Abstract
β-Glucosidases (Bgls) convert cellobiose and other soluble cello-oligomers into glucose and play important roles in fundamental biological processes, providing energy sources in living organisms. Bgls are essential terminal enzymes of cellulose degradation systems and attractive targets for lignocellulose-based biotechnological applications. Characterization of novel Bgls is important for broadening our knowledge of this enzyme class and can provide insights into its further applications. In this study, we report the biochemical and structural analysis of a Bgl from the hemicellulose-degrading thermophilic anaerobe Thermoanaerobacterium saccharolyticum (TsaBgl). TsaBgl exhibited its maximum hydrolase activity on p-nitrophenyl-β-d-glucopyranoside at pH 6.0 and 55 °C. The crystal structure of TsaBgl showed a single (β/α)8 TIM-barrel fold, and a β8-α14 loop, which is located around the substrate-binding pocket entrance, showing a unique conformation compared with other structurally known Bgls. A Tris molecule inhibited enzyme activity and was bound to the active site of TsaBgl coordinated by the catalytic residues Glu163 (proton donor) and Glu351 (nucleophile). Titration experiments showed that TsaBgl belongs to the glucose-tolerant Bgl family. The gatekeeper site of TsaBgl is similar to those of other glucose-tolerant Bgls, whereas Trp323 and Leu170, which are involved in glucose tolerance, show a unique configuration. Our results therefore improve our knowledge about the Tris-mediated inhibition and glucose tolerance of Bgl family members, which is essential for their industrial application.
Highlights
Publisher’s Note: MDPI stays neutralBiomass-derived fuels are believed to play major roles in the global energy future, and technological development driving down costs are important for competing with petroleum and other alternative fuels [1]
Cellulose, a polymer consisting of glucose that is contained in lignocellulose, is inherently recalcitrant for enzymatic degradation and densely packed and arranged, forming a highly crystalline and insoluble structure [4]
The protein was eluted at a molecular mass position of approximately 50 kDa during size exclusion chromatography, which was almost identical to the theoretical molecular weight of TsaBgl (51.728 kDa), indicating that TsaBgl was a monomer in solution (Figure 1a)
Summary
Biomass-derived fuels are believed to play major roles in the global energy future, and technological development driving down costs are important for competing with petroleum and other alternative fuels [1]. Cellulose, a polymer consisting of glucose that is contained in lignocellulose, is inherently recalcitrant for enzymatic degradation and densely packed and arranged, forming a highly crystalline and insoluble structure [4]. Such a microfibril structure prevents cellulolytic enzymes from accessing the β-1,4-glycosidic bonds in cellulose [4]. Thermoanaerobacterium saccharolyticum is a hemicellulose-degrading thermophilic anaerobe and a biological catalyst for the conversion of cellulosic biomass to ethanol [21] This organism grows at temperatures ranging from 30 to 66 ◦ C and at pH values ranging from 3.85 to 6.35 [21]. Our results showed that the favorable properties of TsaBgl could be used in the field of biotechnology and provide useful information to broaden our structural knowledge of the Bgl family
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