listed in Table 2. Different fractions of exopolysaccharides from A. brasiliensis (ABPSG) have been isolated and characterized with b-1,3linkage [32] and b-1,6-linkage [33]. The purified enzyme was
Reaction mixture containing purified enzyme (b-1,3-glucanase 12.5 U/mg). After the reactions, reducing sugars released by enzymatic hydrolysis were determined. The amounts of reducing sugars released per minute are shown as percentages of that measured for laminarin, which was defined as 100%. c ABPSG: the exopolysaccharides isolated from the liquid culture of A. brasiliensis ATCC 76739.
and and and and and
Relative activity of b-1,3-glucanase (%)b b-1,6 b-1,4 b-1,4 b-1,4 b-1,6
100 0.3 24.5 1.7 15 1.5 8 1.1 0 0.0 4 1.3 0 0 0 0
a
b
active only toward glucans containing b-1,3-linkages, such as laminarin, ABPSG and purified cell walls from oat and barley. The enzyme hydrolyzed laminarin more efficiently than purified cell walls (mixed linkages); however, no reaction was observed toward lichenan, chitosan, cellulose, and amylose. A slight activity was recorded with pustulan (linear b-1,6glucan), probably due to the presence of b-1,3-linkages in their branching points [24]. Likewise, fungal b-1,3-glucanases that
Fig. 2. SDS-PAGE patterns of b-1,3-glucanase at various stages of purification. Separation was performed on a 12.5% (w/v) polyacrylamide-SDS. Lane: (M) molecular mass markers (200 mg proteins loaded); (a) proteins precipitated from the culture supernatant with 30–70% saturated (NH4)2SO4 (100 mg proteins); (b) pooled fractions from the phenyl–Sepharose eluate (50 mg proteins).
Fig. 3. Effect of pH on the activity (*) and stability (&) of the purified b-1,3glucanase from A. brasiliensis. The reaction mixtures in suitable buffers were equilibrated and then b-1,3-glucanase (10 U/mg) was added to start the reaction. The pH values were adjusted with the following buffer systems: 50 mM sodium acetate (pH 3.0–5.0), 50 mM phosphate (pH 5.0–7.0), and 50 mM Tris– HCl (pH 7.0–8.0). The enzyme activity was measured at 50 8C for 30 min.
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that laminarin was cleaved randomly. For the first 30 min of hydrolysis, laminaribiose and higher laminari-oligosaccharides were detected but no glucose was formed. Besides, the purified enzyme did not show hydrolytic activity against p-nitrophenyl b-D-glucopyranoside (a typical substrate for exo-glucanases) (Table 2). The result is similar to those of endo-1,3-bglucanases in Candida utilis [28] and in Pyrococcus furiosus [38]. Glucose was generally the first and final product of exoglucanase in the pattern of enzymatic hydrolysis, which was not observed in this study. These results suggested that the purified enzyme in this study was an endo-enzyme. 4. Conclusion Fig. 4. Effect of temperature on the activity (*) and stability (&) of the purified b-1,3-glucanase from A. brasiliensis. The enzyme reaction was carried out in 50 mM acetate buffer (pH 5.0) for 30 min.
could hydrolyze b-1,3-linkages but b-1,6-linkages less efficiently have been observed from other fungi including T. harzianum [34] and Saccharomyces cerevisiae [35,36]. 3.4. Optimum pH and temperature The pH optimum of the 33 kDa b-1,3-glucanase from A. brasiliensis was 4.5 (Fig. 3), which is similar to that of the 32 kDa endo-b-1,3-glucanase produced by A. bisporus [14]. The purified enzyme showed high pH stability within the range of pH 3.5–6.0 (Fig. 3) similar to those of other fungal b-1,3glucanase reported before [21]. The temperature optimum for the enzyme activity was 45 8C at pH 4.5 (Fig. 4). The enzyme was stable up to 50 8C and show a half-life of 30 min when incubated at 55 8C (Fig. 5). The bglucanase of a thermophilic fungus Scytalidium thermophilum exhibited temperature optimum at 60 8C, but showed a half-life of 20 min when incubated at 55 8C [37]. The thermostability of the purified enzyme was higher than that of S. thermophilum. 3.5. Inhibition by metal ions and chemicals The results of the influence of metal ions and organic compounds on the activity of the b-1,3-glucanase showed that the enzyme was strongly inhibited by Hg2+ with 42% residual activity, which implied the presence of sulfur-groups in the enzyme [14]. The denaturing agent SDS, urea, Fe2+, and Cu2+ showed significant inhibitory effects on the activity by 36, 25, 29, and 30%, respectively. However, no significant inhibition (<15%) on the enzyme activity was observed with PMSF, EDTA, Na+, and Mg2+, while slight enhancements were observed in the presence of Ca2+, Zn2+, and K+ with 112%, 109%, and 103%, respectively. 3.6. Characterization of endo-glucanase The mode of action of the purified enzyme was examined by analyzing the corresponding hydrolysis products of laminarin by HPLC. Time-course data of enzymatic hydrolysis revealed
The results of this study have shown that A. brasiliensis could produce an endo-b-1,3-glucanase in the submerged culture. The presence of endo-b-1,3-glucanase and its significant relative activity (24.5%, Table 2) on ABPSG might explain the fall of the yield of bioactive exopolysaccharides in the late stage of the submerged cultures. Besides, the conversion of growth substrate into mushroom fruit bodies in the solid-state systems might be enhanced due to the saprophytic activity of A. brasiliensis on decaying biomass through endo-b-1,3-glucanase. The purified enzyme with relatively good thermostability and acidic pH tolerance might be used in industrial applications in the future. Acknowledgement The authors would like to thank the Council of Agriculture of the Republic of China, Taiwan, for financially supporting this research under Contract No. COA 91 AG-3.1.3-FD-Z3. References [1] Bendjeddou D, Lalaoui K, Satta D. Immunostimulating activity of the hot water-soluble polysaccharide extracts of Anacyclus pyrethrum, Alpinia galanga and Citrullus colocynthis. J Ethnopharmacol 2003;88:155– 60. [2] Ito H, Shimura K, Itoh H, Kawade M. Antitumor effects of a new polysaccharide–protein complex (ATOM) prepared from Agaricus blazei (Iwade strain 101) ‘‘Himematsutake’’ and its mechanisms in tumorbearing mice. Anticancer Res 1997;17:277–84. [3] Mizuno T, Hagiwara T, Nakamura T, Ito H, Shimura K, Sumiya T, et al. Antitumor activity and some properties of water-soluble polysaccharides from ‘‘Himematsutake’’ the fruiting body of Agaricus blazei Murill. Agric Biol Chem 1990;54:2889–96. [4] Kim YW, Kim KH, Choi HJ, Lee DS. Anti-diabetic activity of betaglucans and their enzymatically hydrolyzed oligosaccharides from Agaricus blazei. Biotechnol Lett 2005;27(7):483–7. [5] Delmanto RD, de Lima PLA, Sugui MM, da Eira AF, Salvadori DMF, Speit G, et al. Antimutagenic effect of Agaricus blazei Murrill mushroom on the genotoxicity induced by cyclophosphamide. Mutat Res Genet Toxicol Environ Mutagen 2001;496:15–21. [6] Mizuno M, Kawakami S, Sakamoto Y, Fujitake N. Macrophages stimulated by polysaccharide purified from Agaricus brasiliensis S. Wasser et al. (Agaricomycetidae) enhance mRNA expression of Th1 cytokine including IL-12 and 18. Int J Med Mushrooms 2003;5:383–9. [7] Shu CH, Wen BJ. Enhanced shear protection and increased production of an anti-tumor polysaccharide by Agaricus blazei in xanthan-supplemented cultures. Biotechnol Lett 2003;25(11):873–6.
C.-H. Shu et al. / Process Biochemistry 41 (2006) 1229–1233 [8] Shu CH, Wen BJ, Lin KJ. Monitoring the polysaccharide quality of Agaricus blazei in submerged culture by examining molecular weight distribution and TNF-alpha release capability of macrophage cell line RAW 264.7. Biotechnol Lett 2004;26(4):2061–4. [9] Dong Q, Yao J, Yang X, Fang J. Structural characterization of a watersoluble b-d-glucan from fruiting bodies of Agaricus blazei. Murr Carbohydr Res 2002;337:1417–21. [10] Kawagishi H, Inagaki R, Kanao T, Mizuno T, Shimura K, Ito H, et al. Fractionation and antitumor activity of the water-in-soluble residue of Agaricus blazei fruiting bodies. Carbohydr Res 1989;186:267–73. [11] Kojima T, Tabata K, Itoh W, Yanaki T. Molecular weight dependence of the antitumor activity of Schizophyllan. Agric Biol Chem 1986;50:231–2. [12] Ohno N, Furukawa M, Miura NN, Adachi Y, Motoi M, Yadomae T. Antitumor beta glucan from the cultured fruit body of Agaricus blazei. Biol Pharm Bull 2001;24(7):820–8. [13] Sakagami H, Ikeda M, Konno K. Stimulation of tumor necrosis factorinduced human myelogenous leukemic cell differentiation by high molecular weight PSK subfraction. Biochem Biophys Res Commun 1989; 162:597–603. [14] Galan B, Garcia Mendoza C, Calonje M, Novaes-Ledieu M. Production, purification, and properties of an endo-1,3-b-glucanase from the basidiomycete Agaricus bisporus. Curr Microbiol 1999;38:190–3. [15] Rast DM, Baumgartner D, Mayer C, Hollenstein GO. Cell wall-associated enzymes in fungi. Phytochemistry 2003;64:339–66. [16] Ilmen M, Saloheimo A, Onnela ML, Penttila ME. Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 1997;63:1298–306. [17] Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F. Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol 2002;99:63–78. [18] Penttila ME, Andre L, Saloheimo M, Lehtovaara P, Knowlse JKC. Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae. Yeast 1987;3:175–85. [19] Igarashi K, Tani T, Kawai R, Samejima M. Family 3 b-glucosidase from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium is a glucan 1,3-b-glucosidase. J Biosci Bioeng 2003; 95:572–6. [20] Lymar ES, Li B, Renganathan V. Purification and characterization of a cellulose-binding b-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl Environ Microb 1995;61:2976–80. [21] Pitson SM, Seviour RJ, McDougall BM. Noncellulolytic fungal b-glucanases: their physiology and regulation. Enzyme Microb Technol 1993; 15:178–92. [22] Pitson SM, Seviour RJ, McDougall BM. Purification and characterization of an extracellular b-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in b-glucan degradation. Enzyme Microb Technol 1997;21:182–90.
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[23] West TP, Strohfus B. A pullulan-degrading enzyme activity of Aureobasidium pullulans. J Basic Microbiol 1996;36:377–80. [24] Cruz J, Pintor-Toro JA, Benı´tez T, Llobell A, Romero LC. A novel endobeta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol 1995;177:6937–45. [25] Miller GL. Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal Chem 1959;31:426–8. [26] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [27] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–5. [28] Villa TG, Notario V, Villanueva JR. Occurrence of an endo-1,3-betaglucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity. Biochem J 1979;177:107–14. [29] Aida K, Okada T, Kasahara N, Nikaidou N, Tanaka H, Watanabe T. Comparative studies of b-1,3-glucanase A1 and B of Bacillus circulans WL-12: purification and enzymatic properties. J Ferment Bioeng 1995; 80:283–6. [30] Noronha EF, Ulhoa CJ. Characterization of a 29-kDa b-1,3-glucanase from Trichoderma harzianum. FEMS Microbiol Lett 2000;183:119–23. [31] Prokop A, Rapp P, Wagner F. Production, purification and characterization of an extracellular endo-b-1,3-glucanase from a momocaryon of Schizophyllum commune ATCC 38548 defective in exo-b-1,3-glucanase formation. Can J Microbiol 1994;40:18–23. [32] Mizuno M, Minato K, Ito H, Kawade M, Terai H, Tsuchida H. Anti-tumor polysaccharide from the mycelium of liquid-cultured Agaricus blazei mill. Biochem Mol Biol Int 1999;47:707–14. [33] Kawagishi H, Inagaki R, Kano T, Shimura K, Ito H, Hagiwara T, et al. Formolysis of a potent antitumor (1-6)b-D-glucan–protein complex from Agaricus blazei friting bodies and antitumor activity of the resulting products. Carbohydr Polym 1990;12:393–403. [34] Noronha EF, Ulhoa CJ. Purification and characterization of an endo-b-1,3glucanase from Trichoderma harzianum. Can J Microbiol 1996;42:1039– 44. [35] Hien NH, Fleet GH. Separation and characterization of six b-1,3-glucanases from Saccharomyces cerevisiae. J Bacteriol 1983;156:1204–13. [36] Tanaka H, Phaff JH. Enzymatic hydrolysis of yeast cell wall. I. Isolation of wall-decomposing organisms and separation and purification of lytic enzymes. J Bacteriol 1965;89:1570–80. [37] Zanoelo FF, Polizeli MdLTdM, Terenzi HF, Jorge JA. b-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol Lett 2004;240:137–43. [38] Gueguen Y, Voorhorst WG, van der Oost J, de Vos WM. Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 1997; 272:31258–64.
Short communication
Production, purification and partial characterization of a novel endo-b-1,3-glucanase from Agaricus brasiliensis Chin-Hang Shu *, Chun-Jun Xu, En-Shu Lin Department of Chemical and Materials Engineering, National Central University, Taoyuan, Taiwan, ROC Received 7 September 2005; received in revised form 28 November 2005; accepted 1 December 2005
Abstract An endo-b-1,3-glucanase from Agaricus brasiliensis ATCC 76739 was first produced and purified in the submerged culture. Purified endo-b1,3-glucanase with a 13.1-fold purification and 6.7% yield was prepared by two steps: precipitation with 70% saturation ammonium sulfate and chromatography on hydrophobic interaction chromatography. The molecular mass of the enzyme was estimated to be 33 kDa by SDS-PAGE. The presence of endo-b-1,3-glucanase might explain the fall of the yield of bioactive exopolysaccharides in the late stage of the submerged cultures. The pH optimum for the enzyme was 4.5, and the temperature optimum was 45 8C. The enzyme showed high pH stability within the range of pH 3.5–6.0 and thermostability up to 50 8C, and exhibited a half-life of 30 min at 55 8C, which was better than that of a thermophilic fungus, Scytalidium thermophilum. The enzyme activity was strongly inhibited by HgCl2. # 2005 Elsevier Ltd. All rights reserved. Keywords: Agaricus brasiliensis; Endo-b-1,3-glucanase; Purification; Exopolysaccharides; Thermostable; Characterization
1. Introduction b-Glucans are not only the main structural components of the cell walls of most fungi but also responsible for the antitumor bioactivity [1–3] and the anti-diabetic activity [4] in several animal studies. The production of polysaccharides by the edible and medicinal mushroom Agaricus brasiliensis (previously named Agaricus blazei) has attracted considerable interest due to their strong antimutagenic effect [5,6], and information is available on the culture conditions which are known to affect their production [7,8]. Many studies indicated that the polysaccharides of A. brasiliensis are heteropolysaccharides [9,10]. The b(1 ! 3) backbone and the b(1 ! 6) branch of polysaccharides are probably responsible for their anti-tumor activity [11–13]. Edible mushrooms such as Agaricus bisporus, Lentinula edodes and Pleurotus sajor-caju produce endo-b-1,4-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), and b-glucosidase (EC 3.2.1.21) when cultivated on glucan and cellulose rich materials in either submerged culture or in solid-state systems [14,15]. However, most studies on the production and
regulation of fungal endoglucanases focused on Trichoderma reesei [16–18] and Phanerochaete chrysosporium [19,20], and studies of endoglucanases of edible mushrooms except A. bisporus are limited [14]. Fungal glucanases were commonly responsible for the degradation of exopolysaccharides such as pullulans in the late stage of the submerged cultures [21–23]. Enzymatically hydrolyzed oligosaccharides from A. brasiliensis by a bacterial glucanase have been shown to have twice the anti-diabetic activity in diabetic rats [4]. Thus, characterization of the glucanase of this economically important edible mushroom A. brasiliensis would be essential for a better understanding of its biological efficiency (i.e. conversion of growth substrate into mushroom fruit bodies) in the solid-state systems and the production of the bioactive polysaccharides and oligosaccharides. In this study, we first report the purification and characterization of an extracellular endo-b1,3-glucanase produced by A. brasiliensis when grown in an air-lift bioreactor.
2. Materials and methods 2.1. Microorganism and culture conditions
* Corresponding author at: #300 Jung-Da Road, Chung-Li, Taoyuan, Taiwan, ROC. Tel.: +886 3 4263749; fax: +886 3 4263749. E-mail address: [email protected] (C.-H. Shu). 1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.12.011
A. brasiliensis ATCC 76739 was grown in a culture medium containing (g/l): glucose 10; peptone 5; yeast extract 3; malt extract 3; KH2PO4 3; MgSO47H2O 0.3; Vitamin B1 0.01 [8]. The pH of the medium was adjusted with 1 M HCl and
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0.5 M NaOH to 5.2. A. brasiliensis was cultivated at 28 8C with 0.2 vvm aeration rate in a 2 l air-lift bioreactor for 25 day. Cell mass in the broth was removed by filtration through Whatman paper No. 1 in Millipore filtration apparatus, and the filtrate was extensively dialyzed against water at 4 8C.
2.2. Enzyme purification The purification procedure for endo-b-1,3-glucanase from A. brasiliensis involves two steps. 2.2.1. Step 1: ammonium sulphate precipitation Ammonium sulfate was added to the filtrate to give a concentration of 70% (w/v) saturation at 4 8C [14]. Precipitation was allowed for 10 h, and followed by centrifugation at 6000 rpm in a Hettich MIRO 22R refrigerated centrifuge for 20 min. The precipitate was dissolved in a minimal amount of 50 mM acetate buffer (pH 5) containing 1 mM EDTA, and dialyzed for 24 h with three changes in the same buffer. 2.2.2. Step 2: DEAE–Sepharose column chromatography Crude enzyme was applied to a phenyl–Sepharose (Amersham Bioscience, Piscataway, NJ, USA) column (1.6 cm 20 cm) previously equilibrated with the 50 mM acetate buffer (pH 5) containing 1.0 M ammonium sulfate. After washing with two bed volumes of the initial buffer, elution was performed with a linear gradient of 1.0–0.0 M ammonium sulfate at a flow rate of 60.0 ml/h. Fractions showing b-1,3-glucanase activity were pooled, concentrated by ultrafiltration (Centricon 10, Amicon Division, W.R. Grace & Co.) and stored at 20 8C.
2.3. b-1,3-Glucanase assay and protein determination b-1,3-Glucanase activity was routinely assayed by incubating 5 mg laminarin (b-1,3-glucan, Sigma), in 50 mM potassium acetate buffer, pH 5.0, with 1 ml of enzyme solution appropriately diluted in the same buffer [24]. After 30 min of incubation at 50 8C, the reaction was stopped by heating at 100 8C for 10 min. Then, the reducing sugars contents were determined by using the 3,5dinitrosalicylic acid (DNS) [25]. One unit of b-1,3-glucanase was defined as the amount of enzyme that catalyzes the release of 1 mmol of glucose equivalent per min. Protein concentration was determined by the method of Bradford [26], with bovine serum albumin as the standard.
2.4. Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in Laemmli’s system [27], using 4% acrylamide in the stacking gel and 12.5% acrylamide in the separating gel. Protein bands were visualized by staining with Coomassie R 250 brilliant blue. Low-molecular-mass standard proteins (Amersham Biosciences) were used for molecular mass determination as follows: phosphorylase b (94 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and alactalbumin (14.4 kDa).
component within the pH range of 3.0–8.0: sodium acetate (pH 3.0–5.0), phosphate (pH 5.0–7.0), and Tris–HCl (pH 7.0–8.0). The temperature optimum of the b-1,3-glucanase was determined by performing the standard assay within the temperature range of 25–65 8C. The thermal stability of the purified glucanase was examined by measuring the residual activity after incubation the enzyme mixture at each desired temperature for 30 min.
2.7. Determination of the effect of metal ions The effects of several metal ions and compounds on b-1,3-glucanase activity were investigated. The purified enzyme (12.5 U/mg) was preincubated with each 1 mM metals or organic compounds for 15 min at 50 8C. The b-1,3glucanase activity was determined by the standard assay as described above using laminarin as substrate. 100% activity corresponds to no reagents added.
2.8. Pattern of enzymatic hydrolysis Products released following time-course incubation (0–2 h) of laminarin solution with the purified enzyme, at 50 8C and pH 5.0, were separated and analyzed using NH2-HPLC (Nucleosil 10 NH2, 4.6 250 mm, GL Science, Japan) with RI detector (SFD, RI2000) [29]. The mobile phase was water– acetonitrile mixture (2:8) with a flow rate of 1.0 ml/min.
3. Results and discussion 3.1. Enzyme production Enzyme production was accomplished by using a glucan containing complex medium in an air-lift bioreactor. The activity of the endo-b-1,3-glucanase in the broth increased with mycelial biomass, but the activity reached its maximum, 2.5 U/ml, after 18 days of cultivation in the stationary phase as shown in Fig. 1. The presence of endo-b-1,3-glucanase activity might partially contribute to the fall of the yield of bioactive exopolysaccharides in the late stage of the submerged cultures [8]. 3.2. Purification of glucanase and size determination Purification of an endo-b-1,3-glucanase produced by A. brasiliensis was accomplished by a two-step procedure: precipitation with 70% saturation of ammonium sulfate and hydrophobic interaction chromatography on phenyl–Sepharose. Chromatography on phenyl–Sepharose resulted in the
2.5. Substrate specificity The activity of the purified b-1,3-glucanase was tested on various polymers with a- or b-glycosidic bonds at a final concentration of 5 mg/ml. In each case, degradation was assayed by the production of reducing sugars and measured as described above. Substrate blanks were included in parallel. Besides, a typical substrate for exo-b-glucanases p-nitrophenyl-b-D-glucopyranoside (pNPG) was included, and the enzyme activity toward pNPG was measured by the amount of pnitrophenol liberated from pNPG at 410 nm as described elsewhere [28].
2.6. Determination of pH and temperature optimum and their stability The pH optimum of the b-1,3-glucanase was determined by performing the standard enzyme assay except the appropriate buffers containing 100 mM each
Fig. 1. Extracellular production of endo-b-1,3-glucanase from A. brasiliensis in air-lift bioreactor during growth in 1.0% glucose supplemented culture: (*) extracellular protein (mg/ml); (~) biomass (g/l); (&) b-1,3-glucanase activity (U/ml).
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Table 1 Summary of purification scheme for the b-1,3-glucanase of A. brasiliensis Purification steps
Total protein (mg)
Total activity (U)
Specific activity (U/mg)
Yield (%)
Purification factor
Culture filtrate 30–70%(NH4)2SO4 Phenyl–Sepharose
39.50 32.60 0.20
145.70 136.40 9.70
3.69 4.18 48.50
100 93.6 6.66
1 1.13 13.1
separation of one peak with b-1,3-glucanase activity at a low ammonium sulfate eluant. After the phenyl–Sepharose chromatography, the specific activity of b-1,3-glucanase was about 48.5 U/mg and 13.1-fold purification was achieved with a recovery of 6.7% (Table 1). A single protein band was observed with no trace of contaminants as it was electrophoresed on SDS-PAGE (Fig. 2). The molecular mass of the purified b-1,3glucanase from A. brasiliensis was estimated to be 33 kDa by SDS-PAGE (Fig. 2). The molecular masses of fungal b-1,3glucanases are species dependent [14], and values of 29, 32, and 35.5 kDa have been obtained for T. harzianum, A. bisporus, and Schizophyllum commune [14,30,31], respectively.
Table 2 Substrate specificity of the purified b-1,3-glucanase from A. brasiliensis on a variety of b-glucana Substrate
Main linkage b-1,3 b-1,3 b-1,3 b-1,3 b-1,3 b-1,3 b-1,4 b-1,4 a-1,4
3.3. Substrate specificity
Laminarin ABPSGc Oat glucan Barley glucan Lichenan Pustulan Chitosan Carboxymethylcellulose Amylose p-Nitrophenyl b-D-glucopyranoside
The results of substrate specificity of the purified b-1,3glucanase on various glucan substrates are listed in Table 2. Different fractions of exopolysaccharides from A. brasiliensis (ABPSG) have been isolated and characterized with b-1,3linkage [32] and b-1,6-linkage [33]. The purified enzyme was
Reaction mixture containing purified enzyme (b-1,3-glucanase 12.5 U/mg). After the reactions, reducing sugars released by enzymatic hydrolysis were determined. The amounts of reducing sugars released per minute are shown as percentages of that measured for laminarin, which was defined as 100%. c ABPSG: the exopolysaccharides isolated from the liquid culture of A. brasiliensis ATCC 76739.
and and and and and
Relative activity of b-1,3-glucanase (%)b b-1,6 b-1,4 b-1,4 b-1,4 b-1,6
100 0.3 24.5 1.7 15 1.5 8 1.1 0 0.0 4 1.3 0 0 0 0
a
b
active only toward glucans containing b-1,3-linkages, such as laminarin, ABPSG and purified cell walls from oat and barley. The enzyme hydrolyzed laminarin more efficiently than purified cell walls (mixed linkages); however, no reaction was observed toward lichenan, chitosan, cellulose, and amylose. A slight activity was recorded with pustulan (linear b-1,6glucan), probably due to the presence of b-1,3-linkages in their branching points [24]. Likewise, fungal b-1,3-glucanases that
Fig. 2. SDS-PAGE patterns of b-1,3-glucanase at various stages of purification. Separation was performed on a 12.5% (w/v) polyacrylamide-SDS. Lane: (M) molecular mass markers (200 mg proteins loaded); (a) proteins precipitated from the culture supernatant with 30–70% saturated (NH4)2SO4 (100 mg proteins); (b) pooled fractions from the phenyl–Sepharose eluate (50 mg proteins).
Fig. 3. Effect of pH on the activity (*) and stability (&) of the purified b-1,3glucanase from A. brasiliensis. The reaction mixtures in suitable buffers were equilibrated and then b-1,3-glucanase (10 U/mg) was added to start the reaction. The pH values were adjusted with the following buffer systems: 50 mM sodium acetate (pH 3.0–5.0), 50 mM phosphate (pH 5.0–7.0), and 50 mM Tris– HCl (pH 7.0–8.0). The enzyme activity was measured at 50 8C for 30 min.
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that laminarin was cleaved randomly. For the first 30 min of hydrolysis, laminaribiose and higher laminari-oligosaccharides were detected but no glucose was formed. Besides, the purified enzyme did not show hydrolytic activity against p-nitrophenyl b-D-glucopyranoside (a typical substrate for exo-glucanases) (Table 2). The result is similar to those of endo-1,3-bglucanases in Candida utilis [28] and in Pyrococcus furiosus [38]. Glucose was generally the first and final product of exoglucanase in the pattern of enzymatic hydrolysis, which was not observed in this study. These results suggested that the purified enzyme in this study was an endo-enzyme. 4. Conclusion Fig. 4. Effect of temperature on the activity (*) and stability (&) of the purified b-1,3-glucanase from A. brasiliensis. The enzyme reaction was carried out in 50 mM acetate buffer (pH 5.0) for 30 min.
could hydrolyze b-1,3-linkages but b-1,6-linkages less efficiently have been observed from other fungi including T. harzianum [34] and Saccharomyces cerevisiae [35,36]. 3.4. Optimum pH and temperature The pH optimum of the 33 kDa b-1,3-glucanase from A. brasiliensis was 4.5 (Fig. 3), which is similar to that of the 32 kDa endo-b-1,3-glucanase produced by A. bisporus [14]. The purified enzyme showed high pH stability within the range of pH 3.5–6.0 (Fig. 3) similar to those of other fungal b-1,3glucanase reported before [21]. The temperature optimum for the enzyme activity was 45 8C at pH 4.5 (Fig. 4). The enzyme was stable up to 50 8C and show a half-life of 30 min when incubated at 55 8C (Fig. 5). The bglucanase of a thermophilic fungus Scytalidium thermophilum exhibited temperature optimum at 60 8C, but showed a half-life of 20 min when incubated at 55 8C [37]. The thermostability of the purified enzyme was higher than that of S. thermophilum. 3.5. Inhibition by metal ions and chemicals The results of the influence of metal ions and organic compounds on the activity of the b-1,3-glucanase showed that the enzyme was strongly inhibited by Hg2+ with 42% residual activity, which implied the presence of sulfur-groups in the enzyme [14]. The denaturing agent SDS, urea, Fe2+, and Cu2+ showed significant inhibitory effects on the activity by 36, 25, 29, and 30%, respectively. However, no significant inhibition (<15%) on the enzyme activity was observed with PMSF, EDTA, Na+, and Mg2+, while slight enhancements were observed in the presence of Ca2+, Zn2+, and K+ with 112%, 109%, and 103%, respectively. 3.6. Characterization of endo-glucanase The mode of action of the purified enzyme was examined by analyzing the corresponding hydrolysis products of laminarin by HPLC. Time-course data of enzymatic hydrolysis revealed
The results of this study have shown that A. brasiliensis could produce an endo-b-1,3-glucanase in the submerged culture. The presence of endo-b-1,3-glucanase and its significant relative activity (24.5%, Table 2) on ABPSG might explain the fall of the yield of bioactive exopolysaccharides in the late stage of the submerged cultures. Besides, the conversion of growth substrate into mushroom fruit bodies in the solid-state systems might be enhanced due to the saprophytic activity of A. brasiliensis on decaying biomass through endo-b-1,3-glucanase. The purified enzyme with relatively good thermostability and acidic pH tolerance might be used in industrial applications in the future. Acknowledgement The authors would like to thank the Council of Agriculture of the Republic of China, Taiwan, for financially supporting this research under Contract No. COA 91 AG-3.1.3-FD-Z3. References [1] Bendjeddou D, Lalaoui K, Satta D. Immunostimulating activity of the hot water-soluble polysaccharide extracts of Anacyclus pyrethrum, Alpinia galanga and Citrullus colocynthis. J Ethnopharmacol 2003;88:155– 60. [2] Ito H, Shimura K, Itoh H, Kawade M. Antitumor effects of a new polysaccharide–protein complex (ATOM) prepared from Agaricus blazei (Iwade strain 101) ‘‘Himematsutake’’ and its mechanisms in tumorbearing mice. Anticancer Res 1997;17:277–84. [3] Mizuno T, Hagiwara T, Nakamura T, Ito H, Shimura K, Sumiya T, et al. Antitumor activity and some properties of water-soluble polysaccharides from ‘‘Himematsutake’’ the fruiting body of Agaricus blazei Murill. Agric Biol Chem 1990;54:2889–96. [4] Kim YW, Kim KH, Choi HJ, Lee DS. Anti-diabetic activity of betaglucans and their enzymatically hydrolyzed oligosaccharides from Agaricus blazei. Biotechnol Lett 2005;27(7):483–7. [5] Delmanto RD, de Lima PLA, Sugui MM, da Eira AF, Salvadori DMF, Speit G, et al. Antimutagenic effect of Agaricus blazei Murrill mushroom on the genotoxicity induced by cyclophosphamide. Mutat Res Genet Toxicol Environ Mutagen 2001;496:15–21. [6] Mizuno M, Kawakami S, Sakamoto Y, Fujitake N. Macrophages stimulated by polysaccharide purified from Agaricus brasiliensis S. Wasser et al. (Agaricomycetidae) enhance mRNA expression of Th1 cytokine including IL-12 and 18. Int J Med Mushrooms 2003;5:383–9. [7] Shu CH, Wen BJ. Enhanced shear protection and increased production of an anti-tumor polysaccharide by Agaricus blazei in xanthan-supplemented cultures. Biotechnol Lett 2003;25(11):873–6.
C.-H. Shu et al. / Process Biochemistry 41 (2006) 1229–1233 [8] Shu CH, Wen BJ, Lin KJ. Monitoring the polysaccharide quality of Agaricus blazei in submerged culture by examining molecular weight distribution and TNF-alpha release capability of macrophage cell line RAW 264.7. Biotechnol Lett 2004;26(4):2061–4. [9] Dong Q, Yao J, Yang X, Fang J. Structural characterization of a watersoluble b-d-glucan from fruiting bodies of Agaricus blazei. Murr Carbohydr Res 2002;337:1417–21. [10] Kawagishi H, Inagaki R, Kanao T, Mizuno T, Shimura K, Ito H, et al. Fractionation and antitumor activity of the water-in-soluble residue of Agaricus blazei fruiting bodies. Carbohydr Res 1989;186:267–73. [11] Kojima T, Tabata K, Itoh W, Yanaki T. Molecular weight dependence of the antitumor activity of Schizophyllan. Agric Biol Chem 1986;50:231–2. [12] Ohno N, Furukawa M, Miura NN, Adachi Y, Motoi M, Yadomae T. Antitumor beta glucan from the cultured fruit body of Agaricus blazei. Biol Pharm Bull 2001;24(7):820–8. [13] Sakagami H, Ikeda M, Konno K. Stimulation of tumor necrosis factorinduced human myelogenous leukemic cell differentiation by high molecular weight PSK subfraction. Biochem Biophys Res Commun 1989; 162:597–603. [14] Galan B, Garcia Mendoza C, Calonje M, Novaes-Ledieu M. Production, purification, and properties of an endo-1,3-b-glucanase from the basidiomycete Agaricus bisporus. Curr Microbiol 1999;38:190–3. [15] Rast DM, Baumgartner D, Mayer C, Hollenstein GO. Cell wall-associated enzymes in fungi. Phytochemistry 2003;64:339–66. [16] Ilmen M, Saloheimo A, Onnela ML, Penttila ME. Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 1997;63:1298–306. [17] Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F. Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol 2002;99:63–78. [18] Penttila ME, Andre L, Saloheimo M, Lehtovaara P, Knowlse JKC. Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae. Yeast 1987;3:175–85. [19] Igarashi K, Tani T, Kawai R, Samejima M. Family 3 b-glucosidase from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium is a glucan 1,3-b-glucosidase. J Biosci Bioeng 2003; 95:572–6. [20] Lymar ES, Li B, Renganathan V. Purification and characterization of a cellulose-binding b-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl Environ Microb 1995;61:2976–80. [21] Pitson SM, Seviour RJ, McDougall BM. Noncellulolytic fungal b-glucanases: their physiology and regulation. Enzyme Microb Technol 1993; 15:178–92. [22] Pitson SM, Seviour RJ, McDougall BM. Purification and characterization of an extracellular b-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in b-glucan degradation. Enzyme Microb Technol 1997;21:182–90.
1233
[23] West TP, Strohfus B. A pullulan-degrading enzyme activity of Aureobasidium pullulans. J Basic Microbiol 1996;36:377–80. [24] Cruz J, Pintor-Toro JA, Benı´tez T, Llobell A, Romero LC. A novel endobeta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol 1995;177:6937–45. [25] Miller GL. Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal Chem 1959;31:426–8. [26] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [27] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 1970;227:680–5. [28] Villa TG, Notario V, Villanueva JR. Occurrence of an endo-1,3-betaglucanase in culture fluids of the yeast Candida utilis. Purification and characterization of the enzyme activity. Biochem J 1979;177:107–14. [29] Aida K, Okada T, Kasahara N, Nikaidou N, Tanaka H, Watanabe T. Comparative studies of b-1,3-glucanase A1 and B of Bacillus circulans WL-12: purification and enzymatic properties. J Ferment Bioeng 1995; 80:283–6. [30] Noronha EF, Ulhoa CJ. Characterization of a 29-kDa b-1,3-glucanase from Trichoderma harzianum. FEMS Microbiol Lett 2000;183:119–23. [31] Prokop A, Rapp P, Wagner F. Production, purification and characterization of an extracellular endo-b-1,3-glucanase from a momocaryon of Schizophyllum commune ATCC 38548 defective in exo-b-1,3-glucanase formation. Can J Microbiol 1994;40:18–23. [32] Mizuno M, Minato K, Ito H, Kawade M, Terai H, Tsuchida H. Anti-tumor polysaccharide from the mycelium of liquid-cultured Agaricus blazei mill. Biochem Mol Biol Int 1999;47:707–14. [33] Kawagishi H, Inagaki R, Kano T, Shimura K, Ito H, Hagiwara T, et al. Formolysis of a potent antitumor (1-6)b-D-glucan–protein complex from Agaricus blazei friting bodies and antitumor activity of the resulting products. Carbohydr Polym 1990;12:393–403. [34] Noronha EF, Ulhoa CJ. Purification and characterization of an endo-b-1,3glucanase from Trichoderma harzianum. Can J Microbiol 1996;42:1039– 44. [35] Hien NH, Fleet GH. Separation and characterization of six b-1,3-glucanases from Saccharomyces cerevisiae. J Bacteriol 1983;156:1204–13. [36] Tanaka H, Phaff JH. Enzymatic hydrolysis of yeast cell wall. I. Isolation of wall-decomposing organisms and separation and purification of lytic enzymes. J Bacteriol 1965;89:1570–80. [37] Zanoelo FF, Polizeli MdLTdM, Terenzi HF, Jorge JA. b-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol Lett 2004;240:137–43. [38] Gueguen Y, Voorhorst WG, van der Oost J, de Vos WM. Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus. J Biol Chem 1997; 272:31258–64.