Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab Biochemical characterization of an isolated 50 kDa beta-glucosidase from the thermophilic fungus Myceliophthora thermophila M.7.7 Emily Colferai Bonfá, Marcia Maria de Souza Moretti, Eleni Gomes, Gustavo Orlando Bonilla-Rodriguez⁎ São Paulo State University (Unesp), Institute of Biosciences, Humanities and Exact Sciences (Ibilce), São José do Rio Preto Campus, SP, Brazil A R T I C L E I N F O Keywords: Beta-glucosidase Myceliophthora thermophila Thermophilic fungus A B S T R A C T This study characterized a 50 kDa β-glucosidase (BGL50) produced by the thermophilic fungus Myceliophthora thermophila M.7.7 in solid state cultivation using a mixture of (1:1) sugarcane bagasse and wheat bran. The crude extract zymogram showed two isoforms of β-glucosidase with approximately 50 and 200 kDa, which were separated by gel filtration chromatography. The characterization of BGL50 showed optimum activity at 60 °C and pH 5.0 when 4-nitrophenyl β-D-glucopyranoside (pNPG) was used as the substrate, whereas when using cellobiose, the highest activity was observed at 50 °C and pH 4.5. Several ions and reagents produced different effects on the enzyme activity depending on the substrate and there was complete inhibition with Cu2+ and Fe3+ for both substrates. In addition, nine phenolic compounds showed no inhibitory effects on the enzyme, a sig- nificant feature since β-glucosidase is used for the saccharification of lignocellulosic biomass that generates several phenolic compounds. Kinetic studies revealed competitive inhibition by glucose when pNPG was used, with a Ki value of 1.5 mM and a significantly lower Km (0.52mM) than for cellobiose (8.50mM). The thermo- dynamic parameters showed that BGL50 is very stable at 60 °C displaying a half-life of 855.6 min but it is easily denatured above this temperature. The results emphasize the importance of investigating potential β-glucosi- dases based on cellobiose instead of using only pNPG since, in the industrial process, the enzyme will act on this natural substrate. In addition, understanding the thermostability of the enzyme is an important contribution to enzyme technology. 1. Introduction The World Energy Council considers that oil, natural gas and coal (non-renewable energy sources) contribute with more than 82% of global energy needs and 20% of CO2 emissions are mostly due to oil- based fossil fuels (Shaheen et al., 2013). Therefore, sustainable and renewable alternatives are urgently needed to reduce the dependence on these non-renewable resources. Biomass can be considered as an alternative energy source with increasing importance in the future. First generation ethanol has been an alternative energy source in Brazil, this biofuel being produced by fermenting glucose from sugarcane juice (Kang et al., 2014). However, both agricultural and the food industry generate a significant amount of residues that are a source for fer- mentable sugars that can then sustain the production of second gen- eration ethanol. Sugar cane bagasse and sugar cane straw are the residues from sugar and ethanol industries plants which contain around 75% sugar in the form of cellulose and hemicellulose polymers. Enzymatic saccharification of crystalline cellulose by enzymes from fungi and bacteria to obtain fermentable sugars requires the action of several highly specific enzymes, the final product being mainly free glucose. The cellulolytic complex consists of a variety of hydrolytic and redox enzymes acting synergistically. The hydrolases that can degrade cellu- lose comprise endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.176), exoglucohydrolases (EC 3.2.1.74) and β-glucosidases (EC 3.2.1.21) but they act in combination with hemicellulases and other enzymes (Maitan-Alfenas et al., 2015). Taking into account their sub- strate specificity, β-glucosidases (BGLs) have been classified into three groups: cellobiases (high specificity for cellobiose, a β−1,4-linked glucose dimer), aryl-β-glucosidases (high specificity for substrates such as p-nitrophenyl-β-D-glucopyranoside pNPG), or broad specificity BGLs, the last being the prevailing ones (Sørensen et al., 2013). It is known that microbial cellulolytic enzymes are inhibited by cello-oligosaccharides, cellobiose or the final product of hydrolysis, glucose, and by other monosaccharides as well (Hsieh et al., 2014). β- glucosidases release glucose from the non-reducing terminus of https://doi.org/10.1016/j.bcab.2018.01.008 Received 12 October 2017; Received in revised form 10 January 2018; Accepted 16 January 2018 ⁎ Correspondence to: DQCA, IBILCE-UNESP, Rua Cristovão Colombo 2265, São José do Rio Preto, SP 15054-000, Brazil. E-mail address: gustavo.bonilla@sjrp.unesp.br (G.O. Bonilla-Rodriguez). Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 Available online 31 January 2018 1878-8181/ © 2018 Elsevier Ltd. All rights reserved. T http://www.sciencedirect.com/science/journal/18788181 https://www.elsevier.com/locate/bab https://doi.org/10.1016/j.bcab.2018.01.008 https://doi.org/10.1016/j.bcab.2018.01.008 mailto:gustavo.bonilla@sjrp.unesp.br https://doi.org/10.1016/j.bcab.2018.01.008 http://crossmark.crossref.org/dialog/?doi=10.1016/j.bcab.2018.01.008&domain=pdf cellobiose and cello-oligosaccharides (Sawant et al., 2016). Many commercial enzymes lack good BGL activity, so characterizing new enzymes is essential (Xia et al., 2016) and that is the reason for this study where the biochemical properties of an isolated BGL from My- celiophthora thermophila M.7.7 were analyzed and the inhibitory me- chanisms of glucose and other putative inhibitors were also in- vestigated. Beta-glucosidases hydrolyze soluble cellodextrins and cellobiose to D-glucose, avoiding cellulase inhibition by cellobiose (Karnaouri et al., 2013). 2. Materials and methods 2.1. The microorganism The thermophilic fungus Myceliophthora thermophila M.7.7 was isolated from a compost pile of sugarcane bagasse in a previous work (Moretti et al., 2012) and is part of the working fungal collection at the Laboratory of Biochemistry and Applied Microbiology, IBILCE/UNESP, São José do Rio Preto, SP, Brazil. 2.2. Solid state fermentation and enzyme obtainment To obtain the inoculum, the fungus was incubated at 45 °C for 72 h in 250mL Erlenmeyer flasks containing 50mL of a slanted PDA (potato- dextrose-agar) medium. After this period the spores suspension was done with addition of sterile 100mL of nutrient solution composed of: (g L−1) KH2PO4, 3.0; MgSO4 7H2O 0.5; CaCl2 0.5, Tween 80 (1.0% v/ v), and (1%), yeast extract as a nitrogen source, and pH was adjusted to 5.0. A sterile mixture of sugarcane bagasse and wheat bran (1:1 m/m) was used for solid state fementation (SSF). The choice of these sub- strates was based on previous results (Moretti et al., 2012) in which higher β-glucosidase activity was observed. Polypropylene bags were used containing 5 g of a substrate (2.5 g wheat bran plus 2.5 g of ba- gasse) and 20mL of a nutrient solution containing spores (107 spores g−1), resulting in a final moisture content of 80%. This material was incubated at 45 °C and, after 96 h, 50mL of distilled water (1:10m/v) was added to the fermented material. The mixture was stirred for 30min in a shaker at 100 rpm, filtered and centrifuged at 10,000 × g for 15min at 10 °C for clarification. The supernatant was used as a crude enzyme solution. Total protein was quantified by a classic method (Lowry et al., 1951), using a standard curve of bovine serum albumin (Sigma-Aldrich, St. Louis, USA). 2.3. Enzyme assay For the β-glucosidase activity assay using a chromogenic substrate, 50 mL of enzyme solution were added to a mixture of 250mL of sodium acetate buffer (0.1 mol L−1 pH 5.0) and 250mL of 4mmol L−1 pNPG (Sigma-Aldrich, St. Louis, USA). The reaction was maintained at 60 °C for 5min and halted with 2mL of a 2mol L−1 solution of Na2CO3. The released nitrophenol was quantified by absorbance readings at 410 nm using a standard curve. To determine the β-glucosidase activity using cellobiose as a sub- strate, a sequence of two reactions was performed. In the first reaction, 20 mL of enzyme solution were added to 10mL of 4mmol L−1 cello- biose in a 0.1mol L−1 sodium acetate buffer (pH 5.0). The reaction was maintained at 50 °C for 20min and stopped by immersing the tubes in boiling water for 2min. Then the concentration of glucose released by the enzyme was determined by a glucose kit (Katal, Belo Horizonte, Brazil), adding 1mL of the kit solution to 10mL of the mixture from reaction 1, maintaining at 37 °C for 15min. The absorbance readings were performed at 505 nm and the amount of released glucose was determined from a standard curve. In order to determine the optimum incubation time of the enzymatic assays, enzymatic activities were measured according to the methods described above for pNPG and for cellobiose by only changing the re- action time (2–20min) for each substrate. The maximum incubation time adopted was that which ensured the linear release of the product in relation to the time. The initial reaction rate Vo, was calculated as μmol min−1 (John, 2002). 2.4. Separation and purification of the enzyme isoforms The crude enzyme solution, maintaining its high activity prior to gel filtration chromatography was concentrated using salting-out pre- cipitation with 70% ammonium sulfate. The solution was stirred for 30min at room temperature and subsequently centrifuged at 3000 × g for 40min at 4 °C. The precipitate was re-suspended and filtered using a 0.45 µm membrane and subsequently subjected to gel filtration chro- matography using a 16/100 column filled with Sephacryl S-100 h (GE HealthCare, Amersham, UK) attached to a GE Äkta Purifier 10 FPLC System (GE HealthCare, Amersham, UK) equilibrated with 20mmol L−1 acetate buffer pH 5.0 containing 0.3 mol L−1 NaCl. The linear flow was 6 cm h−1, and 1mL fractions were collected as the absorbance at 280 nm started to increase. The separation of the iso- forms BGL200 and BGL50 was checked by zymography using esculin. The BGL50 isoform was further purified by applying 10mL of the pooled fractions obtained after gel filtration in an XK16 ion-exchange chromatography column packed with a Q-Sepharose resin and con- nected to the Äkta equipment described above. Initially, the proteins were eluted with Tris buffer pH 7.5 (20mmol L−1) at the same linear flow as before (6 cm h−1). After washing with 5 column volumes, a linear salt gradient was started using a similar buffer but also con- taining 0.5mol L−1 NaCl, collecting 1mL per tube. Beta-glucosidase activity was determined using pNPG. 2.5. Enzyme characterization The protein profile and molecular mass estimation was assessed by SDS-PAGE (See et al., 1990) and zymograms using esculin (Kwon et al., 1994). For the estimation of the molecular mass ‘m’ of the isoforms (expressed in kDa) under denaturing conditions, a polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sul- fate (SDS-PAGE) using 10% running gel and 5% stacking gel. The gel was divided into two parts, one named "A" stained with Coomassie Blue and the other labeled "B" stained by the esculin/ferric chloride method after being washed twice in 2.5% Triton X-100. The estimation of the molecular mass of the isoforms was done by comparing the zymogram bands with the migration of standard globular proteins and the loga- rithm of their m. With the purpose of assessing the effect of pH and temperature on the activity and stability of BGL50, the enzyme characterization tests were performed using the same experimental conditions for both sub- strates, only varying the final enzymatic assay used for each one. The behavior of the enzyme activity as a function of pH was studied by incubating the enzyme and substrate in several suitable buffers: ci- trate (pH 3.0), acetate (pH 3.5–5.5), MES (pH 5.5–6.5), HEPES (pH 7.0–8.0) and glycine (pH 8.5–10.5), and measuring the activity at 60 °C. The effect of temperature on the enzymatic activity was evaluated by incubating the reaction mixture at temperatures in the range from 30° to 80°C, and the activity was measured at the optimum pH. The thermostability of the enzyme in the absence of substrate was evaluated maintaining the enzyme solution for one hour at tempera- tures from 30° to 80°C. After this period, samples were taken for an enzymatic activity assay performed under optimum pH and tempera- ture conditions. The effect of pH on enzyme stability was achieved by keeping the enzyme solution for 24 h at 25 °C in buffers between pH 3 and 11, subsequently assaying the enzymatic activity under optimum pH and temperature conditions. The effect of temperature on the conservation of the enzyme was E.C. Bonfá et al. Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 312 also evaluated at temperatures of −80, −10, 10 and 25 °C. Enzyme assays were done as described after 6 h, 1 day, 1 and 2 months. The influence of cations and reagents in the enzyme assay on BGL50 was evaluated by measuring the enzyme activity in the presence of KCl, MgCl2, NaCl, MnCl2, FeCl3, CuCl2, CoCl2, NiCl, AlCl3, BaCl2, ZnCl2, LiCl2, PVA, DMSO, Triton-X-100, Isopropanol, PEG, Ethanol, Acetone, EDTA, DTT, SDS and glucose in a final concentration of 2.5mmol L−1. The results were compared with those of the control sample not ex- posed to those chemicals (with a reference of 100%) using Student's t- test for two independent samples (Zar, 2010), adopting as significant values of p< .05, performed by QtiPlot software version 0.9.9.11 (© Ion Vasilief 2004–2017). The influence of phenolic compounds representative of those that can be released during the pretreatment of lignocellulosic biomass was evaluated for both substrates by the measurement of enzymatic activity in the presence of tannic acid, p-coumaric acid, syringic acid, gallic acid, ferulic acid, 4-hydroxybenzoic acid, vanillin, vanillic acid and syringaldehyde. The phenolics final concentration was 2.5mmol L−1 in the reaction mixture using cellobiose as substrate. However, when used pNPG, due to the coloration of the tannic acid, ferulic acid and syr- ingaldehyde reagents, their concentrations were changed to 0.062, 0.5 and 0.25mmol L−1 respectively, while keeping the other phenolics at 2.5 mmol L−1. The effects were measured by combining the phenols with the enzyme and the substrate immediately at the beginning of the assay. The results were compared with those of the control sample (reference of 100% using Student's t-test, adopting as significant values p< .05). The tests were performed using QtiPlot. The kinetic parameters of BGL50 were determined by varying the concentration of the substrate pNPG (0.2–20mmol L−1) in the absence and presence of glucose (8mmol L−1) with a 5-min incubation. The values of the parameters Km and Vmax were obtained by nonlinear re- gression performed by QtiPlot using the Michaelis-Menten equation, allowing the identification of competitive inhibition. The value of the dissociation constant for the enzyme-inhibitor complex (Ki) for glucose was calculated from the equation Km app = Km (1 + [I]/Ki), where Km app is the apparent Km for competitive inhibition and [I] is the molar concentration of the inhibitor (Wilson and Walker, 2010). The kinetic parameters using the natural substrate (cellobiose) were determined by varying the concentration of cellobiose (0.5–80mmol L−1) with a 3-min incubation and performing the en- zyme assay. The values of Km and Vmax were calculated as previously described. The thermodynamic analysis of BGL50 thermal denaturation was done using pNPG. The calculation of the activation energy Ea, the temperature coefficient Q10, half-life T1/2 parameters of the enzyme, as well as those related to the thermal denaturation (the activation energy Ea(D), ΔH(D), ΔG(D) and ΔS(D)) followed the method proposed in the literature (Saqib et al., 2012, 2010) and done in a previous study (Trindade et al., 2016). It is assumed that the irreversible denatured “I” state is evaluated using N ↔ D → I, where "N" represents the native conformation and "D" the reversible denatured conformation. 3. Results and discussion Two liters of crude enzyme extract were obtained after solid-state cultivation. The protein concentration in the sample determined by the Lowry assay was 2.8 mgmL−1 and β-glucosidase activity 17 U mL−1, a total of approximately 34,000 U, with a specific activity of 6 U mg−1. A zymogram showed distinct bands displaying β-glucosidase activity (Fig. 1) corresponding to two isoforms named according to their ap- parent molecular mass as BGL200 and BGL50 (near 200 and 50 kDa respectively, as estimated from a plot of migration vs. the logarithm of the m of the protein markers). Several filamentous fungi have the property of expressing different β-glucosidase isoforms, depending on the culture conditions or carbon sources (Singhania et al., 2011). The regulation mechanism of the expression of multiple cellulase isoforms is not yet fully elucidated and requires further research on the sequences and different expressions of the isoforms (Badhan et al., 2007). Induc- tion of different isoforms may be related to the metabolites present in the culture media and understanding this regulation would be im- portant in designing culture conditions for the desired overincreased production of isoforms or secondary metabolites. The type of sub- merged or solid state cultivation also influences the expression of the distinct isoforms (Gomes et al., 2009; Nazir et al., 2010; Silva et al., 2007; Willick and Seligy, 1985). Only the lower molecular weight β-glucosidase (BGL50) was par- tially purified and characterized since the enzyme similar to isoform BGL200 has already been studied (Brognaro, 2014). A salting out precipitation of the crude extract using ammonium sulfate concentrated the enzymes to 72.7 U mL−1 for gel filtration chromatography. The separation of β-glucosidase isoforms (Fig. 2) was compatible with a profile for monomeric forms with the previously estimated values of 50 and 200 kDa and was verified by a 10% poly- acrylamide gel zymogram using esculin as substrate and ferric chloride (Fig. 3). For the characterization of BGL50, fractions 35–49 were pooled, since BGL200 was not detected in those samples. The gel filtration was performed several times in order to obtain sufficient activity, always checking the purity by zymography, reaching a purification factor of 11.2 but a low yield of 25.5%, which can be explained in part because the other isoform (BGL200) was discarded and and this activity is compared to the initial activity of the crude extract containing both isoformsthe comparison considers the initial activity of the crude ex- tract. Additional purification steps, namely ion-exchange and hydro- phobic interaction chromatography, were tried but the enzyme yield decreased substantially. Fig. 1. 10% polyacrylamide gel under denaturing conditions. (A) part of the gel stained for proteins with Coomassie Blue showing the molecular mass protein standards and (B) another portion of the same gel stained with the esculin / ferric chloride method (zy- mogram) showing two bands with β-glucosidase activity for a sample of crude extract of M. thermophila M.7.7. E.C. Bonfá et al. Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 313 For the enzyme assays, the release of the product was linear up to five minutes for pNPG and three minutes for cellobiose, these being adopted as the incubation times. All the assays were performed in tri- plicate. Regarding the effect of temperature, most β-glucosidases from me- sophilic fungi exhibit optimum temperatures for activity between 40 and 50 °C (Bhatia et al., 2002). The enzymatic activity profile for BGL50 when varying the assay temperature resulted in optimum temperatures of 60 and 50 °C using pNPG and cellobiose, respectively (Fig. 4A), that can be explained by the different incubation times, being shorter for pNPG. As for thermostability, BGL50 remained stable up to one hour of incubation at 60 °C in the absence of substrates (Fig. 4B). The BGL200 isoform studied by Brognaro (2014) presented maximal activity at 65 °C on pNPG. The high rigidity of thermophilic enzymes may result from the in- terplay of various putative factors: hydrogen-bonds, hydrophobic in- teractions, internal packing, salt-bridges and secondary structural fea- tures (Shiraki et al., 2001). So, the determination of the definitive factor for this property depends on molecular and structural studies. With regard to the pH effect, the literature reports that fungal β- glucosidases exhibit optimum pH values between 4.0 and 6.0 (Bhatia et al., 2002). The optimal pH of BGL50 was 4.5 for cellobiose and pH 5.0 for pNPG (Fig. 5A). For comparison, BGL200 presented optimum pH of 4.5 using pNPG (Brognaro, 2014). Using cellobiose, the activity decreases more significantly around the maximum value when compared to the curve produced using pNPG, probably due to various residues side chains that interact differently with each substrate, as verified in a study of a bacterial β-glucosidase (Rajoka et al., 2015). Those authors demonstrated the different inter- actions of a β-glucosidase (BGLA) from the bacteria Thermotoga mar- itima with cellobiose and pNPG. In the BGLA-cellobiose complex, three hydrogen-bond interactions were obtained involving residues Asn223, Ser229, and His298 while the BGLA-pNPG interacts with active site residues: Glu166, Tyr295, and Asn223. Obviously, the comparison of enzymes from Fungi and Archaea may not be the best approach due to the phylogenetic distance and primary sequence differences but a si- milar explanation could be considered. In terms of the effect of pH on the BGL50 enzyme, it exhibited the highest activity around pH 4.0–4.5 and decreased below 50% only at pH values above 9.5 (Fig. 5B). When the effect of ions and other chemicals on the enzymatic ac- tivity was tested, no ion caused any significant increase in enzymatic activity and it was completely inhibited by Cu2+ and Fe2+ ions when using either substrate (Table 1). Different effects on beta-glucosidases have been reported for those metals, mostly decreasing the enzyme activity by several degrees (Pereira et al., 2017) supposing that at least part of the iron oxidizes to Fe3+, a favorable reaction. Heavy metals, such as Cu2+, exhibit high affinity for thiol groups; usually, these heavy metal ions oxidize the functional groups of the cysteine residues and may inhibit the enzymatic activity of certain proteins (Hayashi et al., 1999). Another possibility could be the redox effect of copper and iron Fig. 2. Chromatographic profile of the enzyme solution produced by the fungus M. thermophila M.7.7 applied to a C16/100 column filled with Sephacryl S-100 h. Protein absorbance values at 280 nm are shown as a dashed line and enzymatic activity of β- glucosidase as a continuous line, expressed as the absorbance at 410 nm of the product p- nitrophenol. Fig. 3. Zymogram of the partially purified β-glucosidase isoforms by gel filtration on Sephacryl S-100. The numbers represent the fraction numbers, and the figure does not show the fractions beyond tube 35, which contained only the BGL50 isoform. Fig. 4. Effect of temperature on BGL50 using cellobiose (filled circles) and pNPG (open circles) as substrate: (A) Variation of BGL50 activity at different temperatures; (B) Residual activity of BGL50 when incubated at different temperatures for 1 h in the ab- sence of substrate. The graphs represent the means (symbols) and standard deviations (bars). E.C. Bonfá et al. Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 314 oxidizing the hemiacetal ends and leading to inhibition, as pointed out for cellulases (Tejirian and Xu, 2010). The Zn2+ ion and dithiothreitol (DTT) showed large differences in enzyme behavior when assayed on both substrates. Using cellobiose a total inhibition of the enzyme was observed, whereas, when using pNPG, the average inhibitions were 37% and 17.5%, respectively (Tables 1 and 2). The effects of zinc and DTT in the cellobiose assay are worth noting because they cause interference in the assay itself. The independence of cofactors and a wide tolerance to the presence of ions observed for M. thermophila BGL50 show a similarity to a ß- glucosidase produced by the fungus Aureobasidium pullulans (Saha et al., 1994). Although none of the tested cations significantly increased BGL50 activity (Table 1), the decrease in activity induced by ethylenediami- netetraacetic acid (EDTA) (Table 2) suggests the need to look for some divalent metal ion not tested here. Thermal and chemical pretreatments of sugar cane bagasse release a series of compounds that can act as potential inhibitors of both the enzymatic hydrolysis and subsequent fermentation. The types of toxic compounds and their concentrations in lignocellulosic hydrolysates depend on both the raw material and the pretreatment operating con- ditions. One class of degradation products, which are potential fer- mentation inhibitors are phenolic derivatives (Palmqvist and Hahn- Hägerdal, 2000). Among the phenols released in the hydrolysis of lig- nocellulosic material, vanillin, syringaldehyde and hydroxybenzoic acid significantly inhibit cellulases, especially β-glucosidases (Ximenes et al., 2010). The results of the present study (Table 3) show a wide resistance of BGL50 to several phenolic compounds in a short preincubation time, showing that the enzyme has a potential application in the hydrolysis of lignocellulosic materials that release these phenolic derivatives. Most of the activities were higher with cellobiose than when tested using pNPG and, due to the high standard deviation, the observed average increase or decrease of the activity in the presence of phenolics was not sig- nificant, the only exceptions being gallic acid using cellobiose and tannic acid using pNPG. Nevertheless, the effects of phenols on β-glu- cosidases present in commercial enzymes from Trichoderma reesei (Spezyme CP®) and Aspergillus niger (Novozyme 188®) showed a sig- nificant inhibition by tannic acid that was evident in short or especially after long (24 h) preincubation times, while other phenols induced different degrees of inhibition (Ximenes et al., 2011), exerting higher inhibition when the enzymes were exposed for longer periods. The kinetic parameters, maximum reaction rate (Vmax) and Michaelis constant (Km), were calculated by nonlinear regression, a more adequate method than the popular Lineweaver-Burk linearization (Helfgott and Seier, 2007; Mason and Lai, 2000); the latter assumes linearity, constant variance and errors with normal distribution, which often do not occur or the operator is unaware of the validity of these assumptions (Helfgott and Moore, 2011). Vmax and Km values for BGL50 using pNPG as substrate (Fig. 6) for the reaction without glucose were 0.42± 0.01 μmol min−1 and 0.52±0.07mmol L−1 respectively. In the presence of the inhibitor, the values were 0.45± 0.02 μmol min−1 and 3.27±0.49mmol L−1, confirming, within the margin of error, that the kinetic parameter af- fected by the presence of glucose is the Km constant, a typical compe- titive inhibition when the ligand has a chemical structure similar to the substrate, being able to bind at the active site but not in a productive Fig. 5. Effect of pH on BGL50 using cellobiose (filled circles) and pNPG (open circles) as substrate: (A) Variation of BGL50 activity at different pH values; (B) Residual activity of BGL50 when incubated at different pHs for 24 h at room temperature in the absence of substrate. The graphs represent the means (symbols) and standard deviations (bars). Table 1 Effect of ions in a final concentration of 2.5mmol L−1 on the enzymatic activity of BGL50. Residual activity % (mean± standard deviation) pNPG ±SD Cellobiose ± SD Controle 100.0 13.3 100.0 0.3 NaCl 70.6* 6.7 57.1* 8.4 MnCl2 49.6* 9.9 67.1* 8.8 KCl 76.2* 5.0 81.9 15.6 FeCl3 0.0* 0.0 0.0* 0.0 BaCl2 105.4 6.9 76.7 11.1 MgCl2 100.0 7.8 62.7* 9.1 AlCl3 83.5 6.9 43.7* 7.2 ZnCl2 63.0 10.6 ND* 0.0 CuCl2 ND* 0.0 ND* 0.0 LiCl2 102.6 2.7 128.4 37.3 * Significant differences (p< .05) in Student's t-test. ND: not detected. Table 2 Effect of reagents in a final concentration of 2.5 mmol L−1 on the enzymatic activity of BGL50. Residual activity % (mean± standard deviation) pNPG ±SD Cellobiose ± SD Control 100.0 13.3 100.0 0.3 DMSO 92.9 5.6 57.0* 1.3 PVA 71.2 3.0 56.8 19.0 Triton 45.0 10.6 57.5* 8.9 Isopropanol 70.1 7.9 66.5 14.9 PEG 8000 67.6 6.2 58.5* 8.3 Ethanol 70.6 15.9 55.6* 5.7 Acetone 76.5 8.9 52.8* 1.3 EDTA 68.3 17.0 62.0* 8.5 DTT 82.5 6.9 ND* 0.0 SDS 82.4 9.2 60.6 27.1 * Significant differences (p< .05) in Student's t-test. ND: not detected. E.C. Bonfá et al. Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 315 way. The rate Km/Km app using pNPG was 0.16, lower than the value of 0.21 reported for a commercial preparation (Novozymes SP188) from A. niger (Chauve et al., 2010), suggesting a stronger inhibition for BGL50. From these data it was also possible to estimate the inhibition constant Ki = 1.5 mmol L−1; accordingly, glucose has a strong com- petitive inhibition effect on M. thermophila BGL50. For the yeast Clavispora NRRL Y-50464, Ki values were approximately 38 and 62mmol L−1 for two isoforms (Wang et al., 2016), but Ki values for β- glucosidase inhibition from other fungi can reach more than 100mmol L−1 (Riou et al., 1998). For comparison, kinetic values of an M. thermophila β-glucosidase, expressed when the BGL3a gene was cloned in Pichia pastoris (Karnaouri et al., 2013) were Km = 0.39±0.12mmol L−1 (close to our estimated value) but Ki was 0.28mmol L−1, lower than for BGL50. Since Vmax depends on enzyme concentration Et (Vmax=kcat.[Et]), usually it is not a reliable parameter for comparing to values in the literature. The values of Vmax and Km obtained for BGL50 using cellobiose as substrate (Fig. 7) were2.55± 0.09 μmol min−1 and 8.50±1.03mmol L−1, respectively. The kinetic values obtained show that BGL50 has a much lower Km for the synthetic substrate than for the natural one, suggesting that BGL50 is an aryl-β-glucosidase. In most cases, β-glucosidases show high catalytic activity and high affinity with the artificial substrate pNPG and MUG (methyl umbelliferyl β-D-glu- coside), compared to cellobiose (Nam et al., 2010). According to those authors, the kinetics of β-glucosidase depends on the configuration of its substrate and cellobiose requires a conformational change for the catalysis. The enzyme has a very rigid structure at the S1 subsite that Table 3 Effect of phenolic compounds on the enzymatic activity of BGL50 with pNPG and cellobiose substrates, measured as residual activity (%). The values represent the arithmetic mean for each substrate± standard deviation of triplicates. mmol L−1 pNPG ±SD mmol L−1 cellobiose ± SD Control 2.5 100.0 13.7 2.5 100.0 8.1 4-hydroxybenzoic acid 2.5 97.5 6.0 2.5 109.7 1.4 ferulic acid 0.5 97.4 3.1 2.5 99.1 4.2 gallic acid 2.5 91.1 14.4 2.5 75.3* 2.3 p-coumaric acid 2.5 70.5 17.4 2.5 118.0 0.9 syringaldehyde 0.25 92.8 36.9 2.5 96.4 2.3 syringic acid 2.5 95.2 18.9 2.5 111.4 2.3 tannic acid 0.0625 ND* ND* 2.5 99.5 12.3 vanillic acid 2.5 87.6 0.1 2.5 125.1 4.7 vanillin 2.5 94.0 1.3 2.5 118.0 4.7 * Significant differences (p< .05) in Student's t-test ND: not detected. Fig. 6. Michaelis-Menten curve for BGL50 using pNPG as substrate in the presence (empty triangles) and absence (filled circles) of glucose (8mmol L−1). The graphs re- present the means (symbols) and standard deviations (bars). Fig. 7. Michaelis-Menten curve for BGL50 using cellobiose as substrate. The graph re- presents the means (symbols) and standard deviations (bars). Fig. 8. Arrhenius plot for the calculation of the activation energy (Ea) and optimum temperature of the BGL50. The graph represents the means (symbols) and standard de- viations (bars). Table 4 Temperature coefficient (Q10) estimate based on the Arrhenius Graph. Temp (°C) Temp (K) Q10 30 303 1.83 40 313 1.76 50 323 1.70 60 333 1.65 70 343 1.60 E.C. Bonfá et al. Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 316 will accommodate glucose from cellobiose but a second glucose will alter the rotation conformation to fit into the substrate binding site. This could be the reason behind the lower efficiency of β-glucosidases with cellobiose than with the synthetic substrate pNPG (Singhania et al., 2013). The released glucose, in turn, may also cause enzyme inhibition. Analysis of irreversible thermal denaturation shows the optimum temperature as 62.6 °C and the activation energy (Ea) of BGL50 as 46.2 kJmol−1 from the Arrhenius curve shown in Fig. 8. The activation energy of BGL50 was three times higher than that reported for A. fu- migatus β-glucosidase (14.8 kJmol−1) (Das et al., 2015) but lower than for Fusarium solani β-glucosidase (53.3 kJmol−1) (Bhatti et al., 2013). The temperature coefficient Q10, which represents the increase in the reaction rate every 10 °C of temperature rise, was also estimated at different temperatures (Table 4). The Q10 values show a decrease as the temperature increases as a result of thermal denaturation. The best parameter to indicate the energy required to change enzyme con- formation is the activation energy for denaturation Ea(d), representing the energy barrier required to bring the enzyme from a native state to the denatured state (Saqib et al., 2010), and its value for BGL50 was calculated as 614.7 kJ mol−1. Comparing to the β-glucosidases of the fungus A. fumigatus (48.80 kJmol−1) (Das et al., 2015), Thermoascus aurantiacus (414 kJmol−1) and A. pullulans (537 kJmol−1) (Leite et al., 2007) BGL50 has higher conformational stability. The half-life of BGL50 at 60 °C is quite high compared to the other temperatures tested, representing a high structural stability when maintained at that temperature, but undergoes a significant denatura- tion before reaching 65 °C. Compared to the results found in the lit- erature for other fungal enzymes, the half-life of BGL50 is much lower; for the β-glucosidase of the fungus F. solani at 65 °C the half-life is 159min (Bhatti et al., 2013), for the β-glucosidase from A. pullulans t1/2 is 90 min at 80 °C and for the enzyme from Thermoascus aurantiacus 30min at 80 °C (Leite et al., 2007). Nevertheless, the experiments performed for up to 2 months for samples kept at −80, −10, 10 and 25 °C showed no significant decrease of the activity (results not shown). The enthalpy variation (ΔHd) indicates the amount of non-covalent bonds that are broken during the protein denaturation process. For BGL50, the values are lower than those reported for a beta-glucosidase from F. solani (50 kJmol−1) (Bhatti et al., 2013), A. pullulans (534 kJmol−1) and T. aurantiacus (411 kJ mol−1) (Leite et al., 2007). The value of ΔGd is proportional to protein stability (Longo and Combes, 1999), and this will depend on the temperature. Therefore, BGL50 shows higher stability at 60 °C, as shown in Table 5. The results indicate that at 65 °C much of the structural organization has already been destroyed. In fact, the irreversible transition between the native and denatured state occurred in a narrow range of temperature, be- tween 60 and 65 °C. The variation of the denaturation entropy at 65 °C was −285.4 Jmol−1 K−1, lower than the result for F. solani β-glucosidase, in the range of −176 Jmol−1 K−1 (Bhatti et al., 2013). Low ΔSd values suggest the exposure of nonpolar side chains, causing ordering of water molecules in the form of clathrates or "cages" (Siddiqui et al., 1997). The thermodynamic parameters for the β-glucosidase produced by the mesophilic fungus A. pullulans (Leite et al., 2007) evaluated at 80 °C showed that the enzyme is more resistant to thermal inactivation than BGL50. Therefore, the complex understanding of enzymatic thermo- stability is not only associated with the thermophilicity of the organism. 4. Conclusion The lower molecular mass beta-glucosidase from M. thermophila (around 50 kDa) was easily purified and the results suggest that it is a catalytically efficient enzyme for cellobiose hydrolysis. The differences in data about enzyme activity between the synthetic (p-PNPG) and the natural substrate (cellobiose) under different reaction conditions shows some important findings since they can be used to define the sacchar- ification procedures. The kinetic and thermodynamic properties and its behavior with phenolic compounds properties suggest that the enzyme would be efficient for industrial purposes. Funding This work was supported by FAPESP (Grants 11/23991-7 and 10/ 12624-0) and CAPES (fellowship for ECB). References Badhan, A.K., Chadha, B.S., Kaur, J., Saini, H.S., Bhat, M.K., 2007. Production of multiple xylanolytic and cellulolytic enzymes by thermophilic fungus Myceliophthora sp. IMI 387099. Bioresour. Technol. 98, 504–510. Bhatia, Y., Mishra, S., Bisaria, V.S., 2002. Microbial beta-glucosidases: cloning, proper- ties, and applications. Crit. Rev. Biotechnol. 22, 375–407. Bhatti, H.N., Batool, S., Afzal, N., 2013. Production and characterization of a novel β- glucosidase from Fusarium solani. Int. J. Agric. Biol. 15, 140–144. Brognaro, H., 2014. Estudo do Potencial Enzimático Hidrolítico e Oxidativo do Microorganismo Myceliophthora thermophila M.7.7 (Ph.D. Thesis). Universidade Estadual Paulista Julio de Mesquita Filho, Instituto de Biociências, Letras e Ciências Exatas. Chauve, M., Mathis, H., Huc, D., Casanave, D., Monot, F., Ferreira, N., 2010. Comparative kinetic analysis of two fungal β-glucosidases. Biotechnol. Biofuels 3, 1–8. Das, A., Paul, T., Ghosh, P., Halder, S., Das Mohapatra, P., Pati, B., Mondal, K., 2015. Kinetic study of a glucose tolerant beta-glucosidase from Aspergillus fumigatus ABK9 entrapped into alginate beads. Waste Biomass-. Valoriz. 6, 53–61. Gomes, E., Leite, R.S.R., da Silva, R., Silva, D., 2009. Purification of an Exopolygalacturonase from Penicillium viridicatum RFC3 produced in submerged fermentation. Int. J. Microbiol. 2009 (2009), 1–8. Hayashi, S., Sako, S., Yokoi, H., Takasaki, Y., Imada, K., 1999. Purification and char- acterization of the intracellular β-glucosidase from Aureobasidium sp ATCC 20524. J. Ind. Microbiol. Biotechnol. 22, 160–163. Helfgott, M., Moore, D., 2011. Introductory calculus for the natural sciences. CreateSpace. Helfgott, M., Seier, E., 2007. Some mathematical and statistical aspects of enzyme ki- netics. J. Online Math. Appl. 7, 1–34. Hsieh, C.C., Cannella, D., Jørgensen, H., Felby, C., Thygesen, L.G., 2014. Cellulase in- hibition by high concentrations of monosaccharides. J. Agric. Food Chem. 62, 3800–3805. John, R.A., 2002. Enzyme assays: a practical approach. In: Eisenthal, R., Danson, M.J. (Eds.), Enzyme Assays: A Practical Approach. Oxford University Press, pp. 49–78. Kang, Q., Appels, L., Tan, T., Dewil, R., 2014. Bioethanol from lignocellulosic biomass: current findings determine research priorities. Sci. World J. 2014, 1–13. Karnaouri, A., Topakas, E., Paschos, T., Taouki, I., Christakopoulos, P., 2013. Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophila. PeerJ 1, 1–20. Kwon, K.S., Lee, J., Kang, H.G., Hah, Y.C., 1994. Detection of beta-glucosidase activity in polyacrylamide gels with esculin as substrate. Appl. Environ. Microbiol. 60, 4584–4586. Leite, R.S.R., Gomes, E., Da Silva, R., 2007. Characterization and comparison of ther- mostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus. Process Biochem. 42, 1101–1106. Longo, M.A., Combes, D., 1999. Thermostability of modified enzymes: a detailed study. J. Chem. Technol. Biotechnol. 74, 25–32. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Maitan-Alfenas, G.P., Visser, E.M., Guimarães, V.M., 2015. Enzymatic hydrolysis of lig- nocellulosic biomass: converting food waste in valuable products. Curr. Opin. Food Sci. 1, 44–49. Mason, G.F., Lai, J.C.K., 2000. Nonlinear determination of Michaelis-Menten kinetics with model evaluation through estimation of uncertainties. Metab. Brain Dis. 15, Table 5 Kinetic and thermodynamic parameters of irreversible thermoinactivation. Temperature (°C) Temperature (K) kd (min−1) t ½ (min) ΔHd (kJ mol−1) ΔGd (kJmol−1) ΔSd (J mol. K−1) 60 328 0.00081 855.6 2.154 101.58 −311.5 63 333 0.01417 48.9 2.179 94.52 −287.8 65 338 0.01897 36.5 2.196 94.28 −285.4 E.C. Bonfá et al. Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 317 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref1 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref1 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref1 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref2 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref2 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref3 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref3 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref4 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref4 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref4 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref4 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref5 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref5 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref6 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref6 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref6 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref7 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref7 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref7 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref8 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref8 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref8 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref9 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref10 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref10 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref11 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref11 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref11 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref12 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref12 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref13 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref13 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref14 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref14 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref14 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref15 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref15 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref15 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref16 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref16 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref16 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref17 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref17 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref18 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref18 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref19 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref19 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref19 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref20 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref20 133–149. Moretti, M.M.S., Bocchini-Martins, D.A., Da Silva, R., Rodrigues, A., Sette, L.D., Gomes, E., 2012. Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation. Braz. J. Microbiol. 43, 1062–1071. Nam, K.H., Sung, M.W., Hwang, K.Y., 2010. Structural insights into the substrate re- cognition properties of β-glucosidase. Biochem. Biophys. Res. Commun. 391, 1131–1135. Nazir, A., Soni, R., Saini, H.S., Kaur, A., Chadha, B.S., 2010. Profiling Differential Expression of Cellulases and Metabolite Footprints in Aspergillus terreus. Appl. Biochem. Biotechnol. 162, 538–547. Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. II: Inhib. Mech. Inhib. Bioresour. Technol. 74, 25–33. Pereira, J., de, C., Giese, E.C., Moretti, M.M., de, S., Gomes, A.C., dos, S., Perrone, O.M., Boscolo, M., Da Silva, R., Gomes, E., Martins, D.A.B., 2017. Effect of metal ions, chemical agents and organic compounds on lignocellulolytic enzymes activities. In: Enzyme Inhibitors and Activators. InTech, pp. 139–164. Rajoka, M.I., Idrees, S., Ashfaq, U.A., Ehsan, B., Haq, A., 2015. Determination of substrate specificities against β-glucosidase A (BglA) from Thermotoga maritime: a molecular docking approach. J. Microbiol. Biotechnol. 25, 44–49. Riou, C., Salmon, J.M., Vallier, M.J., Günata, Z., Barre, P., 1998. Purification, char- acterization, and substrate specificity of a novel highly glucose-tolerant beta-gluco- sidase from Aspergillus oryzae. Appl. Environ. Microbiol. 64, 3607–3614. Saha, B.C., Freer, S.N., Bothast, R.J., 1994. Production, purification, and properties of a thermostable beta-glucosidase from a color variant strain of Aureobasidium pullulans. Appl. Environ. Microbiol. 60, 3774–3780. Saqib, A.A.N., Farooq, A., Iqbal, M., Hassan, J.U., Hayat, U., Baig, S., 2012. A thermo- stable crude endoglucanase produced by Aspergillus fumigatus in a novel solid state fermentation process using isolated free water. Enzym. Res. 2012, 1–6. Saqib, A.A.N., Hassan, M., Khan, N.F., Baig, S., 2010. Thermostability of crude en- doglucanase from Aspergillus fumigatus grown under solid state fermentation (SSF) and submerged fermentation (SmF). Process Biochem. 45, 641–646. Sawant, S., Birhade, S., Anil, A., Gilbert, H., Lali, A., 2016. Two- way dynamics in β- glucosidase catalysis. J. Mol. Catal. B Enzym. 133, 161–166. See, Y.P., Jackowski, G., Creighton, T.E., 1990. Estimating molecular weight of poly- peptides by SDS gel electrophoresis. In: ProteinStructure: A Practical Approach. IRL Press, Oxford, pp. 1–22. Shaheen, M., Choi, M., Ang, W., Zhao, Y., Xing, J., Yang, R., Xing, J., Zhang, J., Chen, J., 2013. Application of low-intensity pulsed ultrasound to increase bio-ethanol pro- duction. Renew. Energy 57, 462–468. Shiraki, K., Nishikori, S., Fujiwara, S., Hashimoto, H., Kai, Y., Takagi, M., Imanaka, T., 2001. Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart. Eur. J. Biochem. 268, 4144–4150. Siddiqui, K., Najmus Saqib, A., Rashid, M., Rajoka, M., 1997. Thermostabilization of carboxymethylcellulase from Aspergillus niger by carboxyl group modification. Biotechnol. Lett. 19, 325–330. Silva, D., Martins, E.S., Leite, R.S.R., Da Silva, R., Ferreira, V., Gomes, E., 2007. Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation. Process Biochem. 42, 1237–1243. Singhania, R.R., Patel, A.K., Sukumaran, R.K., Larroche, C., Pandey, A., 2013. Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol pro- duction. Bioresour. Technol. 127, 500–507. Singhania, R.R., Sukumaran, R.K., Rajasree, K.P., Mathew, A., Gottumukkala, L., Pandey, A., 2011. Properties of a major β-glucosidase-BGL1 from Aspergillus niger NII-08121 expressed differentially in response to carbon sources. Process Biochem. 46, 1521–1524. Sørensen, A., Lübeck, M., Lübeck, P.S., Ahring, B.K., 2013. Fungal beta-glucosidases: a bottleneck in industrial use of lignocellulosic materials. Biomolecules 2013, 612–631. Tejirian, A., Xu, F., 2010. Inhibition of cellulase-catalyzed lignocellulosic hydrolysis by iron and oxidative metal ions and complexes. Appl. Environ. Microbiol. 76, 7673–7682. Trindade, L.V., Desagiacomo, C., Polizeli, M. de L.T. de M., Damasio, A.R. de L., Lima, A.M.F., Gomes, E., Bonilla-Rodriguez, G.O., 2016. Biochemical characterization, thermal stability, and partial sequence of a novel exo-polygalacturonase from the thermophilic fungus Rhizomucor pusillus A13.36 obtained by submerged cultivation. Biomed. Res. Int. 2016, 1–10. Wang, X., Liu, Z.L., Weber, S.A., Zhang, X., Barre, P., Kim, S.-H., 2016. Two new native β- Glucosidases from Clavispora NRRL Y-50464 confer its dual function as cellobiose fermenting ethanologenic yeast. PLoS One 11, 1–19. Willick, G.E., Seligy, V.L., 1985. Multiplicity in cellulases of Schizophyllum commune. Derivation partly from heterogeneity in transcription and glycosylation. Eur. J. Biochem. 151, 89–96. Wilson, K., Walker, J.M., 2010. Principles and Techniques of Biochemistry and Molecular Biology. Cambridge University Press. Xia, W., Bai, Y., Cui, Y., Xu, X., Qian, L., Shi, P., Zhang, W., Luo, H., Zhan, X., Yao, B., 2016. Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1. Sci. Rep. 6, 1–11. Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M., 2011. Deactivation of cellulases by phenols. Enzym. Microb. Technol. 48, 54–60. Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M., 2010. Inhibition of cellulases by phenols. Enzym. Microb. Technol. 46, 170–176. Zar, J.H., 2010. Biostatistical Analysis. Prentice-Hall/Pearson. E.C. Bonfá et al. Biocatalysis and Agricultural Biotechnology 13 (2018) 311–318 318 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref20 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref21 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref21 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref21 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref21 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref22 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref22 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref22 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref23 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref23 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref23 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref24 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref24 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref25 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref25 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref25 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref25 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref26 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref26 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref26 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref27 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref27 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref27 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref28 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref28 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref28 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref29 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref29 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref29 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref30 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref30 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref30 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref31 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref31 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref32 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref32 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref32 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref33 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref33 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref33 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref34 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref34 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref34 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref35 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref35 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref35 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref36 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref36 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref36 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref36 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref37 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref37 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref37 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref38 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref38 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref38 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref38 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref39 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref39 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref40 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref40 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref40 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref41 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref41 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref41 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref41 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref41 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref42 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref42 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref42 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref43 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref43 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref43 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref44 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref44 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref45 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref45 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref45 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref46 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref46 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref47 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref47 http://refhub.elsevier.com/S1878-8181(17)30526-1/sbref48 Biochemical characterization of an isolated 50 kDa beta-glucosidase from the thermophilic fungus Myceliophthora thermophila M.7.7 Introduction Materials and methods The microorganism Solid state fermentation and enzyme obtainment Enzyme assay Separation and purification of the enzyme isoforms Enzyme characterization Results and discussion Conclusion Funding References