PRODUCTION AND CHARACTERIZATION OF A THERMOSTABLE Β-GLUCOSIDASE FROM Myceliophthora heterothallica

: The conversion of biomass from agro-industrial residues into bioproducts is of great interest, especially to Brazil, where bioenergy has a huge potential for development. Enzymes involved in biodegradation of lignocellulosic biomass are those of the cellulase system, of which β-glucosidase is a constituent. The production and characterization of β-glucosidase by the thermophilic fungus Myceliophthora heterothallica by solid-state cultivation on different agro-industrial residues (sugarcane bagasse, sugarcane straw, wheat bran and a mixture of these three materials (1:1:1 w/w) was evaluated. Solid-state cultivation were conducted in 250 mL Erlenmeyer flasks, with 5 g of each substrate. Different culture parameters, such as supplementary nutrient solution to the substrate, supplementary nutrient solution pH, initial substrate moisture and fungus incubation temperature, were evaluated to establish conditions of higher enzyme production by the fungus The greatest production of enzymes occurred in a mixture of wheat bran, sugarcane bagasse and straw bagasse (1:1:1). The activity of β-glucosidase was greater under the following conditions: nutrient solution composed of NH 4 NO 3 , MgSO 4 .7H 2 O and (NH4) 2 SO 4 (0.1%), at pH 4.5 or 6.0, fungus incubation at 40°C or 45°C, initial moisture of substrate at 80%. Enzyme presented optimum pH at pH 5.0 and good pH stability. Best temperature was 65°C and enzyme showed 100% stability for 1h, up to 60°C. The use of agro-industrial residues provided good production of β-glucosidase by fungus, with enzyme having the characteristics desirable from the industrial application.


INTRODUCTION
Solid-state cultivation (SSC) is an increasingly employed process to obtain microbial products for agricultural residues as substrates for microbial growth, with value aggregation (GAO et al., 2008;ASGHER et al., 2016). The application of residues to bioprocesses has become important from the environmental point of view since it reduces problems related to their inadequate management and consequent environmental damages. In addition, the materials´ low cost and high availability make them excellent alternative substrates for obtaining microbial products used in industrial processes (PANDEY et al., 2000).
One of the most prominent sectors in Brazil, especially in the states of São Paulo and Minas Gerais, is the sugar-energy sector, due to the establishment and expansion of sugarcane plants in several municipalities (OLIVEIRA; MENDES, 2014). Since there is a great generation of sugar cane residues, such as straw and bagasse, the latter are potential substrates to obtain bioproducts.
Further, besides the use of biomass residues from sugarcane to generate electricity, industries produce second-generation ethanol with microbial enzymes derived from the residue, reported by scientific studies. However, obtaining fermentable sugars through the degradation of these materials may also enhance the production of several other industrial products, such as biosurfactants, flavoring agents, organic acids and others (SANTOS et al., 2012).
One of the most important groups of enzymes in the lignocellulosic biomass degradation is cellulases, an enzymatic complex whose enzymes act synergistically in the transformation of cellulose into monomers and glucose dimers. They are generally subdivided into three classes: endo-1,4-β-D-glucanases or endoglucanases (which break the glycosidic bonds of cellulose chains and create new terminals); exo-1,4-β-D-glucanases or cellobiohydrolases (responsible for terminal action leading to cellobiosis) and 1,4-β-D-glucosidases (which hydrolyze cellobiose to glucose) (YOON et al., 2014). β-Glucosidases increase the overall yield of fermentable sugars and reduce the inhibitory effect of cellobiose in other cellulolytic enzymes. Consequently, they continue the enzymatic hydrolysis process (RANI et al., 2014). These enzymes have several applications in industrial processes, including the conversion of glycosides from isoflavones to aglycones, which have antioxidant activities, and are more easily absorbed by the human intestine, providing such health benefits as the prevention of certain types of cancer and as risk-reduction factors against cardiovascular diseases, osteoporosis, menopausal symptoms and diabetes. In wine production, the addition of microbial β-glucosidase in vinification processes enhances the release of volatile terpenes (deglycosylated by the enzyme action) and contributes towards the wine´s aromatic composition (SINGHANIA et al., 2013;SANTOS et al., 2016).
Thermophilic microorganisms have been studied to produce generally thermostable enzymes (MARTINS et al., 2013). Enzymatic mixtures used for the degradation of polysaccharides derived from biomass (cellulose, hemicellulose, pectin) are commonly produced by mesophilic fungal strains belonging to the genera Trichoderma and Aspergillus (VAN DEN BRINK; DE VRIES, 2011). However, thermophilic microorganisms are reported to contain enzymes which are more resistant to denaturation and proteolysis NUSSINOV, 2001).
Enzyme thermal stability allow the saccharification of biomass polysaccharides at high temperatures and, consequently, decrease in reaction time, increase in greater mass transfer and the composition of substrate viscosity, which optimizes the processes in which they act, with decrease of costs. Thus, the alternative is the study of other microorganisms that produce thermostable enzymes that degrade plant biomass (BERKA et al., 2011;VAN DEN BRINK et al., 2013).
Myceliophthora is a genus composed of mesophilic and thermophilic fungi, which includes 10 species. M. thermophila, M. heterothallica, M. hinnulea and M. fergusii were described as thermophilic and regarded as industrially interesting enzyme producers due to their high activity and thermostability rates (MAIJALA et al., 2012). Current study evaluated β-glucosidase production by thermophilic fungus Myceliophthora heterothallica in solid-state cultivation using agroindustrial residues as substrates, to determine the effect of different fermentative parameters on enzyme production, and to characterize the enzyme in relation with optimal pH and temperature of performance and the stability to these factors.

Microorganism
The thermophilic fungus Myceliophthora heterothallica F.2.1.4. was isolated from sugarcane bagasse compost provided by the Laboratory of Applied Biochemistry and Microbiology of the Universidade Estadual Paulista (UNESP), Brazil, and maintained in the Laboratory of Microbiology of the Universidade do Estado de Minas Gerais (UEMG), Brazil. The fungus was activated on Agar Malt medium (Acumedia) and incubated at 45°C for 5 days. Culture medium composed of oatmeal (Quaker) 3% and bacteriological Agar 2%, pH adjusted to 5.5, was kept under the same conditions of temperature and culture time for periodic peaks and conservation of pure culture.

Enzyme production
For each solid-state cultivation (SSC) culture, a pre-inoculum of the fungus was placed in a 250 mL Erlenmeyer flask containing 100 mL of a medium composed of oat flour (Quaker) 3% and Agar (2%), pH adjusted for 5.5 with HCl. The fungus was inoculated onto the surface of this medium, by streaking, and incubated at 45°C until complete growth. The microorganism was then suspended with 150 ml distilled water and inoculated into each Erlenmeyer.
The fungus was cultivated in 5.0 g of the following substrates: sugarcane bagasse, sugarcane straw, wheat bran and a mixture of these three materials (1:1:1 w/w) (namely MIX). Bagasse and sugarcane straw were retrieved from sugarcane plants in the municipality of Frutal MG Brazil. Wheat bran was purchased on the local market. Substrates were washed, dried at 60°C and sieved in a 10 mesh.
Solid-state cultivation cultures were conducted in 250 mL Erlenmeyer flasks, with 5 g of each substrate. In cultures for the evaluation of the influence of time on enzyme production, initially sterilized distilled water was used for hydration of the medium at pH 5.0 and the fungus was cultured at 45°C. Samples were taken every 24 hours, up to 240 hours (10 days). Further, 40 mL (wheat bran) and 80 mL (bagasse, cane straw and three-substrate mix) of distilled water were added to each sample. The mixture was homogenized manually and subsequently shaken (50 rpm), for 20 minutes. The material was then filtered on a nylon cloth disc, centrifuged at 10000 xg for 15 min, at 5°C, and the supernatant was used for the determination of enzymatic activities.

Cultivation parameters
Different culture parameters, such as supplementary nutrient solution to the substrate, supplementary nutrient solution pH, initial substrate moisture and fungus incubation temperature, were evaluated to establish conditions of higher enzyme production by the fungus.
The following nutrient solutions were used on the substrate to assess the effect of substrate supplementation on enzyme production: 1-Distilled water 0.1% (control); 2-NH 4 NO 3 (0.1%); 3-(NH 4 ) 2 SO 4 ; 4-NH 4 NO 3 , MgSO 4 .7H 2 O and (NH 4 ) 2 SO 4 (0.1%); 5-Yeast extract (0.1%), so that the initial moisture is at 80%. The pH rates of each solution were evaluated from 4.0 to 6.0 (with a variation of 0.5 in 0.5), at a 5x5 factorial plan. So that the effect of the substrate´s initial moisture could be evaluated, volumes of the nutrient solution (chosen in the previous stage) were added to the inoculum so that initial moisture contents were 60%, 65%, 70%, 75% and 80%. Fermentation temperatures evaluated were 40°C, 45°C, 50°C and 55°C, at a 5x5 factorial plan.

β-glucosidase characterization
The effect of pH on the enzyme activity was evaluated at pH range between 3.0 and 10.0 using 0.1 M buffers: pH 3: sodium citrate; pH 3-5.5: sodium acetate; pH 6.0-6.5: MES; pH 7.0-7.5: HEPES; pH 8.0-10.0: glycine. Temperature effect was assayed by incubating the reaction mixture at a temperature ranging between 35 and 80°C, in optimal pH. Further, the effect of pH on enzyme stability was analyzed by incubating the crude enzyme solution in various buffers with pH ranging between 3.0 and 10.0, during 24h, at 25°C, followed by the determination of -glucosidase residual activity, under optimum conditions of pH and temperature. Thermal stability was determined by incubating the crude enzyme between 40 and 80°C, for 60 min, followed by the determination of βglucosidase residual activity, under optimum pH and temperature conditions.
Effect of glucose on β-glucosidase activity was determined by adding glucose at different concentrations (0-50mM) in the reaction mixture, under optimum pH and temperature conditions. Enzymatic assay and statistical analysis of data β-D-Glucosidase activity was assessed in a mixture of 50 mm sodium acetate buffer, pH 5.0, 450 L of 4 mM L −1 p-nitrophenyl-β-Dglucopyranoside (pNP-Glc) as substrate, and 50 L of the enzyme solution incubated for 10 min, at 60°C. Reaction ceased by adding 2 mL of 2 M Na 2 CO 3 and absorbance read at 410 nm. One unit of β-glucosidase (U) was defined as the amount of enzyme that releases 1 mole of nitrophenol/min in reaction conditions (PALMA-FERNANDEZ et al., 2002).
Analysis of variance of the experiments was carried out with data obtained from the different treatments applied. Scott-Knott test was applied at 5% significance.

RESULTS AND DISCUSSION
Production of β-glucosidase by M. heterothallica on different substrates The highest production rate of initial βglucosidase occurred in the substrate formed by the mixture of wheat bran, sugarcane bagasse and straw (1:1:1 w/w), with initial rates (30.5 U g -1 ) ( Figure  1). Rates were high when compared to results in the literature, since no variation of cultivation parameters had yet been made for its production. As observed, prior to defining cultivation parameters that could increase β-glucosidase production, the activity rate obtained was close to some reports in the literature for fungal β-glucosidases, such as 40.4 U g -1 obtained by A. fumigatus cultivated at 45°C in wheat bran (MORETTI et al., 2012), 41.8 U g -1 obtained by Thermomucor indicae-seudaticae N31 cultivated at 45°C in soybean meal  and 30.5 U g -1 obtained by A. fumigatus cultivated at 45°C in sugarcane bagasse (DE OLIVEIRA RODRIGUES et al., 2017).
β-glucosidase activity was detected as from 2 days of culture, with peaks of activity on the 4 th and 7 th day, indicating that there may also be isoforms of the enzyme in the enzymatic extract. The peak of enzymatic activity occurred in 7 days, with rate presenting statistical difference when compared to the others. In the case of wheat bran, the activity was detected as from day 1 of cultivation, with peaks on the 6 th and 8 th day of culture. In sugarcane straw, it showed a peak between 2 and 5 days, and another between 7 and 10 days. In sugarcane bagasse, the enzymatic activity was also lower than that found in the other substrates (Figure 1).
Results indicate that, in addition to complementing the wheat bran with carbon source and energy, sugarcane bagasse and straw may contribute towards a greater fungus growth (visual data, not quantified in the experiments). This may be due to the fact that the mixture provided a greater contact surface of the fungus to the substrates (SHI et al., 2009), particularly wheat bran which, in an isolated way, becomes more compacted and increases the enzymes production. Effect of culture parameters on β-glucosidase production by M. heterothallica When the effect of different supplemental nutrient solutions to the substrate (MIX) on the βglucosidase production was evaluated, it has been observed that the substrate supplementation increased enzyme production, when compared to control (water only). Statistical analysis of BG activity data with the different nutrient solutions revealed that the conditions in which there was a significant statistical difference comprised the supplementation of the substrate with the solution composed of NH 4 NO 3 , MgSO 4 .7H 2 O and (NH 4 ) 2 SO 4 (0.1%), at pH 4.5 and 6.0 ( Figure 2). When compared to initial rates in control, the supplementation with the solution increased by almost 3 times the maximum β-glucosidase activity rate obtained by the fungus (Figure 2). The above demonstrated the importance of evaluating this factor for enzyme production. As a rule, when studying complementary source of nutrients to the substrate, nitrogen sources are mainly evaluated. Gottschalk et al. (2013) reported that inorganic nitrogen sources are generally more easily assimilated by fungi than organic ones, even though they point out that this depends on fungal lineage, the nature of the element and its concentration. Ahmed et al. (2017) cited that most research works on the microbial β- glucosidase production do not focus on the optimization of supplementary nitrogen sources to the carbon sources in the fermentation. The authors also reinforce the need to study the mechanisms by which nitrogen sources influence the expression of these enzymes. These observations corroborate the importance of the evaluation of different supplementary sources of nutrients on β-glucosidase production.
In the case of the substrate´s initial pH, different species require different pH rates for optimal β-glucosidase production. However, as in the case of temperature, most β-glucosidases research fails to report pH optimization for its production. A random pH rate is used in which these species grow optimally (AHMED et al., 2017). This information reinforces the study of these variables in current analysis. Throughout the microbial culture, in solid-state fermentation, pH rate is not controlled because of the process´s heterogeneity (GARCIA et al., 2015). According to Pandey et al. (2000), the difficulty of monitoring and controlling parameters in solid-state fermentation is perhaps the main disadvantage of the process, and pH variations during the fermentation process may occur due to the metabolic activity of the microorganisms. They may be increased or decreased according to the by-products released or the nutrients consumed during the process.
Under the best conditions established in previous experiments, the effect of the initial moisture of the substrate (MIX) and the incubation temperature of the fungus on βglucosidase production was determined. Incubation of the fungus at 40ºC or 45ºC, with initial substrate moisture at 80%, provided the highest activity of the enzyme, with significant statistical difference. Higher moisture rates (85%) decreased enzymatic activity at all incubation temperatures (Figure 3). Regarding to substrate moisture, when the moisture content is below the required level, the nutrients´ solubility is limited and hinders their effective absorption by the fungi. On the other hand, when it is raised, the substrate particles are surrounded by a thick layer of water, tending to stick together and limiting gas exchanges. Thus, it is essential to establish the adequate level of moisture for microbial growth and obtain its products (YOON et al., 2004;SANTOS et al., 2016).
Cultivation temperature for the production of β-glucosidases varies from species to species. In most cases, it coincides with the optimal temperature of growth of the microorganism. On the other hand, the ideal temperature for enzyme production does not always correspond to the temperature of the microorganism´s natural habitat (AHMED et al., 2017). Consequently, several microorganisms produce the enzyme at temperatures other than its optimum growth, as occurred in current assay with M. heterothallica fungus, where optimum growth temperature was between 45°C and 50°C, although the highest enzymatic activity rate was detected when the cultivation occurred at 40°C. Elyas et al. (2010) reported that the optimal growth temperature of Aspergillus AS58 fungal strain was 30°C, whereas the highest activity rate of its β-glucosidase was detected at 35°C. High or very low temperatures may decrease the microorganism´s growth and thus the formation of the product. Low thermal conductivity of the agro-industrial residues used in solid-state cultivation processes may hinder the dissipation of the metabolic heat generated by microbial growth (PANDEY et al., 2003). Thus, the analysis of culture temperature is highly relevant for delineating a bioprocess to produce enzymes (SANTOS et al., 2016).
Biochemical characterization of β-glucosidase β-glucosidase produced has a better performance at pH rates between 4.0 and 5.5, with activities very close to each other at this pH range, with a peak at pH 5.0 when incubated at 60°C (Figure 4). No enzymatic activity occurred at pH rates above 7.5. Thus, data indicate that the fungus produces a β-glucosidase that may be applied in processes that require higher acidic pH rates. Acid cellulase is more adequate to degrade feedstock cellulose in the bioconversion industry, where biomass undergoes acid pre-treatment. The ability to work in an acidic pH environment is also a requirement for enzymes used in the textile industry in the finishing step where they act on cellulolytic fibers (SHARMA et al., 2016).
With respect to stability when exposed for 24h in the absence of substrate in buffers with different pH rates, it has been observed that the enzyme maintains more than 80% of its activity within a wide pH range (3.5 to 9.5). At optimum pH (5.0), it maintained 88.1% of its activity, after 24h ( Figure 4). According to Baffi et al. (2011), most microbial β-glucosidases present optimum pH between 4.0 and 6.0. Garcia et al. (2015) reported that optimal pH of β-glucosidase produced by Lichtheimia ramosa ranged between 5.0 and 6.0, with peak at pH 5.5. With regard to pH stability, the enzyme also showed high stability (over 90%) over a wide pH range (between 3.0 and 10.0), similar to results in current assay. Santa-Rosa et al. (2018) reported that β-glucosidase from Penicillium sp. presented a higher activity at pH rates between 5.0 and 7.0, with optimum activity at pH 6.0.
In the case of optimum temperature for βglucosidase activity, an increase in enzyme activity was observed up to 65°C, at which temperature (75.4 U g -1 ) was detected. Activity decreases as from this temperature, especially after 75°C ( Figure  5). In the case of the enzyme´s thermostability, results showed that β-glucosidase presented 100.0% and 48.6% stability when respectively exposed at 60ºC and 65ºC. Activity declined in temperatures above 65ºC. Enzyme was not detected when exposed at 70°C, for 1 h ( Figure 5).  reported that β-glucosidase from Myceliophthora thermophila presented optimum activity at 70°C and the enzyme maintained more than 93% of its original activity when incubated at 55°C.
Optimal β-glucosidase temperature of fungus M. heterothallica lies within the optimum temperature range of these enzymes produced by different fungi. Results demonstrate that enzyme has a significant thermostability when compared to enzymes of several microorganisms reported in the literature. Many commercial β-glucosidases of fungi are stable for a short time at high temperatures, denaturing at higher temperatures or time of exposure (LIU et al., 2012). Although enzymes from different organisms vary greatly in properties and functions, cost-effective production, high hydrolytic efficiency and great tolerance to unfavorable conditions are prerequisites of an industrial biocatalyst in various applications. Of particular interest, β-glucosidases from thermophilic fungi are more favorable due to the high-temperature activity and good thermostability (PEI et al., 2011;MALLEK-FAKHFAKH et al., 2016). Thermostability study showed that βglucosidase from M. heterothallica is highly thermostable as it retained above 90% of its original activity after 120 min of incubation up to 60°C. At 65°C, enzyme half-life was approximately 1h. (Figure 6). Figure 6. Effect of different temperatures on β-glucosidase stability.  reported the production of a β-glucosidase of the thermophilic fungus Thermoascus aurantiacus, which showed maximum activity within 70°C and 75°C range. It maintained 75% stability when incubated for 1h at temperatures between 55°C and 75°C. Garcia et al. (2015) reported a β-glucosidase of the mesophilic fungus Lichtheimia ramosa, which presented optimum temperature of 65°C, maintaining 98% and 40% of its activity when exposed for 1h at 55°C and 60°C, respectively. Santa-Rosa et al. (2018) reported the production of a β-glucosidase of Penicillium sp., with an optimal temperature of 60°C, maintaining 95.7% of its activity when incubated for 1h.
The high thermostability of β-glucosidase of M. heterothallica presented in the present work is desirable in industrial processes that require cellulolytic enzymes that maintain activity under high temperatures.
Glucose in the reaction medium caused a decrease in β-glucosidase activity with increasing glucose concentration. Residual activity of the enzyme at concentrations 5, 10, 15, and 20mM was 66.3%, 54.6%, 43.0%, and 29.3%, respectively. It actually decreased progressively, maintaining 4.0% of activity with glucose at 40mM (Fig 7). In order to verify whether the inhibition of the enzyme was competitive or not, a test was performed in which the substrate concentration was increased from Since most microbial β-glucosidases are inhibited by glucose, it becomes a major limitation for the use of these enzymes in industrial processes (LEITE et al., 2008). High glucose concentrations may directly or indirectly interfere with the binding of the substrate to the activated site and reduce the reaction rate (SINGHANIA et al., 2013). Inhibition of β-glucosidase produced by M. heterothallica was completely reversed when the substrate concentration increased but maintained the same glucose concentration. Results demonstrated that enzyme interaction with the inhibitor was competitive. Competitive inhibition may be reversed by increasing the concentration of the substrate. The reversibility of glucose inhibition of β-glucosidase in current study confirmed the potential of the enzyme in applications requiring saccharification processes.

CONCLUSIONS
The use of agro-industrial residues as substrate provided good β-glucosidase production by M. heterothallica, with the enzyme having the desirable characteristics from industrial application, such as good thermostability and stability over a wide pH range.
The high thermostability of β-glucosidase of M. heterothallica presented in the present work is desirable in industrial processes that require cellulolytic enzymes that maintain activity under high temperatures.

ACKNOWLEDGMENTS
The authors wish to thank Programa PIBIC/UEMG for financial support.