Extracellular Enzymes of the White-Rot Fungus Fomes fomentarius and Purification of 1,4-β-Glucosidase
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- Větrovský, T., Baldrian, P. & Gabriel, J. Appl Biochem Biotechnol (2013) 169: 100. doi:10.1007/s12010-012-9952-9
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Production of the lignocellulose-degrading enzymes endo-1,4-β-glucanase, 1,4-β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase, 1,4-β-xylosidase, Mn peroxidase, and laccase was characterized in a common wood-rotting fungus Fomes fomentarius, a species able to efficiently decompose dead wood, and compared to the production in eight other fungal species. The main aim of this study was to characterize the 1,4-β-glucosidase produced by F. fomentarius that was produced in high quantities in liquid stationary culture (25.9 U ml−1), at least threefold compared to other saprotrophic basidiomycetes, such as Rhodocollybia butyracea, Hypholoma fasciculare, Irpex lacteus, Fomitopsis pinicola, Pleurotus ostreatus, Piptoporus betulinus, and Gymnopus sp. (between 0.7 and 7.9 U ml−1). The 1,4-β-glucosidase enzyme was purified to electrophoretic homogeneity by both anion-exchange and size-exclusion chromatography. A single 1,4-β-glucosidase was found to have an apparent molecular mass of 58 kDa and a pI of 6.7. The enzyme exhibited high thermotolerance with an optimum temperature of 60 °C. Maximal activity was found in the pH range of 4.5–5.0, and KM and Vmax values were 62 μM and 15.8 μmol min−1 l−1, respectively, when p-nitrophenylglucoside was used as a substrate. The enzyme was competitively inhibited by glucose with a Ki of 3.37 mM. The enzyme also acted on p-nitrophenylxyloside, p-nitrophenylcellobioside, p-nitrophenylgalactoside, and p-nitrophenylmannoside with optimal pH values of 6.0, 3.5, 5.0, and 4.0–6.0, respectively. The combination of relatively low molecular mass and low KM value make the 1,4-β-glucosidase a promising enzyme for biotechnological applications.
KeywordsCellulose1,4-β-glucosidaseGlycosyl hydrolaseSaprotrophic basidiomycetesWood-rotting fungiFomes fomentarius
The white-rot basidiomycete Fomes fomentarius is widely distributed and has been found in northern and southern Africa, Asia, eastern North America, and Europe . The species most typically grows on hardwoods and is probably a long-term heart rot pathogen that results in significant economical damage. In northern regions, this species is most commonly found on birch trees, while in the south, it is more commonly found on beech trees . This species has been used extensively by humans for many centuries : The black felt-like substance contained in the pouch of the 5,000-year-old Neolithic-man, Ötzi,  was determined to be made of loosely interwoven hyphae from the fruiting bodies of F. fomentarius. The fungus is also known in Chinese traditional medicine where it is used for the treatment of various diseases including oral ulcers, gastroenteric disorders, hepatocirrhosis, inflammation, and various cancers [1, 12]. Water or methanol extracts from the fungus have been tested for pharmacological activity [17, 18, 25]. The fungus produces both intracellular polysaccharides  and exopolysaccharides that have been demonstrated to have antitumor effects . Other studies on the fungus have described the autofluorescence of F. fomentarius , biosorption of several dyes from aqueous solutions , element sorption and distribution in fruit bodies , and the effects of heavy metals on the growth of the fungus under laboratory conditions . F. fomentarius is recognized as a primary colonizer of wood and had been shown to effectively exclude other fungi from the resource . It produces high levels of hydrolytic and oxidative enzymes. Its proficient ability to transforms wood into fungal biomass is indicative of its high-efficiency degradative enzymatic system .
The typical cellulolytic systems of fungi are composed of multiple enzymes. The endoglucanases (EG) hydrolyse cellulose chains internally, cellobiohydrolases (CBH) hydrolyse cellobiose units externally either from the reducing or the nonreducing end of cellulose microfibrils, and 1,4-β-glucosidases (BG) hydrolyse the resulting cellobiose to glucose . Currently, cellulases and related enzymes are used in the food, brewery, wine, animal feed, textile, laundry, pulp and paper industries, and for agriculture, biofuel production, as well as for research purposes. The demand for these enzymes is growing more rapidly than ever before, and developing a more economical supply for this demand has become the driving force for research on cellulases and other related enzymes .
While the production of catalase, superoxide dismutase, and ligninolytic enzymes by F. fomentarius has been studied and described previously [14, 15, 22], there is little knowledge concerning the production of its cellulytic enzymes except for the detection of these enzymes in the fungus-colonized wood . To the best of our knowledge, only one paper, where the effects of enzyme extracts from white-rot fungi on the chemical composition and in vitro digestibility of wheat straw where studied, noted the production of endoglucanase and cellobiohydrolase by F. fomentarius in laboratory cultures . In light of the growing demand for the production of biotechnologically relevant enzymes for cellulose decomposition for the transformation of plant biomass for such applications as biofuels production, the focus of the current study was to characterize the cellulolytic system of the common wood-rotting fungus, F. fomentarius, and to compare its extracellular enzyme production under laboratory conditions with other saprotrophic fungi. The highly-produced1,4-β-glucosidase was purified and characterized with respect to possible future biotechnological applications.
Materials and Methods
Organisms and Growth Conditions for Enzyme Production
The fungal strains Rhodocollybia butyracea (CCBAS 286), Hypholoma fasciculare (CCBAS 281), Irpex lacteus (CCBAS 279), Fomitopsis pinicola (CCBAS 585), Pleurotus ostreatus (CCBAS 476), F. fomentarius (CCBAS 534), Piptoporus betulinus (CCBAS 537), and Gymnopus sp. (CCBAS 287) were obtained from the Culture Collection of Basidiomycetes (the ASCR Institute of Microbiology, v.v.i., Prague, Czech Republic) and were routinely maintained on solid MEA media (20 g l−1 malt extract and 15 g l−1 agar). For enzyme production, fungi were grown at 25 °C for 35 days in 250 ml Erlenmeyer flasks with 40 ml of liquid CLN medium, including modifications from a previously described assay . Each culture was performed in quadruplicate. Samples for enzyme activity assays were collected every 7 days. Activity was measured immediately after sampling. F. fomentarius cultures used for isolation of the 1,4-β-glucosidase were grown for 25 days in fifty 500-ml Erlenmeyer flasks with 100 ml of synthetic CLN medium is an N-limited medium with cellulose as the source of carbon and a high C/N ratio that is typical for wood (5 g l−1 cellulose, 0.2 g l−1 ammonium tartarate, 1 g l−1 KH2PO4, 0.2 g l−1 NaH2PO4, 0.5 g l−1 MgSO4·7H2O, 50 mg l−1 CaCl2, 50 mg l−1 FeSO4·7H2O, 5 mg l−1 ZnSO4·7H2O, 10 mg l−1 CuSO4·5H2O, and 5 mg l−1 MnSO4·4H2O), pH of the medium was adjusted to 6.0 with 5 M NaOH or HCl before autoclaving.
The effects of N addition on the production of extracellular enzymes by F. fomentarius was also studied as previous studies have shown that the activity of hydrolytic enzymes from some saprotrophic basidiomycetes can be up regulated by nitrogen addition [33, 37]. The N content in the CLN medium (0.015 g l−1) was increased to 1.5, 4.5, and 15 g l−1 by the addition of NH4NO3 to the liquid medium (four replicates per treatment). Enzyme activity was observed at weekly intervals over 35 days of incubation.
Extracellular Enzyme Assays and Protein Determination
Laccase (EC 18.104.22.168) activity was measured by monitoring the oxidation of ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) in citrate-phosphate buffer (100 mM citrate, 200 mM phosphate, pH 5.0) at 420 nm .
Manganese peroxidase (MnP, EC 22.214.171.124) activity was measured using a succinate-lactate buffer (100 mM, pH 4.5) . 3-methyl-2-benzothiazolinone hydrazone and 3,3-dimethylaminobenzoic acid were oxidatively coupled by the enzymes. The resulting purple indamine dye was detected using a spectrophotometer at 595 nm. The results were normalized by the enzyme activity of the samples without manganese for MnP or by the substitution of an equimolar amount of EDTA for MnS.
Endo-1,4-β-glucanase (EC 126.96.36.199) and endo-1,4-β-xylanase (EC 188.8.131.52) activities were measured with azo-dyed carbohydrate substrates (carboxymethyl cellulose and birchwood xylan, respectively) following the manufacturer’s guidelines (Megazyme, Ireland). Individual sample reaction mixture contained 0.2 ml of 2 % dye substrate in 200 mM sodium acetate buffer (pH 5.0) and 0.2 ml of fungal sample. Samples were incubated at 40 °C for 60 min, and the reaction was stopped by the addition of 1 ml of ethanol followed by 10 s of vortexing and 10 min centrifugation (10,000 × g) . Released dye amounts were measured at 595 nm. Enzyme activity was calculated according to standard curves that correlated the amount of dye release with the release of reducing sugars.
Cellobiohydrolase (EC 184.108.40.206) activity was measured in microplates using p-nitrophenyl-β-d-cellobioside (pNPC). The reaction mixture contained 0.16 ml of 1.2 mM pNPC in 50 mM sodium acetate buffer (pH 5.0) and 0.04 ml of sample. These reaction mixtures were incubated at 40 °C for 90–120 min. The reaction was stopped by the addition of 0.1 ml of 0.5 M sodium carbonate, and absorbance was read at 400 nm. 1,4-β-Glucosidase (EC 220.127.116.11), 1,4-β-xylosidase (EC 18.104.22.168), and 1,4-β-N-acetylglucosaminidase (chitinase; EC 22.214.171.124) were assayed using p-nitrophenyl-β-d-glucoside, p-nitrophenyl-β-d-xyloside and p-nitrophenyl-N-acetyl-β-d-glucosaminide, respectively, utilizing the same method .
All spectrophotometric measurements were performed in a microplate reader (Infinite, Tecan) or an ultraviolet–visible spectrophotometer (Lambda 11, Perkin-Elmer). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of the reaction product per min.
Purification of 1,4-β-Glucosidase from Fomes fomentarius
Purification of F. fomentarius 1,4-β-glucosidase from the liquid CLN medium
Total protein (μg)
Total activity (mU)
Specific activitya (mU/mg)
DEAE Sepharose, pH 4.5
Mono Q HR 5/5, pH 4.5
Superdex 75 HR 10/30, pH 6.0
Characterization of the Purified 1,4-β-Glucosidase from Fomes fomentarius
The 1,4-β-glucosidase from F. fomentarius was characterized using polyacrylamide gel electrophoresis (SDS-PAGE, 10 % gel) and analytical isoelectric focusing (IEF, 7.5 % gel; Multiphor II electrophoresis system, Pharmacia LKB, Sweden). The IEF gel was prepared using ampholines of pI 2.5–5.0 and pI 3.5–10.0 (Pharmacia LKB, Sweden), and the isoelectric point was estimated using a low pI protein calibration kit, pI 2.5–6.5 (Pharmacia LKB, Sweden) and a surface electrode (Orion, USA). Both SDS-PAGE and IEF gels were stained with the Silver Stain Plus kit (Bio-Rad, USA). For protein identity, IEF gels were also activity-stained with pNPG. Protein concentrations were determined using the Bradford method (Bio-Rad Protein Assay kit) with bovine serum albumin used for standards. The molecular mass of the enzyme was estimated by gel filtration (Superdex 200 HR 10/30 column, Pharmacia LKB) using gel filtration standards (Pharmacia LKB) and by SDS-PAGE using Sigma CK molecular mass markers.
Substrate specificity of F. fomentarius 1,4-βglucosidase
Relative activity (%)
Mean production of extracellular enzymes by saprotrophic basidiomycetes
Purification and Characterization of 1,4-β-glucosidase from Fomes fomentarius
Effects of pH on the hydrolysis rates of various substrates by 1,4-β-glucosidase, purified from F. fomentarius
The enzyme was observed to have activity against multiple substrates that contained different monosaccharides and disaccharides; however, the enzyme’s ability to cleave galactose, xylose and mannose was only 6–15 % of the enzyme activity observed when pNPG was the substrate. The KM for pNPG was determined to be at 62 μM. Activity of 1,4-β-glucosidase was competitively inhibited by the addition of glucose with a Ki value of 3.37 mM.
In this study, eight fungal species belonging to three ecological groups (white-rot, brown-rot, and litter-associated saprotrophic fungi) were tested for extracellular enzyme production. As expected, white-rot species produced high levels of lignin-degrading enzymes, brown-rot fungi produced only cellulolytic and hemicellulolytic enzymes, and litter-colonizing saprotrophic fungi produced both types of enzymes (Table 3). The white-rot basidiomycete F. fomentarius exhibited higher production of laccase, celobiohydrolase, and 1,4-β-glucosidase, greater than any of the other tested fungi. Most importantly, F. fomentarius produced 1,4-β-glucosidase in amounts ten times higher than any other species tested. Thus, F. fomentarius was selected for the purification and characterization of 1,4-β-glucosidase. This enzyme has a broad range of uses in biotechnological applications .
While previous studies on enzyme production in white rote fungi, including Ceriporiopsis aneirina, Ceriporiopsis resinascens and Dichomitus albidofuscus  and H. fasciculare , have shown an increase in cellulolytic enzyme production with nitrogen addition, our study showed a decrease in enzyme activities in F fomentarius with nitrogen addition.
Cellulases are currently the third largest group of industrial enzymes used worldwide according to market size, due to their use in cotton processing, in paper recycling, as detergent enzymes, in juice extraction, as animal feed additives, and as the enzymes used in bioethanol production . Lignocellulosic biomass, a native substrate of fungal extracellular 1,4-β-glucosidases, is the most abundant and inexpensive source of bioethanol production. The capability of various filamentous fungi has been explored for the production of ethanol from this biomass, such as the genera Aspergillus, Rhizopus, Mucor, Monilia, Neurospora, Fusarium, Trichoderma, Mucor, and from the wood-decaying fungi Trametes hirsuta , Phanerochaete chrysosporium , and Gloeophyllum trabeum . Cellulases are also increasingly being used in the textile industry. Their most successful application has been in the production of the stone-washed appearance of denim garments. Other processes that improve fabric appearance by removing fuzz fibers and pills or that have softening benefits have also been explored . The increased use of cellulases is also a result of domestic fabric washing products where they are thought to aid detergency and to clean fiber surfaces, which improve appearance and color brightness . Currently, these finishing and washing applications represent the largest market for cellulase enzymes worldwide. Commercial cellulases that are available for textiles are mainly produced from the fungi Humicola insolens (maximal activity at pH 7) and Trichoderma reesei (maximal activity at pH 5) . The efficiency of the enzyme can be improved by its immobilization on various particles, but determining alternative enzymes that can be produced from various organisms would be desirable, considering the high levels of variability in the enzyme properties [4, 19].
The purified 1,4-β-glucosidase from F. fomentarius exhibited several interesting features that are relevant to its potential biotechnological application. While there are a wide range of molecular masses between fungal β-glucosidases that range from 35 in P. ostreatus to more than 400 in some Phanerochaete chrysisporium and Trametes spp. enzymes [6, 21, 32], the extracellular enzymes are usually smaller. The F. fomentarius enzyme is similar to the small extracellular enzymes produced by Pleurotus or Phanerochaete spp. . The enzyme was observed to have a relatively high pI of 6.4, which is typical for an intracellular enzyme . The optimal pH (6.0) and temperature (45–75 °C) fall within the range observed for other fungal β-glucosidases , but the high enzyme activity at 60 °C may be beneficial for biotechnological applications that require a high process rate. Similar to other β-glycosidases, the β-glucosidase of F. fomentarius has a relatively wide substrate range [9, 34], but the cleavage of 1,4-β-glycosidic bonds in cellobiose is the preferred reaction for the enzyme. More importantly, the enzyme has a low KM of 62 μM. The only lower KM value observed is that of the β-glucosidase of Gloeophyllum trabeum, which has limited commercial applications, due to its high molecular mass .
Compared to other fungal 1,4-β-glucosidases, the combination of a relatively low KM with a high Ki for glucose indicates that the F. fomentarius enzyme is an efficient catalyst. Considering its low molecular mass and high activity at elevated temperatures, this enzyme is potentially suitable for biotechnological applications. This potential may be further explored by investigating the advantages of overexpression, heterologous expression or targeted mutagenesis of the enzyme. The ability of F. fomentarius to produce high titers of 1,4-β-glucosidases as well as laccase and Mn peroxidase suggests that this fungus may have potential in large-scale applications of lignocellulosic degradation (e.g., solid-state fermentation) .
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (OC08050, LA10001).