A Neurospora crassa ÿ-glucosidase with potential for lignocellulose hydrolysis shows strong glucose tolerance and stimulation by glucose and xylose

Abstract Product inhibition of y-glucosidases is one of the principal factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis, particularly at high-solids concentrations. The availability of y-glucosidases with high catalytic efficiency for cellobiose hydrolysis at relatively low temperatures is of major importance for the development of efficient and cost-effective simultaneous saccharification and fermentation (SSF) processes. The gene encoding the y-glucosidase from Neurospora crassa ( gh1-1 ) was cloned and expressed in soluble form in Escherichia coli . The recombinant enzyme (GH1-1) was monomeric (54.2 kDa) and showed optimal temperature and pH of 40⿿45 °C and 5.5⿿6.5, respectively. Moreover, activities around 70% of the maximal were maintained at pH 5.0 and 35 °C. The enzyme was highly stable at pH 5.5⿿8.0 and 35 °C, and showed a half-life of 70 min at 40 °C in water. GH1-1 showed similar apparent affinities for cellobiose (0.21 ± 0.01 mmol L ⿿1 ) and p -nitrophenyl-y- d -glucopyranoside ( p NP-Glc) (0.28 ± 0.01 mmol L ⿿1 ) but hydrolyzed cellobiose with 3.2-fold higher maximal velocity (52.0 ± 3.1 U mg ⿿1 ) and 4.3-fold higher catalytic efficiency (223.8 L mmol ⿿1  s ⿿1 ). Hydrolysis of p NP-Glc by GH1-1 was maximally stimulated 1.8 and 2.0-fold by glucose and xylose at 100 and 150 mmol L ⿿1 concentration, respectively. Moreover, the enzyme was tolerant to glucose up to 950 mmol L ⿿1 and xylose up to 910 mmol L ⿿1 . Xylose (60⿿80 mmol L ⿿1 ) also stimulated the cellobiase activity of GH1-1 about 3.6-fold. Altogether, the characteristics of GH1-1 reveal its excellent potential for application as a component of enzymatic cocktails for the hydrolysis of lignocellulosic biomass, both in separate hydrolysis and fermentation and SSF processes.