Structural basis for glucose tolerance in GH1 ß-glucosidases.

Product inhibition of ß-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some ß-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal ß-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the -1 subsite. The GH1 family ß-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

Characterization of the co-purified invertase and ß-glucosidase of a multifunctional extract from Aspergillus terreus.

The filamentous fungus Aspergillus terreus secretes both invertase and ß-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a ß-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²âº (161 %), Co²âº (68 %) and Mg²âº (61 %) and was inhibited by Al³âº, Ag⁺, Fe²âº and Fe³âº. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K(M) = 22 mM). However, in the presence of Mn²âº, the apparent affinity and V(max) for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V(max) was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and ß-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties.

The thermophilic fungus Chaetomium thermophilum var. coprophilum produced large amounts of extracellular and intracellular beta-glucosidase activity when grown on cellulose or cellobiose as carbon sources. The presence of glucose in the culture medium drastically decreased the level of beta-glucosidase activity, while cycloheximide prevented the induction of the extracellular enzyme activity by cellobiose. An extracellular beta-glucosidase induced by avicel was purified by a procedure involving acetone precipitation and chromatography on two DEAE-cellulose columns. The purified enzyme was a basic protein, with a carbohydrate content of 73%. The deglycosylated enzyme exhibited a molecular mass of 43 kDa, with pH and temperature optima of 5.5 and 65 degrees C respectively. The beta-glucosidase hydrolysed only cellobiose and p-nitrophenyl-beta-D-glucopyranoside, exhibiting apparent Km values of 3.13 mM and 0.76 mM, respectively. The native purified enzyme was stable up to 2 hours at 60 degrees C, and its thermal stability was directly dependent on glycosylation.