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

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2015-12, Vol.122, p.131-140
• Main Authors: Meleiro, Luana Parras, Salgado, José Carlos Santos, Maldonado, Raquel Fonseca, Alponti, Juliana Sanchez, Zimbardi, Ana Lucia Ribeiro Latorre, Jorge, João Atílio, Ward, Richard John, Furriel, Rosa Prazeres Melo
• Format: Article
• Language: English
• Subjects: Glucose- and xylose-stimulation; Lignocellulose saccharification; Neurospora crassa; Recombinant ÿ-glucosidase; Simultaneous saccharification and fermentation
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2015.09.003

[Display omitted] ⿢N. crassa glucose⿿xylose stimulated ÿ-glucosidase (GH1-1) was expressed in E. coli.⿢GH1-1 was stimulated 1.8 to 2-fold by 100mmolL⿿1 glucose or 150mmolL⿿1 xylose.⿢GH1-1 was not inhibited by glucose up to 950mmolL⿿1 or xylose up to 910mmolL⿿1.⿢GH1-1 hydrolyzed cellobiose with high catalytic efficiency, at 40°C.⿢The cellobiase activity of GH1-1was stimulated 3.6-fold by 60mmolL⿿1 xylose. Product inhibition of ÿ-glucosidases is one of the principal factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis, particularly at high-solids concentrations. The availability of ÿ-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 ÿ-glucosidase from Neurospora crassa (gh1-1) was cloned and expressed in soluble form in Escherichia coli. The recombinant enzyme (GH1-1) was monomeric (54.2kDa) 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 70min at 40°C in water. GH1-1 showed similar apparent affinities for cellobiose (0.21±0.01mmolL⿿1) and p-nitrophenyl-ÿ-d-glucopyranoside (pNP-Glc) (0.28±0.01mmolL⿿1) but hydrolyzed cellobiose with 3.2-fold higher maximal velocity (52.0±3.1Umg⿿1) and 4.3-fold higher catalytic efficiency (223.8Lmmol⿿1s⿿1). Hydrolysis of pNP-Glc by GH1-1 was maximally stimulated 1.8 and 2.0-fold by glucose and xylose at 100 and 150mmolL⿿1 concentration, respectively. Moreover, the enzyme was tolerant to glucose up to 950mmolL⿿1 and xylose up to 910mmolL⿿1. Xylose (60⿿80mmolL⿿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.