**4.3** β**-Glycosidase recombinant**

Ramani et al. [32] obtained a β-glucosidase (rBgl4) of *Penicillium funiculosum* successfully expressed in the expression system of *Pichia pastoris* KM71H. The recombinant protein rBgl4, after purified presented a weight of ~130 kDa. The rBgl4 activity test at different pH showed ideal activity at pH 5.0 and temperature of 60°C. The enzyme exhibited a high substrate conversion rate for p-nitrophenylβ-glucosidase and cellobiose, being 3,332 and 2,083 μmol/min/mg, respectively. In addition, rBgl4 demonstrated glucose concentration tolerance of up to 400 mM.

A two-fold increase in glucose yield was observed when supplemented with crude cellulase of *Trichoderma reesei* Rut-C30 in cellulose hydrolysis, suggesting that the recombinant enzyme is a term β-glucosidase and glucose tolerant, and maybe a potential complement to commercial cellulases in cellulose hydrolysis, ensuring profitability in bioethanol production [32].

The gene of a β-glycosidase (bglI) of *Aspergillus niger* NL-1, expressed in *Pichia pastoris,* was obtained by Zhao et al. [30]. The recombinant enzyme showed high activity at pH 4.0 and temperature 60°C and was stable in a pH range of 3.0 to 7.0 and held more than 85% of activity after incubation at 60°C for 30 minutes. The β recombinant glucosidase presented molecular mass of 121 kDa. The authors determined glucose production from avicel compared to recombinant β-glycosidase, where, without the addition of recombinant β-glucosidase, glucose yield was only 49.3%, while with the addition of recombinant β-glucosidase, glucose yield was 63.4%, 70.5% and 78.6%, corresponding to 0.5, 0.75 and 1.0 U/mL, respectively. The results also indicate that BGLI was high glucose tolerant and organic solvent, presenting higher efficiency in the hydrolysis of cellobiose than β-glucosidases.

#### *Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse DOI: http://dx.doi.org/10.5772/intechopen.98363*

This study points to the use of β-glycosidase to improve the enzymatic conversion of cellulose to glucose through synergistic action.

Zhao et al. [31] expressed in *Pichia pastoris* a thermostable beta glycosidase of the thermophilic fungus *Myceliophthora thermophila*. The molecular mass of the enzyme after purification was 130.0 kDa the recombinant enzyme (MtBgl3b) MtBgl3b presented pH 5.0 as the ideal for activity at 60°C, and excellent thermostability at 60 or 65°C. The authors also determined the effects of some metal ions and chemical reagents on the activities of MtBgl3b, where Ca2+, Pb2+, K+ , Mn2+, EDTA, β-ME and Triton X-100 improved the activity by 6.4–29.9%, while Fe3+ completely suppressed the enzymatic activity. In addition, the activities of MtBgl3b were determined in relation to different substrates, for which the enzyme had higher activity against pNPG (258.7 U mg − 1), followed by pNPC (164.5 U mg-1), celotetraosis (125.7 U mg-1), celotriosis (118.0 U mg-1), celobiosis (62.2 U mg-1) and gentilebiosis (63.9 mg U-1). These results indicate that the enzyme presented desirable industrial properties, in addition to thermostability, wide spectrum of substrates and the capacity resistant to ethanol, which makes this protein a great candidate for industrial applications [30].

A β-glucosidase from *A. niger* was successfully expressed in *P. pastoris* and recombinant produced gentileoligosaccharides from glucose. In addition, the main operating parameters of this enzymatic conversion were optimized. At 80% glucose, 60°C, pH 4.5, 1 mmol/ L K +, 60 U of beta-glucosidase per gram of substrate and reaction time of 48 h, the gentiooligosaccharides produced reached 50 g/L [69].
