β-glucosidase (EC 3.2.1.21; BG) cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. In an effort towards designing better BGs, we focused on the role of non-conserved residues across an otherwise homologous BG active site tunnel and designed mutants across the aglycone-binding site (V169C) and the gatekeeper residues (I246A) of the active site tunnel. We expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase (BG) in Halothermothrix orenii. The overexpressed and purified wild-type (B8CYA8) has a high specific activity of 345 μmol/min/mg on pNPGlc and a half-life of 1.13 h when assayed with pNPGlc at pH 7.1 and 70 °C. The specific activities of V169C and I246A were 1.7 and 1.2 times higher than that of wild-type (WT) enzyme with the model substrate pNPGlc, while the activity on the natural substrate cellobiose was slightly higher to the WT. The two mutants were kinetically stable with 4.4- to 11-fold longer half-life compared to the WT enzyme. When the two mutations were combined to generate the V169C/I246A mutant, the specific activity increased to nearly twofold higher than WT on both substrates and the half-life increased fivefold. The two single mutants also show enhanced saccharification of insoluble natural biomass on supplementation of Trichoderma viride cellulase cocktail. These enhanced properties suggest the need for a closer look at the active site tunnel of these enzymes, especially across residues that are not conserved towards improving catalytic efficiencies.

β-Glucosidase (BG) is widely applied in the biofuel's industry, as part of a cellulase cocktail to catalyze the hydrolysis of the β-1,4 linkages that join two glucose molecules in a cellulose polymer. The hydrolysis step is generally recognized as the major limiting step in the development of efficient enzyme-based technologies for the conversion of lignocellulosic biomass to sugars and the production of biofuels due to the accumulation of the reaction product, glucose. Relieving this glucose inhibition of BG is therefore a major challenge. In this study, O08324, a putative BG gene encoded in the hyperthermophilic archaeon Thermococcus sp., was cloned and overexpressed in Escherichia coli. O08324 showed maximum activity between pH 5-6.8 and at 78 °C and was thermostable with a half-life of 860 min at 78 °C in the presence of 1.5 M glucose. O08324 was not inhibited by glucose up to the highest assayable concentration of 4 M and also shows no decrease in activity in the presence of up to 4 M of sodium chloride or potassium chloride. O08324 supplementation of Trichoderma viride cellulase enhanced glucose production by more than 50 % compared to a commercially available BG, when Avicel (10 %, w/v) was used as a substrate at 37 °C. Multiple sequence alignments across previously reported glucose-tolerant BGs shows that many conserved residues previously implicated in glucose tolerance are not conserved in this BG suggesting a need for a relook at understanding the molecular basis of glucose tolerance.

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic β-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of β-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for β-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

A B S T R A C T The cation specific ionophore A23187 (To) is a useful tool for studying the role of intracellular Ca++ (Ca++)i in physiologic processes. The present studies explore the role of (Ca++)i on Na transport in the toad bladder. Scraped

β-Glucosidase (EC 3.2.1.21) plays an essential role in the removal of glycosyl residues from disaccharide cellobiose to produce glucose during the hydrolysis of lignocellulosic biomass. Although there exist a few β-glucosidase that are tolerant to large concentrations of glucose, these enzymes are typically prone to glucose inhibition. Understanding the basis of this inhibition is important for the production of cheaper biofuels from lignocellulose. In this study, all - atom molecular dynamics simulation at different temperatures and glucose concentrations was used to understand the molecular basis of glucose inhibition of GH1 β-glucosidase (B8CYA8) from Halothermothrix orenii . Our results show that glucose induces a broadening of the active site tunnel through residues lining the tunnel and facilitates the accumulation of glucose. In particular, we observed that glucose accumulates at the tunnel entrance and near the catalytic sites to block substrate accessibility and inhibit enzyme activity. The reduction of enzyme activity was also confirmed experimentally through specific activity measurements in the presence of 0–2.5 M glucose. We also show that the increase in glucose concentrations leads to a decrease in the number of water molecules inside the tunnel to affect substrate hydrolysis. Overall, the results help in understanding the role of residues along the active site tunnel for the engineering of glucose-tolerant β-glucosidase.