The enzymatic degradation of cellulose by cellulases has been the focus of several studies for their use in the bioconversions of agricultural wastes, bio-polishing of textiles, the processing of fruit juices, and bioethanol production. Such enzymatic degradation requires the synergistic action between exo-(1, 4)-β-D-glucancellobiohydrolase, endo-(1, 4)-β-D-glucanohydrolase, and β-D-glucosidase . Although numerous microorganisms are known to be able to produce high levels of extracellular cellulases, the best cellulase producers of Trichoderma sp. are fairly deficient in β-glucosidase, causing the accumulation of cellobiose, which produces repression and end product inhibition of the enzymes, both of which limit enzyme synthesis and activity . In the current research, 60 fungal isolates were grown under SSF using wheat bran as a substrate. The highest level of extracellular β-glucosidase activity was observed in the culture filtrate of the fungal isolate DHE7 (81.3 ± 4.2 U/g ds), which was much higher than β-glucosidases produced by Thermoascus aurantiacus (7.0 U/g ds) and Lichtheimia ramosa (0.061 U/g ds) [20, 21] grown under SSF. Based on these results, the isolate DHE7 was genetically identified as a new species of Aspergillus and the nucleotide information is available in GenBank under the accession number of KX950801.
Most microorganisms possess intracellular and cell-wall bound β-glucosidases . One of the earliest reports in this regard is that of Eberhart and Beck  who studied the intracellular localization of β-glucosidase in Neurospora crassa. The filamentous fungus P. decumbens was also found to produce two intracellular β-glucosidases . While Venturi et al.  and Morais et al.  reported the production of extracellular β-glucosidase activity from Chaetomium thermophilum and Pleurotus ostreatus when grown on agro-industrial wastes as substrate. It was reported that the most promising fungi with respect to β-glucosidase production are Aspergillus sp. . In addition, the cellulolytic system of Trichoderma reesei could be successfully supplemented with β-glucosidase from Aspergillus cultures .
The utilization of agro-industrial residues as substrate in SSF has gained great interest due to the low-cost production and the reduction in environmental pollution resulted from the accumulation of these wastes. In this study, Aspergillus sp. DHE7 could grow on various agro-industrial residues as substrate under SSF; however, jojoba meal was estimated to be the most promising fermentable substrate for β-glucosidase production (116.4 + 2.4 U/g ds). This might be due to the nutritional composition of jojoba meal, which is suitable for the growth of various microorganisms as it contains appropriate amounts of carbohydrates, proteins, fats, fiber, and ashes, favoring enzymes production. In this regard, Xin and Geng  reported maximum production of β-glucosidase (61.6 U/g ds) from T. reesei when the fungus grown on woodchips for 192 h at 26 °C. Ng et al.  observed the highest level of β-glucosidase activity from P. citrinum YS40-5 when grown on rice bran for 96 h. While the maximum production of β-glucosidase of 159.3 U/g ds was achieved by Colletotrichum graminicola grown in wheat bran containing medium after 168h of incubation .
The initial medium pH has a great effect on the growth and the production of β-glucosidase by many microorganisms, as it may affect the permeability of cells and other physiological activities. Most filamentous fungi are known for their capability in growing under a broad range of pH using SSF technique, due to the buffering capacity of these solid substrates . The variation of pH values during the fermentation process depends on the microbial by-products released or the consumed nutrients throughout the cultivation process. Generally, pH values greater than 7 were found to have a negative impact on the fungal growth, thus reducing enzyme production . In the present work, optimum initial medium pH for β-glucosidase production by Aspergillus sp. DHE7 was observed in the range of 5.0–7.0 peaking at pH 6.0. This result is closely related to that reported for A. oryzae NRRL 3484, which attained its highest level of β-glucosidase yield at pH 5.5 . This slightly acidic pH value was also reported by many investigators; whereas, Grajek  and Makropoulou et al.  reported that pH 6.0 was the optimal initial pH for β-glucosidases production by Sporotrichum thermophile and Fusarium oxysporum, respectively. Rajoka et al.  also found that the highest yield of this enzyme from Kluyveromyces marxianus was occurred at pH 5.5. Results of the current work demonstrated the sensitivity of the enzyme to lower pH values, hence, at initial medium pH of 4.0 only 44.3 ± 2.4 U/g ds of β-glucosidase activity was detected. This finding is similar to that reported for β-glucosidase production from A. niger . On the other hand, the reduction in β-glucosidase formation versus alkaline pH was much smaller; whereas, at pH of 8.0, β-glucosidase yield of 86.5 ± 2.1 U/g ds was observed in agreement with the results reported by Hoffman and Wood  and Kantham et al. .
In SSF, the initial moisture content (IMC) greatly affects the production and secretion of enzymes, depending on the biomass, the microorganism, and the final product. Generally, IMC between 60 and 78% are often used for the production of β-glucosidase by different filamentous fungi . In the current work, maximum β-glucosidase yield of 141.6 ± 4.6 U/g ds was reported at a moisture level of 70%. Similarly, Brijwani et al.  found that the moisture level of 70% was the best for the highest β-glucosidase production (10.7 U/g ds) during co-cultivation of A. oryzae and T. reesei when grown on wheat bran and soybean peel as substrate. In addition, the optimal β-glucosidase production of 70 U/g ds was reported from Thermoascus aurantiacus grown in wheat bran medium at a moisture content of 60% . Vu et al.  suggested that the presence of free water between the particles of a substrate reduces the substrate porosity; leading to stickiness development and may interfere with gas and heat transfer, while low moisture level tends to reduce nutrient solubility, resulting in an improper swelling that decreases microbial metabolic activity.
Maximum production of β-glucosidase (152.8 ± 4.3 U/g ds) by Aspergillus sp. DHE7 was achieved after 72 h of incubation using jojoba meal as a substrate at an initial medium pH of 6.0 and 70% moisture level. However, no significant activity could be detected in the culture filtrates of Aspergillus sp. DHE7 after 24 h of incubation. It seemed likely that the cell machinery of the organism during this period is directed towards active vegetative growth and mycelium proliferation. The decreased time of cultivation is considered a key improvement in the fermentation process used by Aspergillus sp. DHE7, as the enzyme cost is proportional to the incubation time. In accordance with this result, Mukherjee and Khowala  purified an intracellular β-glucosidase from mycelia of Termitomyces clypeatus grown in a synthetic medium for 4 days. On the other hand, Juhász et al.  reported maximal β-glucosidase production from A. niger on the seventh day of incubation. Gonçalves et al.  reported optimum β-glucosidase productivity from L. ramose after 120 h of incubation. While the highest yield of β-glucosidase of 105.8 U/g ds from A. fumigatus was achieved after 96 h of cultivation in wheat bran medium .
The optimal temperature for β-glucosidase biosynthesis by Aspergillus sp. DHE7 was reported at 35 °C, which is greater than the range of 28–30 °C that is commonly reported for mesophilic microorganisms . This feature promotes the application of this fungal strain in various industrial processes with temperature fluctuations because the control of large-scale fermentation parameters is not as accurate as in the lab. On the other hand, a considerable reduction in β-glucosidase production was observed in cultures grown at 25 °C and 45 °C, which were estimated to be only 55.7% and 46.4% that of the maximum value, respectively. This negative effect might be resulted from the reduction in plasma membrane permeability and metabolic reactions rate at low temperatures, while at high temperatures, the membrane structures collapse and denature structural proteins and enzymes, consequently, resulting in a reduction in enzyme production . Maximum β-glucosidase production of 174.6 ± 5.8 U/g ds from Aspergillus sp. DHE7 was reported at an inoculum size of 2.54 × 107 spores/mL. However, a decline in enzyme production was reported with inoculum levels higher or smaller than the optimum level. Whereas at lower inoculum sizes, microbial biomass cannot proliferate rapidly; therefore, the degradation of substrates is slow, subsequently, affecting metabolite production. On the other hand, the inhibitory impact at higher inoculum size might be attributed to the depletion of nutrients and oxygen form the culture medium .
Aspergillus sp. DHE7 β-glucosidase was purified 29.6-purification fold with 45.2% recovery using 50% ethanol fractionation, followed by DEAE-Sephadex A-50 column then subsequent gel filtration on Sephadex G-100 column. The specific activity of the pure β-glucosidase was 2338.3 U/mg protein, which is much higher than those reported by Chirico and Brown  for Trichoderma reesei (52 U/mg protein), Bhat et al.  for Sclerotium thermophile (89 U/mg protein), and Abdel-Naby et al.  for A. niger A20 (140.35 U/mg protein). The purified enzyme was migrated as only one protein band with an apparent molecular mass of 135 kDa on polyacrylamide gel electrophoresis technique, indicating its homogeneity and purity. This value is favorably comparable to those reported for A. terreus ATCC 52430 and its mutant, UNG1-40 (90 kDa and 95 kDa, respectively) , A. niger KCCM 11239 β-glucosidase (123 kDa)  and Phanerochaete chrysosporium (114 kDa) . However, the molecular mass of Aspergillus sp. DHE7 β-glucosidase is much smaller than those of Sclerotium thermophile (240 kDa)  and Botrytis cinerea (350 kDa) .
Biochemical characterization of the purified Aspergillus sp. DHE7 β-glucosidase indicated that this enzyme displayed its maximum activity at temperature of 60 °C and pH 6.0. A further increase in temperature has resulted in a gradual decrease in enzyme activity, which may result from a conformational change resulting in a loss in the specificity of the active site . It should be noted that temperatures above 50 °C have not been observed routinely for β-glucosidases produced by mesophilic microorganisms. These results are consistent with the findings of Liu et al.  on β-glucosidase activity purified form A. fumigatus Z5. β-glucosidase purified from the thermophilic fungus Chaetomium thermophilum var. coprophilum showed its optimal activity at pH 5.5 and 65 °C . Gueguen et al.  reported that Botrytis cinerea β-glucosidase exhibited optimal catalytic activity at 50 °C and pH 7.0 with citrate-phosphate buffer and 6.5 with phosphate buffer. Optimal temperature of 50–60 °C has also been reported for the enzyme purified from the thermophilic fungus Humicola grisea var. thermoidea at an optimum pH of 6.0 . While Belancic et al.  investigated the optimum activity of Debaryomyces vanrijiae β-glucosidase at a lower temperature of 40 °C. Baffi et al.  reported that most fungal β-glucosidases have optimum activity at temperatures ranging from 40 to 50 °C, while high catalytic activity in enzymes produced by mesophilic microorganisms is not routinely detected at temperatures above 50 °C. Baffi et al.  reported that most fungal β-glucosidases have optimum activity at temperatures ranging from 40 to 50 °C.
The enzyme seems to be also active over a wide range of pH values (4.0–7.0). However, a decline in β-glucosidase activity was observed at pH values below or higher than the optimum pH 6.0. This negative effect may result from the fact that the enzyme is protein in nature and any alteration in the pH value may have a significant effect on the ionic character of its amino or carboxylic groups, which, in turn, may affect the conformation of the enzyme. In addition, the pH reaction may affect the affinity between the enzyme and its substrate . The optimal activity of Pyrococcus furiosus β-glucosidase was also exhibited at pH 6.0 but was inactivated at extreme pH values (pH 4.0 and 9.0) . Fusarium oxysporum β-glucosidase activity was optimal at pH 5.0 and stable at pH between 4.0 and 7.0 . It is also worth mentioning that the retention of maximum activity over a wide pH range of 3.5–7.0 indicates that β-glucosidase from Aspergillus sp. DHE7 may be suitable for the processing of various dairy products.
Measurement of Aspergillus sp. DHE7 β-glucosidase thermostability as a function of time and temperature showed that the enzyme was fully stable at temperatures ranging from 30 to 60 °C up to 60 min. A slight decrease in activity was observed at 70 °C after 30 min of incubation. The enzyme retained about 55% of the initial activity at 80 °C after 60 min of incubation. While about 50% of the activity was observed at 90 °C after 15 min of incubation, then activity decreased rapidly, reaching only 23% at the same temperature after 30 min of incubation. These results showed that Aspergillus sp. DHE7 β-glucosidase was much more stable than β-glucosidase purified from T. harzianum, which remained stable for only 15 min at temperatures below 55 °C and maintained 36% of its initial activity at 60 °C after 15 min . Sporidiobolus pararoseus β-glucosidase was able to maintain its activity at 40 °C for 60 min while retaining only 30% of the initial activity at higher temperatures .
Glycosidases may be divided into three groups on the basis of substrate specificity: (i) aryl-β-glucosidase (which hydrolyzes exclusively aryl-β-glucosides), (ii) cellobiase (which hydrolyzes only cellobiose and short-chain cello-dextrins), and (iii) broad specificity of β-glucosidases (which show activity on both substrate types). However, the relative activity against cellobiose and aryl-β-D-glucosides depends on the source of the enzyme . Plant et al.  reported that β-glucosidase preferred aryl glycoside substrates due to high electrophilicity of aglycone moiety, which improves the stability of the ortho or para-nitrophenoxide anion produced during the initial stage of catalysis. In current research, β-glucosidase purified from Aspergillus sp. DHE7 showed a broad specificity type as it exhibited high reactivity towards p-nitrophenyl-β-D-glucopyranoside, cellobiose, salicin, lactose and p-NP-β-D-galactopyranoside. These results suggested that Aspergillus sp. DHE7 β-glucosidase is a broad spectrum enzyme and may have a potential application in various biotechnological fields. β-glucosidase purified from Talaromyces thermophiles showed affinity against lactose, maltose, and cellobiose with 75, 61, and 6% relative activity, respectively . The broad substrate specificity of β-glucosidase can be attributed to the wide and extended cavity structure surrounding the active-center cavity as determined by homology modelling . Generally, the high specificity of Aspergillus sp. DHE7 β-glucosidase justifies its suitability for the enrichment of cellulolytic complexes defective in β-glucosidase, especially that of Trichoderma.
The effect of substrate concentration on the velocity of the reaction was tested with p-NPG to determine whether or not β-glucosidase is an allosteric enzyme and also to calculate the apparent Km value. Obtaining hyperbolic rather than sigmoid saturation kinetics indicates that Aspergillus sp. DHE7 β-glucosidase does not appear to be an allosteric enzyme. The apparent Km and Vmax values were 0.4 mM and 232.6 U/mL, respectively. The lower Km and higher Vmax of Aspergillus sp. DHE7 β-glucosidase enzyme revealed the high affinity of the enzyme toward p-NPG and the higher catalytic capacity of the enzyme. β-glucosidase produced by thermophilic fungus Chaetomium thermophilum hydrolysed cellobiose and p-NPG with apparent Km values of 3.13 mM and 0.76 mM, respectively . In addition, Saha and Bothast  reported the production of β-glucosidase by Candida peltata which hydrolysed p-NPG and cellobiose with Km values of 2.3 and 66 mM, respectively. Km values between 0.1 and 44 mM p-NPG have been reported for fungal β-glucosidases, and these variations could be related to differences in enzyme assay conditions and substrate preferences .
Among the different metal ions and chemical reagents tested, an increase in enzyme activity was reported with Zn2+, K+, Mg2+, and SH-modifying β-mercaptoethanol reagent, while a slight increase in enzyme activity was observed with Na+, Ca2+, and Fe2+. On the other hand, moderate inhibition was observed by Mn+, Pb2+, Hg2+, Cu2, and Co+. The thiol group inhibitor Ag+ had strongly inhibited B-glucosidase activity, indicating that SH-groups are essential for enzymatic activity and may be located at the active site . The metal chelating agent EDTA did not inhibit β-glucosidase activity, suggesting that the enzyme is not a metalloprotein. In accordance with these results, Shipkowski and Brenchley  found that EDTA treatment had no effect on β-glucosidase produced by Paenibacillus sp. Strain C7. The activity of β-glucosidases purified from different fungi have been was found to be strongly activated by Zn2+  while inhibited by Hg2+, Cu2+, Ag+, Hg2+, Pb2+, and Cd2+ . Zhang et al.  investigated that Ca2+, Mg2+, and Zn2+ had no significant impact on the activity of A. oryzae β-glucosidase while Cu2+ ion strongly inhibited the enzyme activity. In addition, the inhibition of A. ornatus β-glucosidase in the occurrence of Ag2+ and Fe2+ was reported by Yeoh et al. . Peralta et al.  reported that the addition of different metal ions (Mg2+, Ca2+, Co2+, Al2+, Cu2+, Zn2+, and Mn2+) had no effect on β-glucosidase activity, while Hg2+ and Ag2+ had virtually inhibited the activity.
In studies with β-glucosidase, it is important to determine the impact of ethanol on β-glucosidase activity as this enzyme is exposed to significant concentrations of alcohol in a number of industrial applications. In addition, β-glucosidase can catalyze under certain conditions the transglycosylation reaction in the presence of ethanol . In the current study, the β-glucosidase activity on p-NPG was evaluated in the presence of ethanol up to 40% (v/v). Data revealed that concentrations of ethanol up to 15% improved the activity of the enzyme by 25% compared to the original activity. Moreover, β-glucosidase could retain an activity level similar to control at a concentration of 20% ethanol. Taking into consideration that the final concentration of ethanol produced by conventional methods in fermented broths is about 10%, it is suggested that the current Aspergillus sp. DHE7 β-glucosidase is fairly stable for use in ethanol-containing industrial processes and in saccharification processes for bioethanol production . Arévalo et al.  investigated that an improvement in the enzyme catalytic potential in the presence of ethanol might be attributed to the activity of glucosyl transferase. During the enzymatic catalysis, ethanol can enhance the reaction rate by acting as an acceptor of glycosyl residues. Transglycosylation and hydrolysis proceed along the same biochemical pathway, varying only in the final acceptor nature . In addition, Mateo and Di Stefano  reported that changes in medium polarity induced by alcohols could stabilize the conformation of the enzyme.