Characterization of β-Glucosidase Produced by the White Rot Fungus Flammulina velutipes
Characterization of β-Glucosidase Produced by the White Rot Fungus Flammulina velutipes
Journal of Microbiology and Biotechnology. 2015. Jan, 25(1): 57-65
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : January 24, 2014
  • Accepted : September 04, 2014
  • Published : January 28, 2015
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Julieta, Mallerman
Leandro, Papinutti
Laura, Levin

β-Glucosidase production by the white rot fungus Flammulina velutipes CFK 3111 was evaluated using different carbon and nitrogen sources under submerged fermentation. Maximal extracellular enzyme production was 1.6 U/ml, corresponding to a culture grown in sucrose 40 g/land asparagine 10 g/l. High production yield was also obtained with glucose 10 g/land asparagine 4 g/l medium (0.5 U/ml). Parameters affecting the enzyme activity were studied using p -nitrophenyl-β- D -glucopyranoside as the substrate. Optimal activity was found at 50℃ and pHs 5.0 to 6.0. Under these conditions, β-glucosidase retained 25% of its initial activity after 12 h of incubation and exhibited a half-life of 5 h. The addition of MgCl 2 , urea, and ethanol enhanced the β-glucosidase activity up to 47%, whereas FeCl 2 , CuSO 4 , Cd(NO 3 ) 2 , and cetyltrimethylammonium bromide inflicted a strong inhibitory effect. Glucose and cellobiose also showed an inhibitory effect on the β-glucosidase activity in a concentration-dependent manner. The enzyme had an estimated molecular mass of 75 kDa. To the best of our knowledge, F. velutipes CFK 3111 β-glucosidase production is amongst the highest reported to date, in a basidiomycetous fungus.
β-Glucosidases (β-D-glucoside glucohydrolase, E.C., under physiological conditions, catalyze the hydrolysis of β-1,4-glycosidic bonds from the nonreducing termini presented in alkyl- and aryl-β-D-glycosides, as well as different oligosaccharides (containing 2-6 monosaccharides). These enzymes are widespread in nature, occurring in all domains of living organisms, Archaea, Eubacteria, and Eukaryotes, in which they play varied functions [39] . They represent an important group of enzymes because of their potential uses in various biotechnological processes, including biomass degradation [44] , the production of fuel ethanol from cellulosic agricultural residues [22] , release of aromatic compounds in the flavor industry [13] , and synthesis of useful β-glucosides [24] .
Cellulose is the most abundant polymer on Earth, and its complete degradation is accomplished by a cellulolytic complex, which involves the synergistic action of three enzymes: 1,4-β-D-endoglucanase (E.C., 1,4-β-D-cellobiohydrolase (CBH, E.C., and β-glucosidase. It is generally accepted that the endoglucanases and the CBHs act cooperatively and synergistically in depolymerizing cellulose to cellobiose and oligosaccharides, which are then converted by β-glucosidase to glucose [3] . The deficiency in β-glucosidase activity causes the accumulation of the disaccharide cellobiose, leading to the repression of enzyme biosynthesis and end-product inhibition of the upstream enzymes, which result in a limited hydrolysis yield [44] . Therefore, commercially available cellulolytic preparations are often supplemented with β-glucosidase to boost the overall activity, such as that prepared from Trichoderma reesei cellulases [8 , 41] . β-Glucosidase is the rate-limiting factor in the conversion of cellulose to glucose for the subsequent production of fuel ethanol. Product inhibition and thermal inactivation of β-glucosidase constitute two major barriers to the development of enzymatic hydrolysis of cellulose as a commercial process. There is an increasing demand for the identification and production of β-glucosidases, especially those insensitive to product inhibition and with high stability [31] .
The white rot fungus Flammulina velutipes is a xylophagous fungus belonging to the Physalacriaceae family. Recently, F. velutipes strain Fv-1 demonstrated its potential for the conversion of lignocellulosic biomass to ethanol by consolidated bioprocessing, a methodology that combines enzyme production, enzymatic saccharification, and ethanol fermentation in one step, thus reducing the total cost of bioethanol production [22] . The objective of this research was to evaluate the β-glucosidase production by F. velutipes CFK 3111 and to determine the influence of some parameters on the activity and stability of this enzyme.
Materials and Methods
- Organism and Culture Conditions
F. velutipes CFK 3111 ( Physalacriaceae , Agaricales , Basidiomycota ) was obtained from the culture collection of Laboratorio de Micología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. The strain was maintained in maltextract agar (MEA; malt extract 1.2%, glucose 1%, and agar 2%) at 4℃. Growth media consisted of basal medium MgSO 4 ·7H 2 O 0.5 g, KH 2 PO 4 0.5 g, K 2 HPO 4 0.6 g, CuSO 4 ·5H 2 O 0.4 g, MnCl 2 ·4H 2 O 0.09 mg, H 3 BO 3 0.07 mg, NaMoO 4 ·2H 2 O 0.02 mg, FeCl 3 1 mg, ZnCl 2 ·H 2 O 2.5 mg, biotin 0.005 mg, thiamine hydrochloride 0.1 mg, and distilled water up to 1 L. Culture media were supplemented with the indicated carbon source (1 or 4% (w/v)) and nitrogen source (0.4 or 1% (w/v)). Erlenmeyer flasks (125 ml) containing 25 ml of growth media were inoculated with 5 mm diam plugs cut out from the margin of a 7-day-old colony growing on MEA and incubated statically at 28℃. The final pH of all media was adjusted to 6.5. Erlenmeyer flasks were sealed with Parafilmcovered cotton plugs and by a loose cap of aluminum foil in order to avoid excessive water evaporation during extended incubation periods. Cultures were harvested at proper intervals (depicted in Fig. 1 ), 0.5 ml aliquots of the supernatant were collected aseptically, and at day 75, the last sampling day, whole cultures were filtered through a filter paper using a Büchner funnel, dried overnight at 70℃, and weighed. Growth was estimated by measuring the biomass production. When crystalline cellulose was used as a carbon source, the fungal biomass was estimated by N -acetyl glucosamine quantification [29] . Culture supernatant samples were used for β-glucosidase activity, soluble proteins, and consumption of reducing sugars determinations. Unless otherwise stated, all chemicals used were of reagent grade and purchased from Sigma (St. Louis, MO, USA).
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Time course of β-glucosidase production (black bars), extracellular protein (grey bars), and glucose consumption (white bars) during growth in GA medium. Error bars denote standard deviation.
- Enzyme Assay
The β-glucosidase activity was assayed by using p -nitrophenyl-β-D-glucopyranoside ( p NPG; Sigma) as the substrate. First, 0.45 ml of p NPG (0.02% in 50 mM sodium acetate buffer; pH 4.8) was mixed with 0.05 ml of appropriately diluted enzyme solution. The reaction mixture was incubated at 50℃ for 30 min and stopped by the addition of 1 ml of 0.1 M Clark and Lubs buffer (50 ml of 0.1 M H 3 BO 3 in 0.1 M KCl, plus 40.8 ml of 0.1 M NaOH and doubledistilled water up to 100 ml; pH 9.8) [10] . The amount of p -nitrophenol released was determined spectrophotometrically by measuring the absorbance of the solution at 430 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol of p -nitrophenol per minuteunder the assay conditions. Specific activity (U sp) was expressed as U per milligram of dry biomass weight [6] .
- Analytical Determinations
Glucose was measured by the Somogyi-Nelson [26 , 35] method. The extracellular proteins were measured in the culture supernatants using the Bradford method [5] with the BioRad protein assay reagent (Bio-Rad, Hercules, CA) and bovine serum albumin (BSA) as the standard protein.
- Effects of Temperature and pH on β-Glucosidase Activity and Stability
For testing β-glucosidase activity and stability, supernatants from cultures with GA (glucose 10 g/l and asparagine 4 g/l) were assayed. The effect of pH on β-glucosidase activity was determined in the range 2.6-8.0 by incubating the crude enzyme at 50℃ for 30 min in p NPG dissolved in 50 mM citrate phosphate buffer (pHs 2.6-7.0) or 50 mM phosphate buffer (pHs 6.0-8.0). The optimum temperature for β-glucosidase activity was examined after incubating the crude enzyme for 30 min at various temperatures from 30℃ to 70℃ with p NPG in 50 mM sodium acetate buffer (pH 4.8) as substrate. In the experiments testing the effect of pH on the enzyme stability, crude enzyme sample were preincubated for up to 15 h at 50℃ and 60℃ at the pH range 2.6 to 8.0 in the buffers described above. Samples were withdrawn at proper intervals (depicted in Fig. 3 ) for enzyme activity determination at standard assay conditions. Residual enzyme activity was calculated by comparison with non-preincubated supernatants [1] .
- Effects of Various Additives on β-Glucosidase Stability
Crude enzyme sample used in these experiments were desalted against distilled water at 1:1,000 ratio (v/v) using dialysis tubing of molecular weight cut-off 12,000 Da (Sigma-Aldrich, St. Louis, MO, USA), and then concentrated against sucrose for 4 h. The stability of β-glucosidase against urea, maltose, and fructose, selected salts (ZnSO 4 , FeCl 2 , KCl, MgCl 2 , MnCl 2 , Cd(NO 3 ) 2 , Cr(NO 3 ) 2 , SO 4 Cu), chelators (ethylenediaminetetraacetic acid (EDTA) and Azida), and surfactants (sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB)), was tested at 1 and 10 mM. For this, the culture fluid was preincubated with the individual additives at 30℃. Samples were collected after 30 min for enzyme activity determination at standard assay conditions. Control with a preincubation in the absence of any additive was recorded as 100% activity. In addition, the effect of organic solvents on β-glucosidase stability was assayed. Culture filtrate was incubated with methanol (50% (v/v)), acetic acid (50% (v/v)), or ethanol (25, 50, and 75% (v/v)) at 30℃. Samples were collected at selected times and residual activities were determined.
- Effects of Glucose and Cellobiose on β-Glucosidase Activity
The effects of glucose or cellobiose on β-glucosidase activity were conducted using the standard reaction mixture with the addition of the individual sugars at final concentration 50–200 mM. Activity in the control with no sugar added was recorded as 100% [14] .
- Polyacrylamide Gel Electrophoresis
Enzyme extract was analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE), using 9% acrylamide. Proteins separated in the gel were stained with Coomassie Brilliant Blue R-250, and their molecular mass estimated with standard markers: myosin (208 kDa), phophorylase B (114 kDa), BSA (81.2 kDa), ovalbumin (47.9 kDa), carbonic anhydrase (31.5 kDa), trypsin inhibitor (24.8 kDa), and lysozyme (16.6 kDa). The β-glucosidase activity was detected in Native-PAGE in which the lanes of the gel were loaded with 10 mU activity. The bands were visualized by incubating the gel at 50℃ with 0.02% p NPG in 50 mM sodium acetate buffer (pH 4.8) for 2 h. The gel was washed with distilled water, and 0.1 M Clark and Lubs buffer (pH 9.8) was added and incubated until a yellow band appeared; the gel was photographed immediately.
- Statistical Analysis
The data presented are the average of the results of three replicates. Analysis of variance was tested by the software Statgraphics ver. 5.1. The significant differences between treatments were compared by Fisher’s LSD multiple range test at 5% level of probability.
- Effects of the Carbon and Nitrogen Sources on β-Glucosidase Production
The effects of the carbon and nitrogen sources on biomass and enzyme activity are shown in Table 1 . Although all of the liquid media evaluated were able to support mycelial growth of F. velutipes , the biomass production was greatly affected by the culture medium used, and β-glucosidase production showed to be independent of growth. Among the nitrogen sources tested, asparagine was the nutrient that enhanced the highest production of β-glucosidase, where cultures supplemented with this amino acid yielded maximal volumetric and specific activities in all the combinations assayed. Negligible to very low activities were observed when ammonium sulfate was used as the nitrogen source, and intermediate activities were found with glutamic acid. Higher asparagine concentrations in the culture media yielded more biomass (data not shown) but comparable specific activities. Regarding the carbon source used, the highest enzyme activities were detected for glucose, cellobiose, and sucrose, whereas very low yields were achieved with fructose, crystalline cellulose, or lactose. Similar biomass production was observed in cultures grown either with cellobiose or glucose, but slightly higher enzyme activity was achieved with glucose. Overall, of the media assayed, the highest enzyme activity was observed at day 47 in cultures with sucrose 40 g/l and asparagine 10 g/l (1.6 U/ml), and high values were also achieved with GA (0.5 U/ml). As glucose is commonly used as a carbon source, data on fungal growth and production of different enzymes are vast, and thus GA medium was selected for further analysis to facilitate full comparisons with those data reported in the literature.
Effects of carbon and nitrogen sources on β-glucosidase production (mU/ml).
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The values shown correspond to the peak of enzyme production. The numbers above the columns indicate the day these maxima were achieved. Specific activity (U sp) was calculated per milligram of dry biomass. The values are the mean of three replications ± SD. ND, Not determined.
The kinetics of β-glucosidase production and enzyme activity in GA medium are depicted in Fig. 1 . Extracellular proteins and β-glucosidase activity increased steadily up to day 18, when glucose was almost exhausted from the medium (96% of the glucose was consumed by the fungus). Afterwards, enzyme activity increased but at a slower rate until the last sampling day.
- Effects of Temperature and pH on β-Glucosidase Activity and Stability
The effect of temperature on enzyme activity is shown in Fig. 2 A. β-Glucosidase showed maximal activity at 50℃, where at this temperature activity was more than 2-fold higher than that detected at 30℃. The effect of pH was tested in the range 2.6-8.0 ( Fig. 2 B), where β-glucosidase activity was substantially affected, and the enzyme exhibited highest activity at pH 5, although it maintained up to 95% and 44% of its activity at pH 6.0 and 7.0, respectively. No differences owing to buffer composition were detected. Stabilization studies were performed at different pHs ranging from 2.6 to 8.0 at 50℃ and 60℃. The residual activity was quantified at proper intervals. At 50℃, β-glucosidase was highly stable at pHs 5.0 and 6.0, exhibited a half-life of 5 h, and kept 25% of its activity after 12 h of incubation ( Fig. 3 ). The stability as function of pH value was also measured at 60℃. At this temperature, the highest half-life of the enzyme was 9 min at pH 6.0 (data not shown).
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Effects of (A) temperature and (B) pH with citrate phosphate buffer (●) and phosphate buffer (■) on β-glucosidase activity. Error bars are the standard deviations. When not shown, the error bars fall within the symbols.
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pH stability assays, carried out by incubating the enzyme at 50℃ at different pHs from 2.6 to 7.0 with citrate phosphate buffer (white symbols) and from 6.0 to 8.0 with phosphate buffer (black symbols).
- Effects of Various Additives on β-Glucosidase Stability
The effects of various reagents on β-glucosidase stability were investigated ( Table 2 ). Significant inhibition was observed with Cd 2+ and CTAB at 1 mM concentration. When the concentration was increased to 10 mM, potent inhibition (up to 80%) was observed with CTAB, and some inactivation was found with SDS and the metal ions Zn 2+ , Cr 2+ , and Cu 2+ . Fe 2+ almost abolished the β-glucosidase activity. In contrast, Mg 2+ , Mn 2+ , SDS, and urea at 1 mM enhanced the β-glucosidase activity, especially Mg 2+ , which exhibited a potent activation effect with 47.2% of activity increase. No significant effects were found with the sugars maltose and fructose, or with Azida and EDTA.
Effects of additives on β-glucosidase stability.
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The values are the mean of three replications ± SD. aThe activity assayed in the absence of additives was considered as 100%. bResidual activity was evaluated after preincubation for 30 min at 30℃ with the individual additives. ND, Not determined.
The effects of various organic solvents on β-glucosidase stability are depicted in Table 3 and Fig. 4 . Ethanol 50% (v/v) caused an increase of 23% in β-glucosidase activity, whereas a minimum increase of 5% was observed with methanol. Contrariwise, total inhibition was caused by acetic acid. Ethanol activation was concentration-dependent ( Fig. 4 ), and when used at 75% concentration, the enzyme activity increased more than 3-fold after incubation for 30 min, but from this time onward the activity declined.
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Effect of ethanol on β-glucosidase stability. Culture filtrates were incubated with ethanol 0% (●), 25% (■), 50% (▲), and 75% (▼) at 30℃. Samples were collected at selected times and residual activities were determined.
Effects of organic solvents on β-glucosidase stability.
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The values are the mean of three replications ± SD. aThe activity assayed in the absence of additives was considered as 100%. bResidual activity was evaluated after preincubation for 40 min at 30℃ with the individual organic solvents.
- Effects of Glucose and Cellobiose on β-Glucosidase Activity
The carbohydrates glucose and cellobiose inhibited the β-glucosidase activity ( Fig. 5 ). Between them, cellobiose caused the more marked inhibitory effect. When glucose and cellobiose were used at 50 mM, β-glucosidase retained 38.5% and 25.8% of its initial activity, respectively, whereas at higher concentrations (up to 200 mM), the enzyme still retained around 20% of its activity.
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Effects of glucose and cellobiose on β-glucosidase activity. The relative activity was determined by measuring β-glucosidase at 50℃ in 50 mM sodium acetate buffer (pH 4.8), in the presence of glucose (●) or cellobiose (■).
- Polyacrylamide Gel Electrophoresis
Native gel of the crude extract revealed the presence of a single activity band that corresponded with one β-glucosidase with an approximately molecular mass of 75 kDa ( Fig. 6 ). Such band had a coincident Rf with a more intense colored protein band revealed in the SDS-PAGE after Coomassie Blue staining.
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Apparent molecular mass of β-glucosidase from F. velutipes. Activity was revealed after incubating the native gel in p-nitrophenylβ-D-glucopyranoside 0.02% at 50℃.
- Effects of the Carbon and Nitrogen Sources on β-Glucosidase Production
F. velutipes produced high quantities of extracellular β-glucosidase when grown in defined liquid medium with different carbon and nitrogen sources. Cultures with asparagine and sucrose produced the highest volumetric activity yields. High titers were also obtained with asparagine and fructose, cellobiose, or glucose. Enzyme activities were comparable with those yielded by several imperfect fungi species such as Aspergillus , Penicillium , and Trichoderma , which are considered the main β-glucosidase producers [17 , 37] . Although there is growing interest in the search for new fungal β-glucosidases, the vast majority of studies were performed with imperfect fungi, and there are scant reports that deal with basidiomycetous fungi, not only for biotechnological purposes but also for physiological research. Among them, Vìtrovský et al . [38] evaluated the production of lignocellulose-degrading enzymes in several white rot fungi and obtained up to 0.026 U/ml, with Fomes fomentarius being the major producer. Morais et al . [25] studied β-glucosidase production by Pleurotus ostreatus grown in different culture media containing agro-industrial wastes, where the highest activity detected was 0.385 U/ml with straw-pepper-potato extract [37] . In Volvariella volvacea , β-glucosidase activity in supernatants reached 0.13 U/ml [7] , and in Trametes trogii was 0.25 U/ml [21] . Recently, similar titers of β-glucosidase activity (1.5 U/ml) were produced by another strain of F. velutipes [22] in a medium with yeast extract, peptone, and glucose as nitrogen and carbon sources. To the best of our knowledge, F. velutipes CFK 3111 β-glucosidase production is amongst the highest reported to date, in a basidiomycetous fungus.
Differences in enzyme activity as a response to different sources could be explained as being due to a promotion of fungal growth and subsequent major enzyme production. It has been demonstrated in Telephora terrestris that secretion of laccase enzyme is biomass-dependent [18] . Thus, because of the physiological effect that nutrients had on growth, and to avoid interference of possible biomass-dependent activities, it was necessary to calculate the activities per milligram of mycelium. When considering F. velutipes specific activities, cultures with sucrose and asparagine attained maximal titers (10.5-12.1 mU/mg of dry biomass). Similarly, cellobiose, the natural substrate of β-glucosidase, was as efficient as sucrose in enzyme induction.
- Partial Crude Enzyme Characterization
Effects of temperature and pH on β-glucosidase activity and stability. Physicochemical parameters were determined for F. velutipes crude enzyme towards p NPG substrate. Optimal activity conditions were observed at 40℃-50℃ and pHs 5.0-6.0, a range similar to several fungal βglucosidases, such as those from Stachybotrys microspora [33] , Humicola insolens [36] , and Daldinia eschscholzii [19] . β-Glucosidase retained 25% of its initial activity after 12 h of incubation at 50℃, at pHs 5.0 and 6.0, and exhibited a halflife of 5 h. In comparison, β-glucosidase from Fervidobacterium islandicum [15] retained its activity for over 5 h at 50℃ and showed a half-life of 15 min at 100℃. The Scytalidium thermophilum enzyme was thermostable up to 60 min when incubated at 50℃, at pH 6.5, and exhibited a half-life of 20 min when incubated at 55℃ [45] . Pyrococcus furiosus β-glucosidase maintained 80% to 90% of its activity after 30 min of incubation at 100℃, with half-lives of 85 h at 100℃ and 13 h at 110℃ [20] . Microbispora bispora produced a β-glucosidase that was more active at 60℃, retaining 70% of its activity after 48 h of incubation [40] .
- Effects of Various Additives on β-Glucosidase Stability
Up to 92% inactivation was observed with the metal cations Fe 2+ , Cu 2+ , and Cd 2+ ,which may suggest that thiol groups are involved in the catalytic reaction at the active site. A similar effect was observed when studying the β-glucosidase from T. reesei , where the enzyme showed drastic inhibition by these cations; thus, it was speculated that the thiol-group Cys 168 may play a key role in inhibition mechanisms by cations [16] . The surfactant CTAB was another destabilizing compound that also caused noticeable loss of enzyme activity. This molecule commonly used detergent could interfere with hydrophobic sites, causing denaturation and consequently enzyme inactivation. On the other hand, enzyme activity was stimulated by Mg 2+ and Mn 2+ . A previous report showed that β-glucosidase activity from T. reesei was enhanced by up to 226% in the presence of MnCl 2 [16] . The enzyme from F. velutipes was not so strongly enhanced, but this cation provoked the highest enzyme stimulation among compounds assayed in this study. Strong activation by Mg 2+ was observed in the β-glucosidases of phylogenetically distant species such as Neocallimastix patriciarum [9] , D. eschscholzii [19] , and Clostridium cellulovorans [16] , which suggest that Mn 2+ and Mg 2+ ions may assist the enzyme reaction in a highly conserved catalytic site. Ethanol also exerted activity enhancement, especially in short time intervals, with more remarkably effects the more concentrated the solution was. At ethanol 75% (v/v), enzyme activity increased more than 3-fold. It has been proposed that alcohol activation of some β-glucosidases may be due to their glycosyltransferase activities [4 , 28] . In an environment containing high amounts of alcohols and relatively low amounts of water, many β-glucosidases can preferentially use the alcohols as acceptors for the glycosyl moiety during catalysis of p NPG, resulting in elevated reaction rates. There is vast literature where this effect is reported. For example, the β-glucosidase activity of Aureobasidium pullulans was enhanced 15% by ethanol 7.5% (v/v) [31] . Ethanol and methanol at 5% (v/v) increased Stereum hirsutum β-glucosidase activity about 30% [27] , and in Aspergillus oryzae and A. niger, activity was stimulated 30% with ethanol 15% (v/v) [30 , 42] .
- Effects of Glucose and Cellobiose on β-Glucosidase Activity
A decrease in enzyme activity was observed in the presence of glucose and cellobiose, possibly as β-glucosidase is sensitive to product and substrate inhibition, respectively. This effect was described in many other microorganisms [32 , 34] .
- Molecular Mass ofF. velutipesβ-Glucosidase
The molecular mass of β-glucosidase was estimated as 75 kDa. This value falls within the wide range of molecular masses reported for basidiomycetous fungi, varying between 50 and 256 kDa [7 , 23] .
Concluding Remarks
During recent years, studies involving β-glucosidases are becoming more frequent given the key role of this enzyme in ethanol production for biofuels. Most commercial cellulolytic enzymes are obtained from T. reesei , such as the case of Celluclast 1.5L (Novozymes A/S), which has potent endoglucanase and CBH activities, but has to be supplemented with extra β-glucosidase activity from another source in order to improve cellulose hydrolysis; for example, Novozym 188 (Novozymes A/S) that is prepared from A. niger . Recent reports have shown different strategies to reach more efficient processes leading to cellulose hydrolysis, such as immobilization [2 , 11 , 43] , stability enhancement [12] , and manipulation of culture parameters [46] . The production and characterization of novel β-glucosidases, such as the one described in this work, are part of the recent search for enzymes with robustness and high specificity towards cellobiose. Besides contributing with more efficient biotechnological tools in cellulose exploitation, ongoing investigations will expand our knowledge on the physiology of fungal β-glucosidase regulation.
The authors are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and University of Buenos Aires for financial support.
Ahmed SA , El-Shayeb NMA , Hashem AM , Saleh SA , Abdel-Fattah AF 2013 Biochemical studies on immobilized fungal β-glucosidase. Braz. J. Chem. Eng. 30 747 - 758    DOI : 10.1590/S0104-66322013000400007
Alftren J , Hobley TJ 2013 Covalent immobilization of betaglucosidase on magnetic particles for lignocellulose hydrolysis. Appl. Biochem. Biotechnol. 169 2076 - 2087    DOI : 10.1007/s12010-013-0122-5
Bhat M , Bhat S 1997 Cellulose degrading enzyme and their potential industrial application. Biotechnol. Adv. 15 583 - 620    DOI : 10.1016/S0734-9750(97)00006-2
Bhatia Y , Mishra S , Bisaria VS 2002 Microbial beta-glucosidases: cloning, properties and applications. Crit. Rev. Biotechnol. 22 375 - 407    DOI : 10.1080/07388550290789568
Bradford MM 1976 A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72 248 - 254    DOI : 10.1016/0003-2697(76)90527-3
Buswell JA , Cai YJ , Chang ST 1996 Ligninolytic enzyme production and secretion in edible mushroom fungi,InRoyse DJ (ed.).Mushroom Biology and Mushroom Products. Penn State University Pennsylvania 113 - 122
Cai YJ , Buswell JA , Chang ST 1998 β-Glucosidase components of the cellulolytic system of the edible straw mushroom,Volvariella volvacea. Enzyme Microb. Technol. 22 122 - 129    DOI : 10.1016/S0141-0229(97)00151-8
Chauve M , Mathis H , Huc D , Monot F , Ferreira NL 2010 Comparative kinetic analysis of two fungal β-glucosidases. Biotechnol. Biofuels 3 3 -    DOI : 10.1186/1754-6834-3-3
Chen HL , Chen YC , Lu MYJ , Chang JJ , Wang HTC , Ruan SK 2012 A highly efficient β-glucosidase from a buffalo rumen fungusNeocallimastix patriciarumW5. Biotechnol. Biofuels 5 24 -    DOI : 10.1186/1754-6834-5-24
Clark WM , Lubs HA 1916 Hydrogen electrode potentials of phthalate, phosphate and borate buffer mixtures. J. Biol. Chem. 25 479 - 510
Figueira JA , Sato HH , Fernandes P 2013 Establishing the feasibility of using beta-glucosidase entrapped in lentikats and in sol-gel supports for cellobiose hydrolysis. J. Agric. Food Chem. 61 626 - 634    DOI : 10.1021/jf304594s
Ghorai S , Chowdhury S , Pal S , Banik SP , Mukherjee S , Khowala S 2010 Enhanced activity and stability of cellobiase (beta-glucosidase: EC produced in the presence of 2-deoxy-D-glucose from the fungusTermitomyces clypeatus. Carbohydr. Res. 345 1015 - 1022    DOI : 10.1016/j.carres.2010.02.021
Gueguen Y , Chemardin P , Arnaud A , Galzy P 1995 Purification and characterization of an intracellular β-glucosidase fromBotrytis cinerea. Enzyme Microb. Technol. 78 900 - 906    DOI : 10.1016/0141-0229(94)00143-F
Holtzapple M , Cognata M , Shu Y , Hendrickson C 1990 Inhibition ofTrichoderma reeseicellulase by sugars and solvents. Biotechnol. Bioeng. 36 275 - 287    DOI : 10.1002/bit.260360310
Jabbour D , Klippel B , Antranikian G 2012 A novel thermostable and glucose-tolerant β-glucosidase fromFervidobacterium islandicum. Appl. Microbiol. Biotechnol. 93 1947 - 1956    DOI : 10.1007/s00253-011-3406-0
Jen W , Wang W , Lin M , Lin C , Liaw Y , Chang W 2011 Structural and functional analysis of three β-D-glucosidases from bacteriumClostridium cellulovorans, fungusTrichoderma reeseiand termiteNeotermes koshunensis. J. Struct. Biol. 173 46 - 56    DOI : 10.1016/j.jsb.2010.07.008
Jäger S , Brumbauer A , Feher E , Reczey K , Kiss L 2011 Production and characterization of β-glucosidase from differentAspergillusstrains. World J. Microbiol. Biotechnol. 17 455 - 461    DOI : 10.1023/A:1011948405581
Kanunfre CC , Zancan GT 1998 Physiology of exolaccase production byTelephora terrestris. FEMS Microbiol. Ecol. 16 151 - 156    DOI : 10.1111/j.1574-6968.1998.tb12942.x
Karnchanatat A , Petsom A , Sangvanich P , Piaphukiew J , Whalley AJ , Reynolds CD , Sihanonth P 2007 Purification and biochemical characterization of an extracellular betaglucosidase from the wood-decaying fungusDaldinia eschscholzii(Ehrenb.:Fr.) Rehm. FEMS Microbiol. Lett. 270 162 - 170    DOI : 10.1111/j.1574-6968.2007.00662.x
Kengen S , Luesink E , Stams A , Zehnder A 1993 Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeonPyrococcus furiosus. Eur. J. Biochem. 213 305 - 312    DOI : 10.1111/j.1432-1033.1993.tb17763.x
Levin L , Forchiassin F 1997 Efecto de las condiciones de cultivo sobra la producción de celulasas porTrametes trogii. Rev. Argent. Microbiol. 29 16 - 23
Maehara T , Ichinose H , Furukawa T , Ogasawara W , Takabatake K , Kaneko S 2013 Ethanol production from high cellulose concentration by the basidiomycete fungusFlammulina velutipes. Fungal Biol. 117 220 - 226    DOI : 10.1016/j.funbio.2013.02.002
Magalhães PO , Ferraz A , Milagres AFM 2006 Enzymatic properties of two beta-glucosidases fromCeriporiopsis subvermisporaproduced in biopulping conditions. J. Appl. Microbiol. 101 480 - 486    DOI : 10.1111/j.1365-2672.2006.02946.x
Makropoulou M , Christakopoulos P , Tsitsimpikou C , Kekos D , Kolis FN , Macris BJ 1998 Factors affecting the specificity of β-glucosidase fromFusarium oxysporumin enzymatic synthesis of alkyl-β-D-glucosides. Int. J. Biol. Macromol. 22 97 - 101    DOI : 10.1016/S0141-8130(97)00092-5
Morais H , Ramos C , Matos N , Forgacs E , Cserhati T , Almeida V 2002 Liquid chromatographic and electrophoretic characterisation of extracellular β-glucosidase ofPleurotus ostreatusgrown in organic waste. J. Chromatogr. B 770 111 - 119    DOI : 10.1016/S0378-4347(01)00561-8
Nelson NA 1944 Colorimetric adaptation of the Somogyi method for determination of glucose. J. Biol. Chem. 153 376 - 380
Nguyen NP , Lee KM , Lee KM , Kim IW , Kim YS , Jeya M , Lee JK 2010 One-step purification and characterization of a beta-1,4-glucosidase from a newly isolated strain ofStereum hirsutum. Appl. Microbiol. Biotechnol. 87 2107 - 2116    DOI : 10.1007/s00253-010-2668-2
Pemberton JE , Brown RD , Emert GH 1980 The role of β-glucosidase in the bioconversion of cellulose to ethanol. Can. J. Chem. Eng. 58 723 - 729    DOI : 10.1139/v80-111
Plassard CS , Mousain D , Salsac LE 1982 Estimation of mycelial growth of Basidiomycetes by means of chitin determination. Phytochemistry 21 345 - 348    DOI : 10.1016/S0031-9422(00)95263-4
Riou C , Salmon JM , Vallier MJ , Gunata Z , Barre P 1998 Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase fromAspergillus oryzae. Appl. Environ. Microbiol. 64 3607 - 3614
Saha BC , Freer SN , Bothast RJ 1994 Production, purification and properties of a thermostable β-glucosidase from a color variant strain ofAureobasidium pullulans. Appl. Environ. Microbiol. 10 3774 - 3780
Saha BC , Freer SN , Bothast RJ 1995 Thermostable β-glucosidases.InSaddler JN, Penner MH (eds.).Enzymatic Degradation of Insoluble Carbohydrates. ACS Symposium Series 168.
Saibi A , Gargouri A 2011 Purification and biochemical characterization of an atypical β-glucosidase fromStachybotrys microspore. J. Mol. Catal. B Enzym. 72 107 - 115    DOI : 10.1016/j.molcatb.2011.05.007
Schmid G , Wandrey C 1987 Purification and partial characterization of a cellodextrin glucohydrolase (β-glucosidase) fromTrichoderma reeseistrain QM 9414. Biotechnol. Bioeng. 30 571 - 585    DOI : 10.1002/bit.260300415
Somogyi M 1952 Notes on sugar determination. J. Biol. Chem. 195 19 - 23
Souza FHM , Nascimento CV , Rosa JC , Masui DC , Leone FA , Jorge JA , Furriel RPM 2010 Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated β-glucosidase from the thermophilic fungusHumicola insolens. Process Biochem. 45 272 - 278    DOI : 10.1016/j.procbio.2009.09.018
Sørensen A , Lübeck PS , Teller PJ , Ahring BK 2011 β-Glucosidases from a newAspergillusspecies can substitute commercial β-glucosidases for saccharification of lignocellulosic biomass. Can. J. Microbiol. 57 638 - 650    DOI : 10.1139/w11-052
Vìtrovský T , Baldrian P , Gabriel J 2012 Extracellular enzymes of the white-rot fungusFomes fomentariusand purification of 1,4-β-glucosidase. Appl. Biochem. Biotechnol. 169 100 - 109    DOI : 10.1007/s12010-012-9952-9
Woodward J , Wiseman A 1982 Fungal and other β-D-glucosidases. Their properties and applications. Enzyme Microb. Technol. 4 73 - 79    DOI : 10.1016/0141-0229(82)90084-9
Wright R , Yablonsky M , Shalita Z , Goyal A , Eveleigh D 1992 Cloning, characterization and nucleotide sequence of a gene encodingMicrobispora bisporaBglB, a thermostable betaglucosidase expressed inEscherichia coli. Appl. Environ. Microbiol. 58 3455 - 3465
Xiao Z , Zhang X , Gregg DJ , Saddler JN 2004 Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates,InFinkelstein M, McMillan JD, Davison BH, Evans B (eds.).Proceedings of the Twenty-Fifth Symposium on Biotechnology for Fuels and Chemicals. Biotechnology for Fuels and Chemicals Breckenridge, CO 1115 - 1126
Yan TR , Lin CL 1997 Purification and characterization of a glucose-tolerant β-glucosidase fromAspergillus nigerCCRC 31494. Biosci. Biotechnol. Biochem. 61 965 - 970    DOI : 10.1271/bbb.61.965
Yan J , Pan G , Li L , Quan G , Ding C , Luo A 2010 Adsorption, immobilization, and activity of beta-glucosidase on different soil colloids. J. Colloid Interface Sci. 348 565 - 570    DOI : 10.1016/j.jcis.2010.04.044
Zaldívar M , Velásquez JC , Contreras I , Pérez LM 2001 Trichoderma aureoviride7-121, a mutant with enhanced production of lytic enzymes: its potential use in waste cellulose degradation and/or biocontrol. Electron. J. Biotechnol. 4 1 - 7    DOI : 10.2225/vol4-issue3-fulltext-7
Zanoelo FF , Polizeli ML , Terenzi HF , Jorge JA 2004 Betaglucosidase activity from the thermophilic fungusScytalidium thermophilumis stimulated by glucose and xylose. FEMS Microbiol. Lett. 240 137 - 143    DOI : 10.1016/j.femsle.2004.09.021
Zimbardi AL , Sehn C , Meleiro LP , Souza FH , Masui DC , Nozawa MS 2013 Optimization of β-glucosidase, β-xylosidase and xylanase production byColletotrichum graminicolaunder solid-state fermentation and application in raw sugarcane trash saccharification. Int. J. Mol. Sci. 14 2875 - 2902    DOI : 10.3390/ijms14022875