초록
Efficient cellulose saccharification is crucial for the cost-effective production of ethanol from lignocellulosic biomass. Most β-glucosidases are inhibited by glucose, and high enzyme loads are needed to attain acceptable hydrolysis yields, increasing the process costs. Therefore, glucose-stimulated β-glucosidases with high catalytic efficiency for cellobiose hydrolysis are highly interesting, particularly those produced at low cost using convenient expression systems. The bglhi gene from Humicola insolens RP86, which encodes a glucose- and xylose-stimulated β-glucosidase (Bglhi), was cloned and expressed in E. coli. The recombinant Bglhi was expressed in soluble form and dynamic light scattering showed that the protein was monomeric (56.1kDa). The enzyme showed broad substrate specificity, with optima of pH and temperature of 5.0-7.0 and 60<SUP>o</SUP>C, and was stable from pH 4.5 to 8.0, and for 1h at 50<SUP>o</SUP>C in water. The recombinant Bglhi was highly stimulated by glucose and xylose. About 2-fold stimulation of pNP-glucosidase activity occurred at 50mmolL<SUP>-1</SUP> glucose or 100mmolL<SUP>-1</SUP> xylose. In addition, stimulation was observed over 50-370mmolL<SUP>-1</SUP> glucose or 100-1000mmolL<SUP>-1</SUP> xylose. Xylose maximally stimulated (2.3-fold) the cellobiase activity of the enzyme at 80mmolL<SUP>-1</SUP> concentration. The V<SUB>max</SUB> for cellobiose hydrolysis (183.4+/-12.0Umg<SUP>-1</SUP>) was 5-fold higher than that for pNP-Glc (36.4+/-1.4Umg<SUP>-1</SUP>), but a lower K<SUB>M</SUB> for pNP-Glc (0.20+/-0.01mmolL<SUP>-1</SUP>), compared to cellobiose (0.38+/-0.01mmolL<SUP>-1</SUP>), was estimated. More importantly, the recombinant Bglhi presented the highest catalytic efficiency for cellobiose hydrolysis (k<SUB>cat</SUB>/K<SUB>M</SUB>=453.0+/-29.6s<SUP>-1</SUP>mmol<SUP>-1</SUP>L) among all glucose-stimulated and glucose-tolerant β-glucosidases known to date. Together, these properties indicate that the recombinant Bglhi is an attractive candidate to include in enzymatic cocktails for application in industrial processes for the saccharification of lignocellulosic materials.