Glutantßase: a database for improving the rational design of glucose-tolerant ß-glucosidases.

Β-glucosidases are key enzymes used in second-generation biofuel production. They act in the last step of the lignocellulose saccharification, converting cellobiose in glucose. However, most of the ß-glucosidases are inhibited by high glucose concentrations, which turns it a limiting step for industrial production. Thus, ß-glucosidases have been targeted by several studies aiming to understand the mechanism of glucose tolerance, pH and thermal resistance for constructing more efficient enzymes. In this paper, we present a database of ß-glucosidase structures, called Glutantßase. Our database includes 3842 GH1 ß-glucosidase sequences collected from UniProt. We modeled the sequences by comparison and predicted important features in the 3D-structure of each enzyme. Glutantßase provides information about catalytic and conserved amino acids, residues of the coevolution network, protein secondary structure, and residues located in the channel that guides to the active site. We also analyzed the impact of beneficial mutations reported in the literature, predicted in analogous positions, for similar enzymes. We suggested these mutations based on six previously described mutants that showed high catalytic activity, glucose tolerance, or thermostability (A404V, E96K, H184F, H228T, L441F, and V174C). Then, we used molecular docking to verify the impact of the suggested mutations in the affinity of protein and ligands (substrate and product). Our results suggest that only mutations based on the H228T mutant can reduce the affinity for glucose (product) and increase affinity for cellobiose (substrate), which indicates an increment in the resistance to product inhibition and agrees with computational and experimental results previously reported in the literature. More resistant ß-glucosidases are essential to saccharification in industrial applications. However, thermostable and glucose-tolerant ß-glucosidases are rare, and their glucose tolerance mechanisms appear to be related to multiple and complex factors. We gather here, a set of information, and made predictions aiming to provide a tool for supporting the rational design of more efficient ß-glucosidases. We hope that Glutantßase can help improve second-generation biofuel production. Glutantßase is available at http://bioinfo.dcc.ufmg.br/glutantbase .

Purification and characterization of ginsenoside-beta-glucosidase from ginseng.

The ginsenoside-beta-glucosidase that hydrolyzes the beta-(1-->2)-glucoside of the ginsenoside Rg3 sugar moiety to ginsenoside Rh2 was isolated from the ginseng root, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 59 kDa. The optimum temperature of the ginsenoside-beta-glucosidase was 60 degrees C, and the optimum pH was 5.0. Ca2+ ion had positive effect on ginsenoside-beta-glucosidase, while Cu2+ had negative effect on it. The ginsenoside-beta-glucosidase may be a special beta-glucosidase that is different from the original exocellulase such as beta-glucosidase (EC 3.2.1.21).

Preparation and properties of thermoreversible, phase-separating enzyme-oligo(N-isopropylacrylamide) conjugates.

A thermoreversible N-isopropylacrylamide (NIPAAm) oligomer with an N-hydroxysuccinimide (NHS) ester functional end group has been prepared for coupling to an enzyme, beta-D-glucosidase, to form a thermoreversible, phase-separating polymer-enzyme conjugate. This conjugate can be used for separation, recovery, and recycle of an enzyme simply by applying small temperature changes to the reaction medium. In contrast to the random polymer-enzyme conjugates previously reported by us and others in the literature, in this study the conjugate is formed by a single, end attachment of each oligomer chain to the enzyme. Preliminary studies show that the conjugated enzyme exhibits very high retention of activity, even higher than native enzyme, and shows improved thermal stability compared to native enzyme.